EP1474405A2

EP1474405A2 - Process for the preparation of 4-hetero-substituted phenylalanine derivatives

Info

Publication number: EP1474405A2
Application number: EP03739500A
Authority: EP
Inventors: Jason William Beames Cooke; Douglas Hayes; Richard Anthony Henson; Stephen Andrew Hermitage; Richard Anthony Ward; Andrew Jonathan Whitehead
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2002-02-15
Filing date: 2003-02-14
Publication date: 2004-11-10
Also published as: WO2003068725A2; JP2005517703A; AU2003210296A1; AU2003210296A8; WO2003068725A3; GB0203665D0

Abstract

A process for preparing 4-substituted phenylalanine ester derivatives is described. Compounds of this type are known to be useful in the preparation of compounds having mixed ACE-NEP inhibitor activity.

Description

Process

Field of the Invention

The present invention relates to a process for the preparation of 4-substituted phenylalanine ester derivatives. Compounds of this type are known to be useful in the preparation of compounds having mixed ACE-NEP inhibitory activity.

Background of the Invention

Angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) are two zinc metalloproteases which involved in the metabolism of a variety of regulatory peptides and particularly those involved in the control of blood pressure and fluid homeostasis (Fournie-Zaluski et al. (1994) J. Med. Chem. 37:1070-1083). ACE, a zinc-containing carboxydipeptidase, converts the inactive precursor angiotensin I into angiotensin II, a peptide which promotes vasoconstriction and sodium retention and thereby leads to an increase in blood pressure. Compounds with angiotensin converting enzyme (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 degredation of the vasorelaxant peptide bradykinin at its endothelial and epithelial sites of action respectively. Therefore, as ACE and NEP exert their action on the cardiovascular system with different mechanisms of action, compounds with mixed ACE-NEP inhibitory activity are generally used, alone or in combination, in the treatment of hypertension, renal failure, congestive heart failure and ischemic cardiopathologies. WO97/24342 describes certain N-mercaptoacyl phenylalanine deriatives which have mixed ACE-NEP inhibitory activity and are useful in the treatment of cardiovascular diseases, such as hypertension and congestic heart failure.

Processes for the preparation of N-mercaptoacyl phenylalanine deriatives are described in WO 97/24342. Important intermediates in the preparation of N- mercaptoacyl phenylalanine derivatives are 4-substituted phenylalanine ester derivatives e.g. 4-(2-thiazolyl)phenylalanine methyl ester and 4-(1- pyrazolyl)phenylalanine methyl ester.

The problem to be solved by the present invention is the provision of an alternative process for the preparation of 4-substituted phenylalanine ester derivatives. In particular, a process without the use of iodination chemistry was desired. Summary of the Invention

Accordingly, the present invention provides a process (Route A) for the preparation of a compound of formula (IA):

(IA) or a salt thereof, wherein:

R¹ represents hydrogen, C_1-4alkyl or benzyl; R² represents hydrogen or a protecting group -COH or COC_1-4alkyl; and

R⁶ represents a 5- or 6- membered aromatic heterocycle with one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur; from a compound of formula (II):

(II) wherein:

R¹ represents hydrogen, d^alkyl or benzyl; R² represents a protecting group -COH or COC_1-4alkyl; and R⁷ represents -CN or -NH-NH₃+; by heterocycle formation, optionally followed by removal of the protecting group.

A further aspect of the invention is the use of the process of the invention in the preparation of a mixed ACE-NEP inhibitor or a pharmaceutically acceptable derivative thereof. Detailed Description of the 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.

As used herein, the term "heterocycle" means optionally substituted rings containing one or more heteroatoms selected from: nitrogen, sulphur and oxygen atoms. The heterocycle is aromatic i.e., unsaturated. Examples of 5-membered groups include thienyl, furanyl, pyrazolyl, pyrrolidinyl thiazolyl, oxazolyl and imidazolyl. Examples of 6-membered groups include pyridyl and pyrimidinyl.

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 or carbamate, or salt or solvate of such a prodrug, 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. Preferred pharmaceutically acceptable derivatives are salts, solvates and esters. Particularly preferred pharmaceutically acceptable derivatives are salts and solvates.

Examples of salts of a compound of formula (I), are the salts with alkali or alkali-earth metals and the salts with pharmaceutically acceptable organic bases. Examples of salts of a compound of formula (IA) are mineral acid salts such as hydrochloric or hydrobromic, particularly hydrochloric salts.

Preferably, R¹ represents C^alkyl, more preferably methyl.

Preferably, R² represents hydrogen, - COH or -COCH₃.

Preferably, R^β represents a 5-membered aromatic heterocycle, more preferably thiazolyl or pyrazolyl.

In another aspect, the present invention provides a process for the preparation of a compound of formula (IA) or a salt thereof having the formula (IB): or a salt thereof, wherein:

R¹ represents hydrogen, or benzyl; and

R² represents hydrogen or a protecting group -COH; from a compound of formula (II) having the formula (IIB):

(IIB) wherein:

R¹ represents hydrogen, or benzyl; and R² represents a protecting group -COH; by formation of a thioamide, followed by thiazole formation, optionally followed by removal of the protecting group.

In another aspect, the present invention provides a process for the preparation of a compound of formula (IA) or a salt thereof having the formula (IC):

or a salt thereof, wherein: R¹ represents hydrogen, or benzyl; and

R² represents hydrogen or a protecting group -COCH₃; from a compound of formula (II) having the formula (IIC):

(IIC) wherein: R¹ represents hydrogen, C_1-4alkyl or benzyl; and R² represents a protecting group -COCH₃; by formation of a pyrazole, optionally followed by removal of the protecting group.

In a preferred aspect, the present invention provides a process for the preparation of a compound of formula (IB) from a compound of formula (IIB) as defined above comprising: (i) thioamide formation by reaction with

(a) NH₄CI, and

(b) NaSH.2H₂O, or hydrogen sulfide and an organic or inorganic base (e.g. isopropylamine, triethylamine, sodium carbonate, sodium sulfide) in the presence of a strong dipolar aprotic solvent e.g. dimethylacetamide

(DMAC), DMF and/or DMSO, preferably DMAC; (ii) thiazole formation by reaction with an acetal of bromoacetaldehyde e.g. bromoacetaldehyde dimethylacetal or bromoacetaldehyde diethylacetal, in the presence or absence of an acid e.g. p-toluenesulfonic acid (p-tosic acid), at elevated temperature, suitably at 60-100°C; optionally followed by (iii) removal of the protecting group under standard conditions, e.g. with a strong acid e.g. HCI (such as acetyl chloride, thionyl chloride, HCI gas, preferably acetyl chloride) in the presence of an alkanol e.g. MeOH, at a temperature below the reflux temperature of the alcohol, preferably below 10°C.

In another preferred aspect, the present invention provides a process for the preparation of a compound of formula (IC) from a compound of formula (IIC) as defined above comprising: (i) pyrazole formation by reaction with malondialdehyde bis(dimethylacetal) under reflux at elevated temperature; optionally followed by (ii) removal of the protecting group under standard conditions, e.g. with a strong acid e.g. HCI (such as acetyl chloride, thionyl chloride, HCI gas, preferably acetyl chloride) in the presence of an alkanol e.g. MeOH, at a temperature below the reflux temperature of the alcohol, preferably below 10°C.

In the following description the substituents R¹, R², R⁶ and R⁷ are as defined above unless stated otherwise.

A compound of formula (II) may be prepared from a compound of formula (III):

(III)

A compound of formula (IIB) may be prepared from a compound of formula (III) by (i) treatment with strong inorganic acid e.g. HCI, in the presence of a source of nitrite ions e.g. sodium nitrite (NaNO₂), at a temperature below room temperature, preferably below 20°C, more preferably in the range 0-20°C, in a suitable solvent e.g. aqueous acetonitrile; followed by (ii) treatment with a source of copper +1 e.g. CuCI or CuCN and source of free cyanide e.g. KCN or NaCN, preferably potassium coppercyanide (K₂CuCN₃) is used as the source of copper +1 and cyanide, in a suitable solvent e.g. water, acetonitrile and/or DMF, in the presence or absence of potassium bicarbonate or sodium bicarbonate, preferably potassium bicarbonate, preferably at a temperature of 20-40°C.

A compound of formula (IIC) may be prepared from a compound of formula (III) by: (i) treatment with a strong inorganic acid e.g. HCI in the presence of a source of nitrite ions e.g. sodium nitrite (NaNO₂) at a temperature below room temperature, preferably below 20°C, more preferably in the range 0-20°C, in a suitable solvent e.g. aqueous acetonitrile; followed by:

(ii) treatment with a reducing agent e.g. sodium sulfite (Na₂SO₃) at a temperature below room temperature, preferably below 20°C, more preferably in the range 0- 20°C; followed by: (iii) treatment with a strong inorganic acid e.g. HCI and heating to 50-100°C, preferably approximately 60°C. A compound of formula (III) may be prepared from a compound of formula (IV):

(IV)

by reduction of the nitro group using standard methods e.g. under hydrogen using a suitable catalyst, such as palladium on carbon in a suitable solvent such as water, DCM, ethyl acetate, MeOH and/or EtOH, preferably water, ethyl acetate or DCM, preferably at a temperature in the range 20-30°C.

A compound of formula (IV) may be prepared from a compound of formula (V):

(V)

by treatment with the appropriate acid e.g. formic acid (HCOOH) or acetic acid (CH₃COOH) in the presence of a C_2-5acid anhydride, preferably acetic anhydride (Ac₂O), at a temperature preferably in the range 10-30°C.

The process of the invention can be used in the preparation of mixed ACE-NEP inhibitors by any method known in the art, for example as described in WO97/24342 and as shown in the Examples. A further aspect of the invention is therefore the use of the process of the invention in the preparation of a mixed ACE-NEP inhibitor, such as, an N-mercaptoacyl phenylalanine derivative. In other words, a mixed ACE-NEP inhibitor, such as an N-mercaptoacyl phenylalanine derivative, may be prepared using a process comprising the process of the invention. For example, a compound of formula (IA) or a salt thereof may be used in the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof:

(I)

wherein:

R³ is a straight or branched C₂-C alkyl group or an aryl or arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a 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 by one or more substituents, the same or different, selected from the group consisting of hydroxy groups, alkoxy, alkyl, alkylthio, alkylsulphonyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C C₃ alkyl groups containing one or more fluorine atoms, carboxy groups, 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 alkyl moiety; R⁴ is a R⁵COSCH₂ group convertible to a mercaptomethyl group; R⁵ is a straight or branched CrC alkyl or a phenyl group;

R⁶ is a 5- or 6- membered aromatic heterocycle with one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur.

Preferably, the mixed ACE-NEP inhibitor is N-[(S)-2-(Mercaptomethyl)-1-oxo-3- phenylpropyl]-4-(2-thiazolyl)- -phenylalanine or N-[(S)-2-(Mercaptomethyl)-3- methylbutanoyl]-4-(1 H-pyrazol-1-yl)-L-phenylalanine or pharmaceutically acceptable derivatives thereof.

A compound of formula (I) may be prepared from a compound of formula (IA) or a salt thereof by a process comprising:

(i) reaction with a compound of formula (VI):

^R θH R⁴

(VI) wherein R³ 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 a 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 by one or more substituents, the same or different, selected from the group consisting of hydroxy groups, alkoxy, alkyl, alkylthio, alkylsulphonyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C C₃ alkyl groups containing one or more fluorine atoms, carboxy groups, 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 alkyl moiety;

R⁴ is a R⁵COSCH₂ group convertible to a mercaptomethyl group; R⁵ is a straight or branched C C₄ alkyl or a phenyl group; and

(iii) treatment with a suitable base e.g. sodium hydroxide under nitrogen in a suitable solvent e.g. aqueous ethanol or methanol.

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

The steps comprising the reaction of a compound of formula (II) to form a compound of formula (IA) or a salt thereof may be carried out separately or in situ. The reaction is preferably carried out in situ.

In a further aspect of the invention (Route B), the group R² represents a compound of formula (VI) e.g. a side chain (A):

(A)

Accordingly, a compound of formula (IA) or a salt thereof in which R² represents (A) may be prepared from a compound of formula (II) in which R² represents (A) by heterocycle formation according to the methodology previously described. For example, where R⁶ is thiazole heterocycle formation may be performed by formation of a thioamide followed by thiazole formation. Preferably, the reaction comprises: (a) thioamide formation by reaction with thioacetic acid at elevated temperature, preferably 50-90°C more preferably at 60-80°C; followed by

(b) thiazole formation by reaction with an acetal of bromoacetaldehyde e.g. bromoacetaldehyde dimethylacetal or bromoacetaldehyde diethylacetal, in the presence or absence of an acid e.g. p-toluenesulfonic acid or hydrobromic acid, at elevated temperature, suitably at 60-100°C.

A compound of formula (II) in which R² represents (A) may be prepared from compounds of formula (III) in which R² represents (A) according to the methodology previously described. A compound of formula (III) in which R² represents (A) may be prepared from compounds of formula (IV) in which R² represents (A) according to the methodology previously described.

A compound of formula (IV) in which R² represents (A) may be prepared by reacting a compound of formula (VII):

with compound (V) in the presence of a suitable base e.g. aqueous sodium or preferably potassium carbonate or aqueous sodium or preferably potassium bicarbonate or triethylamine or tributylamine and in a suitable solvent e.g. DCM or EtOAc at reduced temperature, preferably 0-20°C.

A compound of formula (VII) may be prepared from a compound of formula (VIII):

by reaction with chlorinating agent such as oxalyl chloride or thionyl chloride in an inert solvent preferably DCM.

Compounds of formula (V), (VI) and (VIII) are known compounds and can be prepared by processes well known in the art. General amino chemistry is described in "The Practice of Peptide Synthesis" M Bodanszky and A Bodanszky Springer- Verlag 1984 ISBN 3-540-13471-9 and ISBN 0-387-13471-9 pp 33-34).

Compounds of formula (II) are novel compounds and hence form a further 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, such as trimethylsilyl or tert-butyldimethylsilyl; alkyl ethers such as tetrahydropyranyl or tert-butyl; or esters such as 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:

Generalised Scheme:

EXAMPLE 1 : Preparation of N-f(S)-2-(Mercaptomethvn-1 -oxo-3-phenylpropyn-4-(2- thiazolyl)-Z.-phenylalanine

Stage 2a

Stage 4

Conditions by Stage. 1 : HCOOH, Ac₂O. 2: (a) H₂, Pd/C. (b) HCI, NaNO₂, K₂Cu(CN)₃. 3: (a) NaSH, NH₄CI, DMAC. (b) BrCH₂CH(OMe)₂. (c) MeOH, AcCI.

4: (S)-ATPA, DCM, SOCI₂ then 4-(2-thiazolyl)phenylalanine methyl ester dihydrochloride, K₂CO₃. 5: NaOH, water, IMS, then HCI.

Stage 1 : Preparation of (S)-4-Nitro-N-formylphenylalanine methyl ester Acetic anhydride (200ml, 2.75eq) was added to formic acid (200ml, 7eq) keeping the temperature below 10°C and then warmed to 18-20°C over at least 15 minutes. The mixture was stirred at 18-20°C for at least 25 minutes. DCM (100ml) was added to the mixed anhydride and the mixture cooled to 10-15°C. Sodium formate (57.4g, 1.1eq) was then added and the mixture stirred for 5-10 minutes. (S)-4-Nitro- phenylalanine methyl ester hydrochloride (200g) was added keeping the temperature at 10-15°C. The reaction was warmed to 18-20°C over 20-30 minutes. Water (4 volumes) was added over 5-10 minutes to give a thick slurry. The slurry was cooled to 10°C over 1 hour and aged at this temperature for a further 1 hour. The solid was collected by filtration and washed with water (2 x 200ml) and dried in vacuo at 40°C to give the title compound as a white, crystalline solid 182g, 94% theory yield.

MS: 253 (MH⁺, 30%), 225 (40%), 193 (80%), 165 (100%).

Rotamers are apparent in the NMR spectra, data quoted for the major rotamer:

¹H NMR (250MHz, CDCI₃) δ: 8.21 (s, 1 H), 8.17 (d, J = 9, 2H), 7.30 (d, J = 9, 2H),

6.12 (br d, J = 8, 1 H), 5.02 (dt, J = 7, 6, 1H), 3.78 (s, 3H), 3.35, 3.22 (AB part of ABX,

J = 6, 14, 2H).

¹³C NMR (100MHz, CDCI₃) δ: 36.66, 51.74, 52.50, 123.59, 130.89, 145.67, 146.79.

161.45, 171.52.

Stage 2: Preparation of (S)-4-Cyano-N-formylphenylalanine methyl ester (S)-4-Nitro-N-formylphenylalanine methyl ester (20.2g) was suspended in water (80ml) and Palladium-Carbon catalyst (5%Pd, 50% water wet, 2g) added. The resulting slurry was heated to 40 to 45°C and hydrogenated until the uptake of hydrogen ceased. The warm (35°C) suspension was filtered through a glass fibre paper and the flask and filter washed with further water (20ml) to give a solution of (S)-4-amino-N-formylphenylalanine methyl ester.

MS: 223 (MH\ 50%), 178 (80%), 163 (100%).

Rotamers are apparent in the NMR spectra, data quoted for the major rotamer: ¹H NMR (250MHz, CDCI₃) δ: 8.15 (s, 1H), 6.88 (d, J = 8, 2H), 6.60 (d, J = 8, 2H), 6.10 (br d, J = 7, 1H), 4.89 (dt, J = 8, 6, 1H), 3.74 (s, 3H), 3.44 (v br s, 2H), 3.03 (d, J = 6, 2H). ¹³C NMR (100MHz, CDCI₃) δ: 37.32, 52.42, 52.80, 115.74, 125.49, 130.51, 145.98, 161.10, 172.22.

The aniline solution was cooled to below 20°C and treated with concentrated HCI (2.2eq, 15.3ml) then the solution was further cooled in an icebath and stirred until any residual solids dissolve. Sodium nitrite (1.2eq, 6.58g) in water (18ml) was added maintaining the temperature below 5°C.

In a separate flask potassium coppercyanide (1.0eq, 17.8g) and potassium bicarbonate (1.Oeq, 8g) were dissolved in water (90ml) at 20°C then treated dropwise with the diazonium solution over approximately 20-30 minutes while stirring rapidly.

The mixture was stirred at 20°C for 30-40 minutes then cooled to <5°C and aged at this temperature for a further 1 hour. The solid was collected by filtration and the cake washed with cold water (2 x 35ml) and sucked dry. The cake was dissolved in DCM (90ml) and dried over MgSO₄. The solids were removed by filtration and washed with DCM (2 x 35ml). The combined filtrates were then concentrated to give a brown solid.

The brown solid was dissolved in DCM (60ml) by heating to 30°C and the stirred solution treated dropwise with cydohexane (60ml) keeping the batch temperature at 30°C. After holding at 30°C for 20mins, the solution was seeded and held at this temperature for a further 1hour. The batch was slowly cooled to approximately 5°C over 1.5hours and aged at this temperature overnight (16 hours). The solid was collected by filtration and the cake washed with 1:1 DCMxyclohexane (2 volumes, 30ml) pre-cooled to approximately 10°C. The cake was pulled dry on the filter then further dried in vacuo to give the title compound as a pale brown solid 12.7g, 60.2% theory yield.

MS: 233 (MH\ 10%), 205 (20%), 173 (60%), 145 (100%).

Rotamers are apparent in the NMR spectra, data quoted for the major rotamer: ¹H NMR (250MHz, CDCI₃) δ: 8.20 (s, 1H), 7.60 (d, J = 8, 2H), 7.22 (d, J = 8, 2H), 6.19 (br d, J = 7, 1H), 4.99 (dt, J = 7, 6, 1 H), 3.77 (s, 3H), 3.29, 3.17 (AB part of ABX, J = 6, 14, 2H).

¹³C NMR (100MHz, CDCI₃) δ: 38.32, 51.98, 53.13, 111.58, 119.02, 130.60, 132.72, 141.80, 161.01, 171.45.

Stage 3: Preparation of (S)-4-(2-thiazolyl)phenylalanine methyl ester dihydrochloride.

(S)-4-Cyano-N-formylphenylalanine methyl ester (2.0g) was dissolved in DMAC (8ml) to give an orange solution. NH₄CI (1.1g) was then added followed by NaSH.2H₂O (0.97g). The reaction mixture was stirred for 3 hours and then 9:1 H₂O:AcOH (10ml) was added slowly over 5 minutes. The mixture was then extracted with DCM (3 x 10ml) and the combined organic extracts evaporated under reduced pressure. The resultant yellow/orange oil was then dried azeotropically with toluene (2 x 10ml) to give 4-thiocarboxamidophenylalanine methyl ester as a yellow oil (a solution in - 8ml DMAC).

A purified sample for characterisation was prepared by chromatography but was still a mixture (thioamide: DMAC 45:55 mol%):

MS: 267 (MH⁺, 100%), 250 (30%).

Rotamers are apparent in the NMR spectra, data quoted for the major rotamer: ¹H NMR (250MHz, CDCI₃) δ: 8.18 (s, 1 H), 8.02 (s, 1 H), 7.82 (d, J = 8, 2H), 7.61 (br s, 1H), 7.16 (d, J = 8, 2H), 6.11 (br s, 1H), 5.00 (dt, J = 7, 6, 1H), 3.78 (s, 3H), 3.27, 3.16 (AB part of ABX, J = 6, 14, 2H).

¹³C NMR (100MHz, CDCI₃) δ: 21.63 (DMAC), 35.16 (DMAC), 37.39, 38.13 (DMAC), 52.02, 52.38, 127.82, 128.99, 138.11 , 140.28, 161.49, 170.70 (DMAC), 171.52, 201.67.

The solution was treated with p-tosic acid (0.2g) and bromoacetaldehyde dimethylacetal (1.3ml) and then heated to 80°C for 4 hours.

Data for N-formyl Phe-thiazole prepared by formylation of Phe-thiazole:

MS: 291 (MH\ 100%), 231 (40%).

Rotamers are apparent in the NMR spectra, data quoted for the major rotamer: ¹H NMR (250MHz, CDCI₃) δ: 8.17 (s, 1H), 7.88 (d, J = 8, 2H), 7.84 (d, J = 3, 1H), 7.32 (d, J = 3, 1H), 7.19 (d, J = 8, 2H), 6.47 (br d, J = 7, 1 H), 4.99 (dt, J = 8, 7, 1 H), 3.75 (s, 3H), 3.23, 3.14 (AB part of ABX, J = 6, 14, 2H).

¹³C NMR (100MHz, CDCI₃) δ: 36.08, 50.24, 51.05, 117.38, 125.27, 128.39, 131.09, 136.21 , 142.17, 159.13, 166.41, 169.89.

The reaction was cooled to 0°C and MeOH (20ml) was added followed by acetyl chloride (1.8ml) keeping the temperature under 10°C. The reaction mixture was heated to 50°C for 3 hours then heated to 80°C and concentrated by distillation to remove MeOH and MeOAc (15ml). The slurry was diluted with toluene (20ml) then cooled to 0°C over 1 hour. The solid was collected by filtration, washed with 2:1 toluene: DMAC (2 x 4ml) and then dried in vacuo at 60°C to give the title compound as an off-white solid 2.1g, 73% theory yield. MS: 263 (MH⁺, 80%), 203 (100%).

¹H NMR (250MHz, DMSO-d6) δ: 8.97 (br d, J = 3, 3H), 7.98 (d, J = 3, 1H), 7.95 (d, J = 8, 2H), 7.85 (d, J = 3, 1H), 7.43 (d, J = 8, 2H), 4.30 (q, J = 5, 1H), 3.69 (s, 3H), 3.34, 3.22 (AB part of ABX, J = 6, 7, 14, 2H).

¹³C NMR (100MHz, DMSO-d6) δ: 35.81, 52.96, 53.41 , 121.10, 126.84, 130.74, 132.02, 137.68, 143.67, 167.37, 169.54.

Stage 4: Preparation of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4-(2- thiazolyl)-L-phenylalanine methyl ester

A solution of (S)-2-(acetylthiomethyl)-3-phenylpropanoic acid (10.86g) in DCM (22ml) was treated with thionyl chloride (3.66ml, 1.1 eq) and heated to reflux for 4 hours. The solution was cooled and the solvent evaporated to give (S)-2-(acetylthiomethyl)-3- phenylpropanoic acid chloride as a yellow oil 11.66g, 99.7% theory yield.

A suspension of 4-(2-thiazolyl)phenylalanine methyl ester dihydrochloride (13.8g), potassium carbonate (11.4g, 2eq) and DCM (100ml) was stirred and cooled to 1°C. Water (50ml) was added and the mixture stirred for 20 minutes. A solution of (S)-2- (acetylthiomethyl)-3-phenylpropanoic acid chloride (10.6g, 1.0eq) in DCM (15ml) was added dropwise over 5 minutes and rinsed in with DCM (15ml). The mixture was stirred at 1°C for 2h then warmed to 22°C and diluted with DCM (30ml). The phases were separated and the DCM solution was washed with 2M HCI (45ml) followed by water (45ml). The DCM solution was concentrated to 80ml by distillation at atmospheric pressure then diluted with isopropanol (100ml) and further distillation continued to remove most of the DCM and give a slurry. The slurry was cooled to 20°C and the solid collected by filtration then washed with isopropanol (2 x 45ml) and dried in vacuo at 50°C to give the title compound as an off-white solid 15.9g, 80.0% theory yield.

MS: 483 (MH⁺, 98%), 441 (100%).

¹H NMR (400MHz, CDCI₃) δ: 7.85 (d, J = 8, 2H), 7.84 (d, J = 3, 1 H), 7.31 (d, J = 3, 1H), 7.27-7.12 (m, 7H), 5.82 (d, J = 7, 1H), 4.78 (dt, J = 8, 6, 1H), 3.62 (s, 3H), 3.17- 3.02 (m, 4H), 2.91, 2.84 (AB part of ABX, J = 6, 9, 14, 2H), 2.61-2.54 (m, 1H), 2.32 (s, 3H).

¹³C NMR (100MHz, CDCI₃) δ: 30.61. 31.16, 37.84, 38.53, 49.53, 52.30, 53.01, 118.76, 126.58, 126.67, 128.50, 128.85, 130.00, 132.46, 138.00, 138.36, 143.69, 168.01 , 171.13, 172.48, 195.71. Stage 5: Preparation of N-[(S)-2-(Mercaptomethyl)-1-oxo-3-phenylpropyl]-4-(2- thiazolyl)- -phenylalanine

Aqueous sodium hydroxide (10M, 17ml, 3.3eq) was added, under nitrogen to a cooled, (0-5°C) stirred, slurry of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4- (2-thiazolyl)- -phenylalanine methyl ester (25g) in a mixture of denatured ethanol (IMS 125ml) and water (75ml) keeping the temperature between 0 and 5°C. The resulting slurry was stirred at 0-5°C for 24 hours and acidified to pH=7-7.5 by dropwise addition of concentrated hydrochloric acid (6ml) keeping the temperature at 0-5°C. Tributylphosphine (0.6ml) was added and the hazy solution clarified by vacuum filtration through a filter paper. The solution was then warmed to 15-20°C and stirred under nitrogen at this temperature for 45-60 minutes. The solution was acidified to pH=5 by dropwise addition of cone, hydrochloric acid (4.5ml) keeping the temperature at 15-20°C. The solution was then stirred, under nitrogen, at 15-20°C for 20-30 minutes to allow crystallisation to initiate. The slurry was acidified to pH=2 by dropwise addition of further concentrated hydrochloric acid (4.0ml) keeping the temperature at 15-20°C. The slurry was then cooled, under nitrogen, to 0-10°C and stirred at this temperature for 60-90 minutes. The solid was collected by filtration, washed with IMS/water (1 :1 , 2 x 50ml) and dried in vacuo at 40°C to give the title compound as a white solid 19.8g, 90% theory yield.

MS: 427 (MH\ 100%).

¹H NMR (400MHz, DMSO-d6) δ: 12.75 (br s, 1 H), 8.35 (d, J = 8, 1H), 7.90 (d, J = 3, 1H), 7.84 (d, J = 8, 2H), 7.75 (d, J = 3, 1 H), 7.35 (d, J = 8, 2H), 7.24 (t, J = 7, 2H), 7.18-7.13 (m, 3H), 4.54 (m, 1 H), 3.15, 2.94 (AB part of ABX, J = 4, 10. 14, 2H), 2.84 (m. 1H), 2.71-2.50 (m, 3H, partially obscured by DMSO), 2.30 (m, 1H), 1.84 (t, J = 8, 1 H).

13 ³/C NMR (100MHz, DMSO-d6) δ: 25.83, 36.95, 37.54, 50.88, 53.41, 120.51 , 126.41, 126.43, 128.55, 129.24, 130.43, 131.79, 139.50, 140.39, 144.13, 167.45, 172.90, 173.10.

EXAMPLE 2: Preparation of N-f (S)-2-(Mercaptomethyl)-1 -oxo-3-phenylpropyll-4-(2- thiazolyl)-Z.-phenylalanine

Stage 3a

Stage 1: Preparation of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4- nitro- -phenylalanine methyl ester

A solution of (RS)-2-(acetylthiomethyl)-3-phenylpropanoic acid (10.86g) in DCM (22ml) was treated with thionyl chloride (3.66ml, 1.1 eq) and heated to reflux for 4 hours. The solution was cooled and the solvent evaporated to give (RS)-2- (acetylthiomethyl)-3-phenylpropanoic acid chloride as a yellow oil 11.66g, 99.7% theory yield.

A solution of 4-nitro-L-phenylalanine methyl ester hydrochloride (5.86g, 22.5mmol), aqueous sodium carbonate (1M, 45ml, 45mmol, 2eq) and DCM (90ml) was stirred and cooled to 1°C. A solution of (RS)-2-(acetylthiomethyl)-3-phenylpropanoic acid chloride (5.77g, 22.5mmol, 1.0eq) in DCM (12ml) was added dropwise over 6 minutes and rinsed in with DCM (12ml). The mixture was stirred at 1°C for 10 minutes then warmed to 22°C and diluted with water (18ml) to dissolve all the solids. The phases were separated and the aqueous phase extracted with DCM (12ml). The combined DCM solutions were washed with 2M HCI (24ml) followed by water (24ml).

The DCM solution was concentrated to 50ml by distillation at atmospheric pressure then diluted with isopropanol (70ml) and seeded with the title compound. The slurry was cooled to 1°C, stirred for 1 hour and the solid collected by filtration then washed with chilled isopropanol (24ml then 12ml) and dried in vacuo at 50°C to give the title compound as an off-white solid 4.28g, 9.64mmol, 42.8% theory yield.

MS: 445 (MH\ 100%).

Anal Calcd for C^H^N^S: C 59.45, H 5.44, N 6.30, S 7.21. Found: C 59.43, H 6.29, N 5.93, S 7.10.

¹H NMR (250MHz, CDCI₃) δ: 8.11 (d, J = 9. 2H), 7.29-7.11 (m, 7H), 5.77 (d, J = 7, 1H), 4.75 (dt, J = 7, 6, 1H), 3.63 (s, 3H), 3.25, 3.09 (AB part of ABX partially obscured, J = 6. 6, 14, 2H), 3.06 (d, J = 7, 2H), 2.88 (d, J = 7, 2H), 2.55 (pent, J = 7, 1H), 2.35 (s, 3H).

³C NMR (100MHz, DMSO-d6) δ: 30.38, 30.60, 36.59, 38.12, 46.91, 52.36, 52.86. 123.58, 126.56, 128.53, 129.22, 130.94, 138.93, 146.01 , 146.67, 171.66, 172.77, 195.12.

Stage 2: Preparation of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4- cyano- -phenylalanine methyl ester

N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4-nitro- -phenylalanine methyl ester (5.22g, 11.7mmol), 10% dry Pd/C (1.0g, 0.2wt) and DCM (50ml) were stirred under an atmosphere of hydrogen at 20°C until the theoretical amount had been consumed. The catalyst was removed by filtration through a glass microfibre filter paper, washed with DCM (2 x 10ml) and the filtrate concentrated to a thick oil. This was dissolved in a mixture of acetonitrile (25ml) and water (25ml) at 15-20°C and 11.6M hydrochloric acid (2.22ml, 25.8mmol; 2.2eq) was added. Sodium nitrite (0.97g, 14.1 mmol, 1.2eq) dissolved in water (5ml) was added to the solution at 15-20°C over 5 minutes.

Potassium copper cyanide (3.88g, 17.6mmol, 1.5eq) and potassium bicarbonate (2.58g, 25.8mmol; 2.2eq) were dissolved in a mixture of water (20ml); acetonitrile (5ml) and DMF (5ml) at 15-20°C and treated with the diazonium solution at 20-25°C over 1.5 hours. The resulting pale brown slurry was aged at 20-25°C for 30-60min, cooled to 0-5°C and aged for 30-60 minutes. The slurry was collected by filtration, washed with acetonitrile/water (1 :1 ) (2 x 10ml), followed by water (2 x 15ml) and dried in a vacuum oven at 50°C for 18 hours to give the title compound as a pale brown solid 4.29g, 10.1 mmol, 86% yield.

MS: 425 (MH⁺, 100%), 383 (60%).

Anal Calcd for C₂₃H₂₄N₂O₄S: C 65.07, H 5.70, N 6.60, S 7.55. Found: C 65.64. H 5.65, N 6.26, S 7.56.

¹H NMR (400MHz, DMSO-d6) δ: 8.48 (d, J = 8, 1H), 7.75 (d, J = 8, 2H), 7.41 (d, J = 8. 2H), 7.29-7.14 (m, 5H). 4.62-4.56 (m. 1H). 3.59 (s. 3H), 3.16, 2.93 (AB part of ABX, J = 5, 10, 14, 2H), 2.85-2.59 (m, 5H), 2.24 (s, 3H).

¹³C NMR (100MHz. DMSO-d6) δ: 30.35, 30.74. 36.83, 38.13, 46.89, 52.33, 52.90, 109.72, 119.32, 126.57, 128.54, 129.22, 130.69, 132.43, 138.94, 143.67, 171.73, 172.76, 195.17.

Stage 3: Preparation of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4-(2- thiazolyl)-L-phenylalanine methyl ester A suspension of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4-cyano-L- phenylalanine methyl ester (101mg) in thioacetic acid (0.5ml) was heated to 80°C for 3 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure to give an orange oil, which was purified by chromatography on silica gel eluting with iso-octane/ethyl acetate 1 :2 to give the thioamide as an orange solid 76mg. HPLC and NMR indicated a mixture of thioamide and S-acetylthioamide products: 77.0%area and 17.7%area respectively.

The mixture of thioamide and S-acetyl products (74mg) were dissolved in acetic acid (1ml), treated with bromoacetaldehyde diethylacetal (32microL, 41 mg, 1.3eq) and p- toluenesulfonic acid (4mg) then heated at 60°C for 2 hours. The solvent was evaporated and the residue partitioned between saturated aqueous sodium bicarbonate (5ml) and ethyl acetate (5ml). The ethyl acetate solution was dried (MgSO₄) and concentrated to give the title compound as a brown solid 83mg, 71% theory overall yield.

Stage 4: Preparation of N-[(S)-2-(Mercaptomethyl)-1-oxo-3-phenylpropyl]-4-(2- thiazolyl)- -phenylalanine

Aqueous sodium hydroxide (10M, 17ml, 3.3eq) was added, under nitrogen to a cooled, (0-5°C) stirred, slurry of N-[(S)-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-4- (2-thiazolyl)- -phenylalanine methyl ester (25g) in a mixture of denatured ethanol (IMS 125ml) and water (75ml) keeping the temperature between 0 and 5°C. The resulting slurry was stirred at 0-5°C for 24 hours and acidified to pH=7-7.5 by dropwise addition of cone, hydrochloric acid (6ml) keeping the temperature at 0-5°C. Tributylphosphine (0.6ml) was added and the hazy solution clarified by vacuum filtration through a filter paper. The solution was then warmed to 15-20°C and stirred under nitrogen at this temperature for 45-60 minutes. The solution was acidified to pH=5 by dropwise addition of cone, hydrochloric acid (4.5ml) keeping the temperature at 15-20°C. The solution was then stirred, under nitrogen, at 15-20°C for 20-30 minutes to allow crystallisation to initiate. The slurry was acidified to pH=2 by dropwise addition of further cone, hydrochloric acid (4.0ml) keeping the temperature at 15-20°C. The slurry was then cooled, under nitrogen, to 0-10°C and stirred at this temperature for 60-90 minutes. The solid was collected by filtration, washed with IMS/water (1:1 , 2 x 50ml) and dried in vacuo at 40°C to give the title compound as a white solid 19.8g, 90% theory yield.

MS: 427 (MH⁺, 100%).

¹H NMR (400MHz, DMSO-d6) δ: 12.75 (br s, 1H), 8.35 (d, J = 8, 1H), 7.90 (d, J = 3, 1 H), 7.84 (d, J = 8, 2H), 7.75 (d, J = 3. 1H), 7.35 (d, J = 8, 2H), 7.24 (t, J = 7, 2H), 7.18-7.13 (m, 3H), 4.54 (m, 1H), 3.15, 2.94 (AB part of ABX, J = 4. 10. 14, 2H), 2.84 (m, 1H), 2.71-2.50 (m, 3H, partially obscured by DMSO), 2.30 (m, 1H), 1.84 (t, J = 8, 1H).

¹³C NMR (100MHz, DMSO-d6) δ: 25.83, 36.95, 37.54, 50.88, 53.41, 120.51. 126.41. 126.43. 128.55, 129.24, 130.43, 131.79, 139.50, 140.39, 144.13, 167.45, 172.90,

173.10. Example 3: Preparation of N-f(S)-2-(Mercaptomethyl)-3-methylbutanoyl-4-(1-H- pyrazol-1 -yl)-L-phenylalanine

Stage 1 Stage 2

Stage 1: Preparation of (S)-N-Acetyl-4-Nitrophenylalanine methyl ester

(S)-4-Nitrophenylalanine methyl ester hydrochloride (5.89g, 22.6mmol) and sodium acetate (1.1eq, 2.04g, 24.9mmol) were suspended in acetic acid (4 volumes, 24ml) and treated with acetic anhydride (1.5eq, 3.2ml, 33.9mmol) at 25°C. The slurry was stirred for 1h then diluted with water (10 volumes, 59ml) to give a solution. The product crystallised within a few minutes. The resulting slurry was stirred for 1 hour and the solid was collected by filtration, washed with water (2 x 12ml) and dried in a vacuum oven at 50°C to give the title compound as a white crystalline solid 3.77g, 62.7% yield. A further crop of crystals was harvested from the filtrate: 0.61g, 10.1% yield. Combined yield 72.8%. LCMS 3.60min, m/z 267 MH⁺.

Stage 2: Preparation of (S)-N-Acetyl-4-Aminophenylalanine methyl ester

(S)-N-Acetyl-4-Nitrophenylalanine methyl ester (3.73g, 14.0mmol) and 10% w/w Pd/C (0.05wt, 0.18g) were mixed with ethyl acetate (10 volumes, 37ml) and stirred under a hydrogen atmosphere for 2 hours. The catalyst was removed by filtration and the solution concentrated to give the title compound as a viscous oil 4.20g. NMR shows residual ethyl acetate approximately 19%w/w. Corrected yield 103%. LCMS 1.37min, m z 237 MH⁺.

Stage 3: Preparation of (S)-N-Acetyl-4-(1-pyrazolyl)-phenylalanine methyl ester

(S)-N-Acetyl-4-Aminophenylalanine methyl ester (517mg, 2.19mmol uncorrected for solvent) was dissolved in water (5 volumes, 2.5ml), cooled to 5°C and treated with concentrated HCI (2.6eq, 0.5ml, 5.7mmol) followed by sodium nitrite (1.05eq, 159mg, 2.30mmol) in water (2 volumes, 1 ml). The very pale yellow solution was stirred at 0- 5°C for 15 minutes then added slowly to a cold solution of sodium sulfite (3eq, 830mg, 6.6mmol) in water (10 volumes, 5ml). The solution immediately became bright yellow then orange. The solution of diazonium salt was washed in with water (1ml) then the orange solution heated to 60°C. Once the solution reached approximately 60°C it was treated with concentrated HCI (4eq, 0.8ml, 8.8mmol) and stirred for approximately 6 hours.

The aqueous solution was concentrated to dryness in vacuo. The resulting yellow solid was suspended in MeOH (100 volumes, 50ml) and heated to reflux. Malondialdehyde bis(dimethylacetal) (1eq, 0.36ml, 2.2mmol) was added and the mixture stirred at reflux for 17 hours. The suspension was cooled to 20°C, filtered and concentrated to a solid. The solid was partitioned between water (50ml) and ethyl acetate (50ml) then basified to pH 12-13 with 2M potassium carbonate. The layers were separated and the aqueous extracted with ethyl acetate (50ml). The combined ethyl acetate solutions were concentrated in vacuo to give the title compound as an orange solid 490mg, 78.0% yield. LCMS 3.54min, m/z 288 MH⁺. Stage 4: Preparation of (S)-4-(1-pyrazolyl)-phenylalanine methyl ester hydrochloride

(S)-N-Acetyl-4-(1-pyrazolyl)-phenylalanine methyl ester (480mg, 1.67mmol) was dissolved in methanol (100 volumes, 50ml) and treated with acetyl chloride (10eq, 1.22ml, 17mmol) then allowed to stand at 20°C for 9 days. The solution was heated at reflux for 23 hours then allowed to cool and treated with more acetyl chloride (10eq, 1.22ml, 17mmol). The solution was heated at 55°C for 3 days then cooled and concentrated to dryness. The oily solid was dissolved in methanol (4 volumes, 2ml) at reflux and diluted with tetrahydrofuran (16volumes, 8ml). The suspension was cooled then concentrated in vacuo to give the title compound as a brown solid 462mg, 98.0% yield. LCMS 2.82min, m/z 246 MH⁺.

Stage 5: Preparation of N-[(2S)-2-(acetylthiomethyl)-3-methylbutanoyl]-4-(1H- pyrazol-1 -yl)-L-phenylalanine Methyl ester Preparation of Acid Chloride

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 (2min) RT: 1.33 minutes acid, 1.61 minutes Me ester). The acid chloride solution was cooled to 20-25°C and used directly in the next step.

Amide Coupling: 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, 25ml) 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 (2min method) RT 1.64 min

Stage 6: Preparation of N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1H- pyrazol-1 -yl)-L-phenylalanine N-[(2S)-2-(acetylthiomethyl)-3-methylbutanoyl]-4-( 1 H-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 (3.5eq, 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 (0.02eq, 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) HCI (2.7eq, 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 (1.9eq, 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, 100ml) 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. LCMS RT 2.98min., MH⁺ 362

Generic HPLC Methods

Mobile phase A: A 0.05%v/v solution of trifluoroacetic acid (TFA) in water. Typically add 0.5ml TFA to 1000ml of water.

Mobile phase B: A 0.05%v/v solution of TFA in acetonitrile. Typically add 0.5ml TFA to 1000ml of acetonitrile.

This method was run on HP (also known as Agilent) 1100 HPLC system.

8 Minute Method also used by LCMS

2 Minute Method

Claims

Claims:

1. A process for the preparation of a compound of formula (IA):

(IA) or a salt thereof, wherein:

R¹ represents hydrogen, C_1-4alkyl or benzyl;

R² represents hydrogen or a protecting group -COH or -COC^alkyl; and R⁶ represents a 5- or 6- membered aromatic heterocycle with one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur from a compound of formula (II):

(II) or a salt thereof wherein: R¹ represents hydrogen, C_1-4alkyl or benzyl;

R² represents a protecting group -COH or -COC_1-4alkyl; and

R⁷ represents -CN or -NH-NH₃+; by heterocycle formation, optionally followed by removal of the protecting group.

A process as claimed in claim 1 wherein R⁶ represents thiazolyl or pyrazolyl.

3. A process as claimed in claim 1 or claim 2 wherein R₂ represents hydrogen, COH or -COCH₃.

4. A process for the preparation of a compound of formula (IA) or a salt thereof as claimed in any of claims 1-3 wherein R⁶ represents thiazole and R⁷ represents CN comprising:

(i) thioamide formation by reaction of a compound of formula (II) with (a) NH₄CI and

(b) NaSH.2H₂O, or hydrogen sulfide and an organic or inorganic base, in the presence of a strong dipolar aprotic solvent;

(ii) thiazole formation by reaction with an acetal of bromoacetaldehyde, in the presence or absence of an acid, at elevated temperature; optionally followed by,

(iii) removal of the protecting group under standard conditions.

5. A process for the preparation of a compound of formula (IA) or a salt thereof as claimed in any of claims 1-3 wherein R⁶ is pyrazole and R⁷ represents -NH-NH₃+ comprising:

(i) pyrazole formation of a compound of formula (II) by reaction with malondialdehyde bis(dimethylacetal); optionally followed by, (ii) removal of the protecting group under standard conditions.

6. A process as claimed in claim 4 wherein the compound of formula (II) wherein R⁷ is -CN is prepared from a compound of formula (III):

(III) wherein: R¹ represents hydrogen, C^alkyl or benzyl; and R² represents -COH or -COC^alkyl; by (i) treatment with strong inorganic acid in the presence of a source of nitrite ions at a temperature below room temperature in a suitable solvent, followed by (ii) treatment with a source of copper +1 and source of free cyanide ions in the presence or absence of potassium bicarbonate or sodium bicarbonate.

7. A process as claimed in claim 5 wherein the compound of formula (II) wherein R⁷ is -NH-NH₃+ is prepared from a compound of formula (III):

(HI) wherein:

R¹ represents hydrogen, C_1-4 alkyl or benzyl; and R² represents -COH or -COC_1-4alkyl; by (i) treatment with a source of nitrite ions at a temperature below room temperature in a suitable solvent, followed by (ii) treatment with a reducing agent.

8. A process as claimed in claim 6 or claim 7 wherein the compound of formula (III) is prepared from a compound of formula (IV):

(IV) wherein:

R¹ represents hydrogen, C_1-4alkyl or benzyl; and R² represents -COH or -COC_1-4alkyl; by reduction of the nitro group.

9. Use of a process as claimed in any one of claims 1-8 in the preparation of a mixed ACE-NEP inhibitor.

10. Use of a process as claimed in claim 9 in the preparation of a compound of formula (I) or a pharmaceutically acceptable derivative thereof:

(I)

wherein:

R³ is a straight or branched C₂-C alkyl group or an aryl or arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a 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 by one or more substituents, the same or different, selected from the group consisting of hydroxy groups, alkoxy, alkyl, alkylthio, alkylsulphonyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C C₃ alkyl groups containing one or more fluorine atoms, carboxy groups, 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 alkyl moiety; R⁴ is a R⁵COSCH₂ group convertible to a mercaptomethyl group; R⁵ is a straight or branched C C₄ alkyl or a phenyl group. R⁶ is a 5- or 6- membered aromatic heterocycle with one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur.

11. Use of a process as claimed in claim 9 or claim 10 wherein the mixed ACE- NEP inhibitor is N-[(S)-2-(Mercaptomethyl)-1-oxo-3-phenylpropyl]-4-(2-thiazolyl)- - phenylalanine or N-[(S)-2-(Mercaptomethyl)-3-methylbutanoyl]-4-(1H-pyrazol-1-yl)-L- phenylalanine or pharmaceutically acceptable derivatives thereof.

12. A process as claimed in claim 1 wherein R² represents a group (A):

(A)

13. A process for the preparation of a compound of formula (I) as claimed in claim 12 wherein R⁶ represents thiazole comprising: (i) thioamide formation by reaction with thioacetic acid at elevated temperature, (ii) thiazole formation by reaction with an acetal of bromoacetaldehyde, in the presence or absence of an acid, at elevated temperature.

14. A compound of formula (II):

(II) wherein R¹ represents hydrogen, C_1-4alkyl or benzyl; and R² represents -COH or COC_1-4alkyl.