N-MERCAPTOACYL PHENYLALANINE DERIVATIVES, PROCESS FOR THEIR PREPARATION, AND PH ARMACEUTICAL COMPOSITIONS CONTAINING THEM Field of the Invention The present invention relates to thioi 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 regulator 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 11, 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 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 endotheliai 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. wherein: R is a mercapto group or a R4COS group convertible in an organism to a mercapto group;

R, 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, C1-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 C1-C4alkyl 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 Ri ; R4 is a straight or branched C1-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, 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 C1 6alkyl ; R2 represents pyrazol or pyrimidine ; or a pharmaceutical acceptable derivative thereof.

Further aspects of the invention are: - A pharmaceutical composition comprising a compound of the invention together with a pharmaceutical 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 therapeutical 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 C1 4alkyl, more preferably C3 4alkyl, most preferably isopropyl.

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

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 pharmaceutical 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, 5th 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-y l-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 pharmaceutical 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-phenylalanine ; N-[(2S)-2-(mercaptomethyl)-4-methylpentanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine ; N-[(2S)-2-(mercaptomethyl)-3-methyibutanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine ; N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-4-yl)-L-phenylalanine ; and pharmaceutical 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 pharmaceutical acceptable derivative thereof in association with a pharmaceutical 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 pharmaceutical 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 pharmaceutical 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 pharmaceutical 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:

wherein R'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. 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 P'represents C1 6alkyl, 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 Fournie-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) :

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 (bill) : 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 C1 6alkyl, 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 P'is an oxygen protecting group e. g. methyl, B represents boron, and p2 is an amino protecting group, such as Boc, by treatment with pyrazol 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 pyrazol may be prepared from compounds of formula (V):

wherein P'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. PdCis, 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)

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 (Vil) 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 (Vil) 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 HCI 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. Pic12. 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 P'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 (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. Pic12, 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): 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 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 (XV) may be prepared by reaction of compounds of formula (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) : R2 H (XVIl) wherein R2 is pyrazol or pyrimidine, preferably pyrazol, 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 P'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) :

by reaction with X, e. g. 12, in the presence of peracetic acid and an acid e. g. H2SO4 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. SOC2 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. 12, 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, chlorotrityl). 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 ter-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-butoxvcarbonyl)-4- (l H-pyrazol-1-vi)-L-phenvialaninate A mixture of the boronic acid* (1) (1. 0g, 3. 09mol), pyrazol (0.2g, 3. 09mmol), copper acetate (0.82g, 4. 64mmol), pyridine (0. 5ml, 6. 18mmol), TEMPO (0.482g, 3. 09mol), 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. l. Lazarova; J. Org. Chem. , 1999,64, 2976 Intermediate 3: Methyl 4- (1 H-pyrazol-1-yl)-L-phenylalaninate 4M HCL in dioxane (1 Oml) 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 30mi), 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-butoxvcarbonyl)- 4-pyrimidin-5-yl-L-phenylalaninate The boronic acid* (1) (3. 0g), 5-bromopyridine (1.77g), PdCl2 [dppf] (0.39g) and potassium carbonate (5.1g) were mixed in degassed DME (45moi). 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-pvrimidin-5-yl-L-phenyialaninate The carbamate (4) (1.12g) was dissolved in dichloromethane (20ml) and treated with 4M HCI 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-ff2- (trimethylsilyl) ethoxylmethyll-1 H-pyrazole To the protected pyrazol&num (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 (10m1), 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), Mu+ 325 &num N. Fugina, W. Holzer, M. Wasicky; Heterocycles, 1992,34, 303

Intermediate 8: Methyl N-(tert-butoxycarbonyl)-4-(1-{[2-(trimethylsilyl)ethoxy]meth yl}-1H-pyrazol-5-yl)-L- phenylalaninate To a degassed solution of the pyrazol intermediate (7) (0.5g, 1. 54mmol) and the boronic acid* (1) (0.415g, 1. 28mol), potassium carbonate (0.89g, 6. 43mmol) in DME (5ml) was added PdC12 [dppf] (53mg, 0. 06mol). 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. l. Lazarova; J. Org. Chem. , 1999,64, 2976 Intermediate 9: Methyl 4-(1 H-pvrazol-5-yl)-L-phenvlalaninate To the phenylalanine derivative (8) (0.38g, 0. 8mmol) was added ethanol (6ml) and 2N HCI (12moi). 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-butoxvcarbonyl)-4- (1-trityl-1H-pvrazol-4-vl)-L-phenVlalaninate To a mixture of bromopyrazole (11) (1. 0g, 2. 56mol), 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. 1mmol). 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 13: Methyl 4- (1 H-pyrazol-4-vl)-L-phenvlalaninate The carbamate (12) (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 Synthesis of Intermediates for Examples 1-5: Intermediate 15: (R)-4-Benzvl-3- (2-hydroxymethyl-3-methvl-butanoyl)-oxazolidinone 4-Benzyl-3- (3-methylbutanoyl)-oxazolidinone (14) * (26. 1g, 0.1mol) 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. OM 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-hyd roxymethyl-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, 0. 8mol). 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, 160mol) 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 (500mi). The aqueous phase was extracted with a further 2 x 200ml of chloroform then acidified with 5M HCI (200ml). The product was extracted with ethyl acetate (1 x 400ml, 2 x 250moi), 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 300m followed by drying and solvent removal gave 8. 1 g (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-Acetvlthiomethvl-3-methvlbutanoic 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-methylbutanoic acid (8.09g, 61 mmol) and thiolacetic acid (13. 1ml, 183 mmol) in THF (100moi) 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 HCI. The product was extracted into DCM (3 x 300ml), the solution dried over Na2SO4 and evaporated to give an orange oil (9.6g, 83%).

Similarly prepared was (S)-2-Acetylthiomethyl-4-methylpentanoic acid Intemediate 18: Methyl N-f (2S)-2-r (acetylthio) methyll-3-methylbutanoyl)-4- (1 H-pyrazol-1-yl)-L-<BR> phenvlalaninate To (2S)-2- (acetylthio)-3-methylbutanoic acid acid (4.37g, 23mmol) in dichloromethane (25ml) was added, dropwise, thionyl chloride (2. 2moi, 30mmol) and the solution stirred at

room temperature under nitrogen for 20 hours, then concentrated in vacuo, azeotroping with further dry dichloromethane (1 Oml).

The amine hydrochloride (6g) was suspended in a stirred biphasic mixture of dichloromethane (80moi) and water (50ml) and potassium carbonate (14.5g, 105mol) 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 30minutes, 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 HCI, 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+ 418 Similarly prepared were: Intermediate 19 : Methyl N-f (2S)-2-r (acetylthio) methyll-3-methylbutanoyll-4-pyrimidin-5-vl)-L-phenylalaninat e LCMS RT=2.88min MH+ 430 Intemediate 20: Methyl N-{(2S)-2-[(acetylthio)methyl]-4-methylpentanoyl}-4-(1H-pyra zol-5-yl)-L- phenvlalaninate LCMS RT=3.15min M+ 432 Intermediate 21: Methyl N-f (2S)-2-[ (acetvithio) methyll-3-methylbutanoyl-4- (1 H-pyrazol-5-vl)-L- phenvlalaninate LCMS RT=2.76min M+ 418 Intermediate 22: Methyl N-f (2S)-2-j (acetvlthio) methyll-3-methylbutanoylT-4-(1 H-pvrazol-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. 45volumes) was heated at 80 + 5°C for 2 hours. Upon completion of the reaction the contents were acidified with conc HCI (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 905°C for 16 hours. The solution was cooled to 205°C and conc HCI (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 405°C. Thioacetic acid (0.36 Wt) was added and the mixture was stirred for at least 12 hours at 405°C. The reaction mixture was cooled to 205°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 conc HCI (1.18 Wt) then the acidified layer was extracted with isopropyl acetate (2x2volumes). The combined organic layers were then washed with water (1volumes) and the mixture concentrated to 2volumes by vacuum distillation then isopropyl acetate (4. 9volumes) was added.

(+) -Ephedrine hydrochloride (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, 0. 5equivalents) added with stirring. L- Phenylalanine (1wt) was added. The mixture was heated to 555°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 205°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 1hour and at such a rate that the temperature did not exceed 60°C. The mixture was then heated to 555°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 (1volume), 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 (1volumes) 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 (1volumes). The organic layers were combined and distilled under vacuum to give a mobile oil. Toluene (0. 5volumes) was added and the organic solution was used without further treatment in the next step.

Formic acid (1.02wt, 0. 84volumes) and toluene (0. 5volumes) were stirred at 123°C and acetic anhydride (0.86wt, 0. 8volumes) 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 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 (5volumes) and washed sequentially with water (4volumes) which was adjusted to pH9 by the addition of 0.880 ammonia solution and then water (5volumes).

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 1hour (seeded if necessary) and then to 7°C over a further 1hour. The crystallised product was aged for at least a further 1hour 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 (8minute method) RT 4.30 minutes

N-formyl-4-iodo-L-phenvlalanine Intermediate 27: N-Formyl-4-iodo-L-phenylalanine methyl ester (1wt) was suspended in methanol (5volumes) and water (5volumes) and treated with 2M sodium hydroxide (1.73 Wt) at 223°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 312°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; 82-86% w/w.

HPLC (2minute method) RT 1.31 min N-Formvl-4-(1 H-pyrazol-1-vl)-L-phenylalanine Intermediate 28: To a solution of N-formyl-4-iodo-L-phenylalanine (1wt) in NMP (2. 5volumes) stirred at 20°C was added anhydrous potassium carbonate (1.52wt, 3. 5equivalents) in portions. The batch temperature was increased to 40°C. Pyrazol (0.26wt, 1. 2equivalents) was added, followed by copper (I) iodide (0.015wt, 0. 025equivalents) and racemic trans-1, 2-Diaminocyclohexane (0.036wt, 0. 1equivalents). 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 (10volumes) was added, followed by DCM (5volumes). 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 (5volumes).

The aqueous phase was cooled to 3 3°C, and acidified by slow addition of concentrated HCI (1. 5volumes) over about 45 minutes then aged for 15minutes. The remaining concentrated HCI (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, 57-65% w/w.

HPLC (2minute method) RT 1.13 min

Methyl 4- (1 H-pyrazol-1-yl)-L-phenvlalanine hydrochloride Intermediate 29: N-Formyl-4- (1 H-pyrazol-1-yl)-L-phenylalanine (1wt) was suspended in methanol (10volumes) and stirred at 22 3°C. Acetyl chloride (0.94 wt, 3equivalents) was added dropwise at 20-50°C over about 15minutes. The resulting solution was warmed to 50 5°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 5volumes at <30°C. Isopropyl acetate (10volumes) was added and the mixture concentrated in vacuo to about 5volumes at <30°C. Isopropyl acetate (10volumes) was again added and the mixture reconcentrated in vacuo to about 5 volumes at <30°C. Isopropyl acetate (10volumes) was then added and the mixture was cooled to 0-5°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 55 5°C.

Expected yield : 85-95% theory; 92-103% w/w.

HPLC (2minuite method) RT 1.03 min 1H NMR (d6-DMSO) 3.15, 3.23 (ABX, J 6,7Hz, 2H), 3.70 (s, 3H), 4.32 (t, J 6Hz, 1H), 6.55 (dd, J 2,2. 5Hz, 1H), 7.38 (d, J 8.5Hz, 2H), 7.74 (d, J 2Hz, 1H), 7.81 (d, J 8.5Hz, 2H), 8.50 (d, J 2. 5Hz, 1 H), 8.70 (br s, 3H).

Methyl N-f (2S)-2-f (acetylthio) methyll-3-methylbutanoyl}-4- ( 1 H-pyrazol-1-yl)-L- phenylalaninate Intermediate 30: Preparation of Acid Chloride (method 1): A suspension of (S)-2-Acetylthiomethyl-3-methylbutanoic acid (+)-ephedrine salt (1.31wt) in toluene (10volumes) was treated with aqueous hydrochloric acid (1M, 6. 6volumes) and stirred vigorously for 15 minutes. The phases were separated and the (upper) organic layer was washed with water (5volumes). 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 (1ml), aged for 20minutes and analysed by HPLC (2minutes) RT: 1.33minutes acid, 1. 61 minutes Me ester/toluene). The acid chloride solution was cooled to 20-25°C and used directly in the next step.

Preparation of Acid Chloride (method 2): A solution of (S)-2-Acetylthiomethyl-3-methylbutanoic acid (3.75g) 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 (1ml), aged for 20 minutes and analysed by HPLC (2minutes) RT: 1.33 minutes acid, 1.61 minutes methyl ester). The acid chloride solution was cooled to 20-25°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 (5volumes) and treated with 2M K2CO3 (10 volumes) then stirred at 20-25° until all the solids had dissolved (up to one hour). The phases were separated and the lower aqueous layer extracted with ethyl acetate (5 volumes). The combined organic layers were treated with charcoal (0.25wt) for about 2 hours at 20-25°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 biphasic solution was treated with the toluene solution of the acid chloride (prepared above) over 20minutes keeping the temperature below 25°C. The acid chloride was rinsed in with toluene (0. 1volumes). The solution was stirred rapidly for 30minutes and the phases separated. The upper organic layer was washed with water (5 volumes) and concentrated to about 8volumes by atmospheric distillation. The solution was held at 75-80°C and slowly diluted with 2,2, 4-trimethylpentane (TMP, iso-octane, 16volumes) over about 30minutes and held at 70-80°C for 15minutes to allow crystallisation to develop. The thin slurry was cooled to 0-5°C over at least 2hours. The solid was collected by filtration, washed with cold (5°C) ethyl acetate/TMP (1: 3, 4volumes) then cold TMP (4volumes) and dried in a vacuum oven at 55°C to give the title compound as an off-white solid.

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 (5. 0g) was suspended in ethyl acetate (75ml) and treated with 2M K2CO3 (33ml) then stirred at 20-25°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 25°C. The solution was stirred rapidly for 30 minutes and the phases separated. The upper organic layer was washed with 1 M HCI (33ml) and water (33ml) then concentrated to approximately 35ml by atmospheric distillation. The solution was held at 75-80°C and slowly diluted with

2,2, 4-trimethylpentane (TMP, iso-octane, 85ml) over approximately 30 minutes and held at 70-80°C for 15 minutes to allow crystallisation to develop. The thin slurry was cooled to 0- 5°C over at least 2 hours. The solid was collected by filtration, washed with cold (5°C) ethyl acetate/TMP (1: 4, 25mi) then cold TMP (30ml) and dried in a vacuum oven at 55°C to give the title compound as an off-white solid 6.4g 87% yield.

HPLC (2minute method) RT 1.64 min Examples Example 1: N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1H-pyrazol-1 -yl)-L-phenylalanine (method 1) Methyl N-{(2S)-2-[(acetylthio) methyl]-3-methylbutanoyl}-4-(1 H-pyrazol-1-yl)-L- phenylalaninate (8.17g, 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, 196mol) 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°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 (400mut), 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. 98minutes, MH+ 362 N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1H-pyrazol-1 -yl)-L-phenylalanine (method 2) N-[(2S)-2-(acetylthiomethyl)-3-methylbutanoyl]-4-(1H-pyrazol -1-yl)-L-phenylalanine methyl ester (33.5g) 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 (8 volumes, 270ml) and stirred at 20°C under nitrogen. 32% w/w (10. 8M) NaOH (3. 5equivalents, 0.78 volumes, 26ml) was added over 10 minutes and the mixture stirred at 20°C for 60 minutes.

Once all the starting material had been consumed, tributylphosphine (0. 02equivalents, 0.012 volumes, 0. 4moi) 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) HCI (2. 7equivalents, 0.57 volumes, 19ml) over 15 minutes and aged for 15 minutes to allow crystallisation to develop. The slurry was acidified to pH 1.2 by addition of 36% w/w (11. 6M) HCI (1. 9equivalents, 0.39 volumes, 13ml). The slurry was cooled to 0-5°C and aged for 30 minutes then the solid collected by filtration, washed with cold (0-5°C) 1: 1 MeOH/water (2 x 3 volumes, 100m1) and dried in a vacuum oven at 50°C to give the title compound as a pale yellow to brown powder. 28.04g, 96.7% yield.

Similarly prepared were: Example 2: N-f (2S)-2- (mercaptomethyl)-3-methylbutanoyll-4-pVrimidin-5-vl-L-phenvl alanine from Methyl N-{(2S)-2-[(acetylthio) methyl]-3-methylbutanoyl}-4-pyrimidin-5-yl)-L- phenylalaninate LCMS RT 2.66minutes, MH+ 373 Example 3: N-i (2S)-2- (mercaptomethyl)-4-methylpentanoyll-4- (1 H-pvrazol-5-yl)-L-phenylalanine from Methyl N-{(2S)-2-[(acetylthio) methyl]-4-methylpentanoyl}-4-(1 H-pyrazol-5-yl)-L- phenylalaninate LCMS RT 2.94minutes, MH+ 376 Example 4: N-r (2S)-2- (mercaptomethyl)-3-methylbutanoyll-4- (1 H-pyrazol-5-vl)-L-phenylalanine from Methyl N-{(2S)-2-[(acetylthio) methyl]-3-methylbutanoyl}-4-(1 H-pyrazol-5-yl)-L- phenylalaninate LCMS RT=2.79min M+ 362 Example 5:

N-r (2S)-2- (mercaptomethyl)-3-methylbutanoyll-4- (1 H-pyrazol-4-vl)-L-phenylalanine from Methyl N- { (2S)-2- [ (acetylthio) methyl]-3-methylbutanoyl}-4- (1 H-pyrazol-4-yl)-L- phenylalaninate LCMS RT 2. 72minutes, MH | 362 Alternative process for Example 1: Methyl N-{(2S)-2-[(acetylthio) 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 (8volumes) and stirred at 20°C under nitrogen. 32% w/w (10. 8M) NaOH (3. 5equivalents, 0. 78volumes) was added over 5minutes and the mixture stirred at 20°C for 60minutes.

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 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. 5 by addition of 36% w/w (11. 6M) HCI (3. 4equivalents, 0. 70volumes) and aged for 10minutes to allow crystallisation to develop. The slurry was acidified to pH 1 by addition of 36% w/w (11. 6M) HCI (1. 2equivalents, 0. 24volumes). The slurry was cooled to 0-5°C and aged for 30minutes then the solid collected by filtration, washed with cold (0-5°C) 1: 1 MeOH/water (2 x 3volumes) and dried in a vacuum oven at 50°C to give N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]- 4-(1 H-pyrazol-1-yl)-L-phenylalanine as a pale yellow to brown powder.

Expected yield: 92-95% th, 80-82% w/w HPLC (2minute method) RT 1.45 min 1H NMR (d6-DMSO) 0.81, 0.86 (2d, J 7Hz, 6H), 1.58 (dd, J 7,9Hz, 1H), 1.72 (octuplet, J 7Hz, 1H), 2.15 (m, 1H), 2.42-2. 59 (m, 2H), 2.91, 2.94 (ABX, J 10,14Hz, 1H), 3.11, 3.14 (ABX, J 5,14Hz, 1H), 4.59 (m, 1H), 6.51 (t, J 2Hz, 1H), 7.38 (d, J 8Hz, 2H), 7.72 (m, 3H), 8.26 (d, J 8Hz, 1H), 8.43 (d, J 2Hz, 1H), 12.7 (brs, 1H).

Analytical Methods 8 Minute Method Parameter Value Analytical Column 50mm x 2. 0mm Luna C18 (2), 3um Mobile Phase Mobile phase A: 0.05% v/v solution of TFA in water Mobile phase B: 0.05% v/v solution of TFA in MeCN Gradient Mobile phase Mobile phase Time A (%) B (%) (minutes) 100 0 0.00 5 95 8.00 100 0 8.01 100 0 10.00 Flow Rate 1. 0ml/minute Temperature 40°C Detection UV, at 220nm Injection Volume 1µl Approximate Run Time 10 minutes 2 Minute Method Parameter Value Analytical Column 50mm x 2. 0mm Luna C18 (2), 3pm Mobile Phase Mobile phase A: 0.05% v/v solution of TFA in water Mobile phase B: 0.05% v/v solution of TFA in MeCN Gradient Mobile phase Mobile phase Time A % B (%) (minutes) 100 0 0.00 5 95 2.00 100 0 2.01 100 0 3.00 Flow Rate 2. Oml/minute Temperature 600C Detection UV, at 220nm Injection Volume 1µl Approximate Run Time 3 minutes

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 : 5pl Temp: RT UV Detection Range: 215 to 330nm Solvents: A: 0. 1% Formic Acid + 10molar Ammonium Acetate.

B: 95% Acetonitrile + 0.05% Formic Acid Gradient: Time (minutes) A% B% 0. 00 100 0 0.70 100 0 4.20 0 100 5.30 0 100 5.50 100 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 fluorescence at 320nm excitation/400nm emission.

1 jl1 of the test compound solution plus TCEP (1: 2.5 compound: TCEP) in acetonitrile/water (1: 1) was mixed with 15 jl1 of the substrate solution (176yam in 10µM TCEP solution) and 15 ; J human ACE in buffer (200pM in 50mM HEPes pH 7.4, 150mM NaCI soln, 1pLM zinc acetate, pH to 7.4 using 1N NaOH) with 101lM TCEP (final concentrations were approx. lOOpM human ACE and MCA substrate at 88uM). After 60 minutes incubation, fluorescence was read using a Tecan SpectraFluor Ultra fluorescence plate reader or equivalent at 320nm excitation/400nm emission.

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 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.

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

(i) Compound Human ACE Human Rat plasma Rabbit NEP pKi plasma ACE ACE pKi pKi pKi 1 9.0 7.1 7.1 >8. 5 2 8. 1 6.9 6.1 >8. 5 3 9. 2 6. 9 7. 1 >8. 5 4 9.0 7.4 7.2 >8. 5 5 8. 6 7. 1 6. 9 >8. 5 X* 8. 0 5. 1 5. 8 >8. 5 Y* 8. 5-->8. 5 (ii) Compound Human ACE Human Rat plasma Rabbit NEP pKi plasma ACE ACE pKi pKi pKi 1 8. 8 7. 0 7. 1 8. 8 2 8. 1 6. 9 6. 1 >8. 5 3 9.2 6.9 7.1 >8. 5 4 9. 0 7. 4 7. 2 >8. 5 5 8. 6 7. 1 6. 9 >8. 5 X* 7. 8 5. 2 5. 8 9. 1 Y* 8.5 - - >8. 5

*Compound X: N- (3-mercapto-2-phenylmethylpropionyl)-4- (2-thiazolyl)-phenylalanine Compound Y: N- (3-mercapto-2-phenylmethylpropionyl-4- (5-pyrimidinyl)-phenylalanine Note: Compounds X and Y can be prepared according to processes provided in W097/24342.

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.