WO1993019059A1

WO1993019059A1 - Antiviral peptides

Info

Publication number: WO1993019059A1
Application number: PCT/EP1993/000597
Authority: WO
Inventors: Colin William Greengrass; Stephen Derek Albert Street; Peter John Whittle
Original assignee: Pfizer Limited; Pfizer Inc.
Priority date: 1992-03-25
Filing date: 1993-03-13
Publication date: 1993-09-30
Also published as: BR9306138A; FI944428A; CZ229594A3; HU9402744D0; NO943540D0; RU94041225A; EP0632808A1; FI944428A0; JPH07501556A; SK114094A3; KR950700901A; AU3748393A; CA2131154A1; NO943540L

Abstract

Compounds of formula (I) and pharmaceutically acceptable salts thereof and bioprecursors therefore wherein R1 is C1-C6 alkyl, C3-C8 cycloalkyl, aryl, heterocyclyl or CONR?9R10; R2 is C¿1-C6 alkyl, C3-C8 cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl or heterocyclyl(C1-C4)alkyl; R3 is C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl, aryl(C2-C4)-alkenyl, heterocyclyl(C1-C4)alkyl or heterocyclyl(C2-C4)-alkenyl; R4 is C1-C6 alkyl, C3-C8 cycloalkyl, aryl or heterocyclyl; each of R?5, R6, R7 and R8¿ is independently H, C¿1?-C6 alkyl or C3-C8 cycloalkyl; or R?5 and R6, or R7 and R8¿ may be joined together to form a 3 to 8 membered carbocyclic ring; X is a 4-10 membered mono or bicyclic heterocyclic group containing carbon ring atoms and one ring nitrogen atom through which the group is attached to the adjacent carbonyl group; the group may be saturated or partially unsaturated and, in addition to the -(CR7R8)m-Het substituent, it may be substituted by up to 4 further substituents independently chosen from F, C1-C6 alkyl, C3-C8 cycloalkyl, OR?11 or NR9R10¿; Het is an imidazolyl or triazolyl group either of which may optionally be substituted by C¿1?-C6 alkyl, C3-C7 cycloalkyl, NR?9R10¿ or CONR9R10, each of R?9 and R10¿ is independently H, C¿1?-C6 alkyl or C3-C8 cycloalkyl, or R?9 and R10¿ may be joined together to form, with the nitrogen to which they are attached, a 4 to 8 membered nitrogen-containing heterocyclic group, R11 is H, C¿1?-C6 alkyl or C3-C8 cycloalkyl; n and m are each independently 0, 1 or 2; wherein any alkyl or cycloalkyl group included in the aforementioned definitions may optionally be fully or partially substituted by fluorine; are inhibitors of retroviral proteases of utility in the treatment and prophylaxis of human retroviral infections.

Description

ANTIVIRAL PEPTIDES

The present invention relates to certain peptide derivatives containing a heterocyclic group which are useful in the treatment or prophylaxis of human

retroviral infections.

The human immunodeficiency virus (HIV), a

retrovirus, is the causative agent of a variety of clinical conditions, the most serious of which are commonly termed AIDS (Acquired Immunodeficiency

Syndrome), and ARC (AIDS-Related Complex). Infection with HIV is characterised by progressive breakdown of the immune system and CNS dysfunction. Severely immune deficient patients suffer from a wide range of

opportunistic infections (e.g. pneumocystis carinii, human cytomegalovirus, or Candida), and cancers such as Kaposi's sarcoma. Loss of cells, particularly CD4⁺ lymphocytes, following infection with HIV is an

important factor in the progressive impairment of immune function. The infection of cells of

monocyte/macrophage lineage with HIV also contributes to the observed pathology. Thus, successful infection of CD4⁺ cells by HIV is a key step in the disease process.

HIV is a retrovirus; it encodes its genetic information in RNA, which is converted into DNA after the virus enters the host cell. An essential step in the retroviral replication cycle is the processing of an initial polypeptide precursor into mature structural and replicative proteins. This processing is carried out by a virus-coded protease and, in the absence of this enzyme activity, viral replication is blocked.

We have discovered that certain peptide

derivatives linked to a heterocyclic group are potent inhibitors of retroviral proteases, both in a cell-free assay and in infected cells and, in addition, show antiviral activity in tissue culture assays. This activity renders such compounds useful for the

treatment and prophylaxis of retroviral infections, in particular, those caused by HIV.

Thus, the present invention provides compounds having the formula

and pharmaceutically acceptable salts thereof and bioprecursors therefor, wherein-:

R¹ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, aryl,

heterocyclyl or CONR⁹R¹⁰;

R² is C₁-C₆ alkyl, C₃-C₈ cycloalkyl(C₁-C₄) alkyl, aryl (C₁-C₄) alkyl or heterocyclyl (C₁-C₄) alkyl;

R³ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₈

cycloalkyl(C₁-C₄) alkyl, aryl(C₁-C₄) alkyl, aryl(C₂-C₄)-alkenyl, heterocyclyl(C₁-C₄) alkyl or heterocyclyl (C₂-C₄)-alkenyl;

R⁴ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl,

aryl or heterocyclyl;

each of R⁵, R⁶, R⁷ and R⁸ is independently H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl; or R⁵ and R⁶, or R⁷ and R⁸ may be joined together to form a 3 to 8 membered

carbocyclic ring;

X is a 4-10 membered mono- or bicyclic

heterocyclic group containing carbon ring atoms and one ring nitrogen atom through which the group is attached to the adjacent carbonyl group; the group may be saturated or partially unsaturated and, in addition to the -(CR⁷R⁸)_m-Het substituent, it may be substituted by up to 4 further substituents each independently chosen from F, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, OR¹¹ or NR⁹R¹⁰;

Het is an imidazolyl or triazolyl group either of which may optionally be substituted by C₁-C₆ alkyl, C₃-C₈ cycloalkyl, NR⁹R¹⁰ or CONR⁹R¹⁰,

each of R⁹ and R¹⁰ is independently H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl, or R⁹ and R¹⁰ may be joined together to form, with the nitrogen to which they are attached, a 4 to 8 membered nitrogen- containing heterocyclic group,

R¹¹ is H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl;

and n and m are each independently 0, 1 or 2;

wherein any alkyl or cycloalkyl group included in the aforementioned definitions may optionally be fully or partially substituted by fluorine.

In the above definition of R¹, R², R³ and R⁴, heterocyclyl means a 4 to 6 membered heterocyclic group containing as heteroatoms up to four nitrogen atoms, or an oxygen or sulphur atom optionally with one or two nitrogen atoms. The ring may be aromatic, or fully or partially saturated and may optionally be benzo-fused or substituted by C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₂-C₅ alkanoyl, C₁-C₄ alkoxy, halo, hydroxy, oxo or aryl.

Preferred heterocyclyl groups are pyridyl, pyrimidinyl, thienyl, isoquinolyl and tetrazolyl.

In the above definitions of R¹, R², R³ and R⁴, aryl means phenyl optionally substituted with from 1 to 3 substituents each independently selected from C,-C₆ alkyl, C₃-C₈ cycloalkyl, C₁-C₄ alkoxy, C₂-C₅ alkanoyl, hydroxy, halo, C₁-C₄ alkyl fully or partially

substituted by fluorine, C₁-C₄ alkoxy fully or partially substituted by fluorine, phenyl, phenoxy, benzyl, benzoyl, phenylSO₂-, pyridyl, tetrazolyl,

phenyltetrazolyl, NR⁹R¹⁰ or CONR⁹R¹⁰; wherein R⁹ and R¹⁰ are as previously defined. Halo means fluoro, chloro, bromo or iodo.

The imidazolyl or triazolyl group, Het, may be linked either by a ring carbon or ring nitrogen atom and may be unsubstituted or mono-, di or or tri-substituted. Triazolyl includes both 1,2,3 and 1,2,4-triazolyl groups.

Alkyl and alkoxy groups containing 3 or more carbon atoms may be branched or straight-chain. Any alkyl, alkoxy or cycloalkyl group included in the above definitions may optionally be fully or partially substituted by fluorine.

The term bioprecursor in the above definition means a pharmaceutically acceptable biologically degradable derivative of the compound of formula (I) which, upon administration to an animal or human being, is converted in the body to produce a compound of the formula (I). Examples include ester derivatives formed between the free hydroxy group in the compound of formula (I) and, for example, an amino acid (such as L-valine).

It will be appreciated that the compounds of formula (I) have a number of asymmetric carbon atoms and the invention includes all possible stereoisomers whether separated or not.

In one particular and preferred aspect of the invention there are provided compounds having the stereochemistry-:

In the above formula heavy bonds are used to indicate that the group lies above the plane of the molecule while a broken bond is used to indicate that the group lies below.

In the definition of R¹, aryl is preferably phenyl and heterocyclyl is preferably oxetan-3-yl or 1,1-dioxothietan-3-yl. R¹ is preferably t-butyl,

isopropyl, oxetan-3-yl or 1,1-dioxothietan-3-yl and (CR⁵R⁶)_n is absent; or R¹ is phenyl and (CR⁵R⁶)_n is CH₂; or R¹ is H₂NCO-, CH₃NHCO- or (CH₃)₂NCO- and (CR⁵R⁶)_n is CH₂ or CH(CH₃). Particularly preferred are compounds wherein R¹ is t-butyl, isopropyl or oxetan-3-yl and n is 0, most particularly where R¹(CR⁵R⁶)_n- is t-butyl.

In the definition of R², aryl is preferably phenyl and heterocyclyl is for example pyridyl, pyrimidinyl or thienyl. R² is preferably aryl (C₁-C₄) alkyl; benzyl is particularly preferred.

In the definition of R⁴, aryl is preferably phenyl and heterocyclyl is preferably pyridyl, pyrimidinyl or thienyl. R⁴ is preferably C₁-C₆ alkyl; particularly preferred are isopropyl and sec-butyl (valine or isoleucine derivatives).

The heterocyclic group X is preferably a 4-6 membered saturated or monounsaturated group and is most preferably an azetidine, pyrrolidine,

tetrahydropyridine or piperidine group; piperidine being particularly preferred.

R⁷ and R⁸ are preferably H and m is preferably O or 1.

In the definition of R³, aryl is phenyl,

unsubstituted or substituted as defined in the term aryl above, and heterocyclyl is for example pyridyl, pyrimidinyl, isoquinolyl or thienyl. R³ is preferably aryl (C₁-C₄) alkyl or aryl (C₂-C₄) alkenyl; R³ is most preferably benzyl optionally substituted in the phenyl ring by fluoro, chloro, iodo, methyl, trifluoromethyl or trifluoromethoxy, or R³ is 3-phenyl-prop-2-enyl or 3-phenylpropyl.

Particular and preferred individual compounds include:

1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazol-1-yl)azetidine,

1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(3-phenylprop-2-en-1-yl)hexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(R)-2-(4-chlorobenzyl)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazol-1-yl)azetidine and

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl]-4-(imidazol-1-yl)piperidine.

In a second aspect of the present invention, there is provided a compound of formula (I), or a

pharmaceutically acceptable salt thereof or

bioprecursor therefor, for use as a medicament, especially for use in the treatment or prophylaxis of human retroviral infections, in particular HIV

infections. The invention also includes the use of a compound of the formula (I), or of a pharmaceutically acceptable salt thereof or bioprecursor therefor, for the manufacture of a medicament for use in the

prophylaxis or treatment of retroviral infections.

The invention further includes a pharmaceutical composition comprising a compound of the formula (I), or a pharmaceutically acceptable salt thereof or bioprecursor therefor, and a pharmaceutically

acceptable diluent or carrier.

The antiviral activity of the compounds of general formula (I) is established using in vitro assay

systems. For example, the compounds of formula (I) are able to completely protect human T-cell line H9 for 7 days from the progressive effects of HIV infection.

Untreated virus-infected cells display typical

cytopathic effects such as formation of syncytia and cell death. In addition, virus particles produced from virus-infected cells treated with a compound of formula (I) are non-infectious.

Examples of infections which may be treated or prevented by the compounds of formula (I) include those caused by human or animal retroviruses, especially HIV-1. Clinical conditions which may therefore be treated or prevented include AIDS, ARC, and HIV related

dementia. The compounds may also be used to block disease progression in symptomless infected

individuals.

The compounds of formula (I) can be prepared using the coupling and protection techniques which are familiar to those skilled in the art of peptide

chemistry.

The process is illustrated, by way of example, by the routes shown below for the preparation of compounds of formula (la):

The procedure outlined in Scheme A, starts with a protected lactone (II). This is alkylated, using for example n-butyllithium or lithium hexamethyldisilazide followed by addition of a compound of formula R 3Br and separation of the desired isomer to give the product

(III). The lactone ring is then opened by treatment with dilute alkali to give the corresponding hydroxy-acid and the hydroxy group is subsequently protected, for example as the t-butyldimethylsilyl derivative, by reaction with t-butyldimethylsilyl chloride in N,N- dimethylformamide, followed by hydrolysis of the protected ester to give the intermediate (IV).

Coupling of this intermediate with the other fragment (VIII), to produce the compounds of formula (la), is illustrated in Scheme B. In this process, the protecting group on the intermediate (V) is removed (in the case of t-butoxycarbonyl by treatment with HCl) and the resulting compound (VI) is reacted with the N-protected amino acid Boc-NHCH(R⁴) CO₂H, using an amide coupling reagent, to give the intermediate (VII). The N-protecting group from (VII) is then removed and the amine product (VIII) is coupled to intermediate (IV) to give intermediate (IX). The hydroxy protecting group X¹ is then removed from (IX) to give the final product of formula (la). In an alternative procedure the intermediate (IX) can be produced by coupling of the intermediate (X) with the intermediate (VI). The intermediate (X) can be prepared from intermediate (IV) by reaction with a carboxyl protected amino acid

H₂NCH(R⁴) CO₂R and subsequent basic hydrolysis.

SCHEME A

Protecting group, X^' : tButyldimethylsilyl is preferred R¹-(CR^sR⁶)_n: t-Butyl or benzyl are preferred

Amide coupling reagents:- e.g., 1-(3-dimethylaminopropyl)-3-ethyIcarbodiimide Protecting groups:- P; t-butoxycarbonyl or benzyloxy carbonyl are preferred

X'; t-butyldimethylsilyl is preferred.

R; methyl or ethyl are preferred In another alternative procedure illustrated in Scheme C, removal of the t-butoxycarbonyl group from the intermediate (XI), by treatment with acid, for example trifluoroacetic acid, and subsequent reaction with a carbonate derivative of formula (XII) gives products of formula (XIII) in which the R¹(CR⁵R⁶)_n group is other than t-butyl. For example, reaction of products of formula (XI) with 3-oxetanyloxy- carbonyloxysuccinimide gives the products (XIII) where R¹- is 3-oxetanyl and n is 0.

SCHEME C

X^' is either H or a protecting group, e.g., t-butyldimethylsilyl.

Y is a group which is susceptible to nucleophilic displacement - succinimidooxy is preferred. The starting materials of formula (V) required for the procedure described above are in some cases

literature compounds or they can be prepared by routine synthetic procedures from readily available starting materials. Thus for example reaction of 1(N-t-butoxycarbonyl)-3-hydroxy-azetidine with

methanesulphonyl chloride followed by reaction with a compound of formula Het-H in the presence of a base gives the required compounds of formula (V) wherein X is an azetidine ring. Alternatively, for example, 1-(N-benzyloxycarbonyl)-4-ketopiperidine is converted to the 4-amino derivative by reaction with sodium

cyanoborohydride in the presence of ammonium acetate. Subsequent reaction with dimethylformamide azine gives the 4-(1,2,4-triazol-4-yl) derivative.

Reaction of 1-(N-t-butoxycarbonyl)-4-ketopiperidine with the anion derived by reacting 1-diethoxymethyl)imidazole with n-butyllithium followed by treatment of the product with methanesulphonyl chloride in the presence of a base gives the N-protected-4-imidazol-2-yl(1,2,5,6-tetrahydropyridine) intermediate. Catalytic hydrogenation yields the corresponding N-protected-4-(imidazol-2-yl)-piperidine. Reaction of the above keto compound directly with a heterocyclic compound, e.g. imidazole, in the presence of thionyl chloride gives compounds of formula (V) wherein X is a tetrahydropyridine group, reduction again gives the piperidine derivative.

The intermediates of formula (V) in Scheme B where m does not equal 0 can be prepared by standard

transformations from the appropriate precursors using, for example, nucleophilic opening of an epoxide group to introduce the Het group. Thus for example reaction of 1-(N-t-butoxycarbonyl)-4-ketopiperidine with

trimethylsulphonium iodide in the presence of a base gives the 4-spiro-2'-oxirane. Reaction of this product with imidazole followed by elimination of the 4-hydroxy group gives 1-(N-t-butoxycarbonyl)-4-(imidazol-1-yl)methyl-1,2,5,6-tetrahydropyridine; reduction gives the corresponding piperidine derivative.

The N-t-butoxycarbonyl (BOC-) derivatives of the naturally occurring amino acids used in the synthesis of the compounds of the formula (VII) are commercially available as are their hydroxysuccinimido esters. The corresponding intermediates derived from unnatural amino acids can be prepared by standard procedures (see, for example, M. J. O'Donnell et al, J. Amer.

Chem. Soc., 1989, 111, 2353). The compounds of formula (II) can be prepared from the corresponding t-butyloxycarbonyl-protected amino-aldehydes (see D. H. Rich et al, J. Org. Chem., 1978, 43, 3624 and Y. Hamada et al, Chem. Pharm. Bull., 1982, 30(5), 1921)

by reaction with ethyl propiolate (see A. H. Fray et al, J. Org. Chem., 1986, 51, 4828), followed by

reduction to give the 5-t-butyloxycarbonylamino-4-hydroxy-6-phenylhexanoate. Cyclization by refluxing in toluene then affords the lactones of formula (II), as mixtures of diastereomers which can be separated by standard procedures.

In the above routes and specific Examples

presented herein, certain hydroxy and amino-protecting and carboxy-activating groups are required. It will be apparent to those skilled in the art that the coupling and protection procedures described could be carried out by any standard method for peptide synthesis and these procedures are therefore included in the scope of the invention. The choice of a particular protecting group is dependent to a great extent upon the

availability of the necessary reagent, its effect upon solubility of the protected compound, its ease of removal and the presence of other groups which might be affected by its use. For example, it is necessary in the above process to protect and deprotect particular amino groups in order to permit further reaction at the regenerated amino group and the choice of protecting group for a given amino group will depend upon the role of said amino group in the overall reaction scheme.

Amino protecting groups having varying levels of lability can be used. Such groups are known in the art and attention is directed to the reviews by Bodansky et al., "Peptide Synthesis", 2nd Ed., John Wiley & Sons, N.Y. (1976); Greene, "Protective Groups in Organic Synthesis", John Wiley & Sons, N.Y. (1981); McOmie, "Protective Groups in Organic Chemistry", Plenum Press, N.Y. (1973); and to Sheppard in "Comprehensive Organic Chemistry, The Synthesis and Reactions of Organic

Compounds", Pergamon Press, N.Y. (1979), edited by E. Haslam, Part 23.6, pages 321-339.

Representative amino-protecting groups include, but are not limited to, aminoxycarbonyl such as

benzyloxycarbonyl; substituted or unsubstituted aralkyl such as benzyl, trityl, benzhydryl and 4-nitrobenzyl; benzylidene; arylthio such as phenylthio, nitro-phenylthio and trichloro- phenylthio; phosphoryl derivatives such as dimethylphosphoryl and O,O-dibenzylphosphoryl; trialkylsilyl derivatives such as trimethylsilyl; and others as are described in U.S.

Pat. No. 4,322,341. The preferred amino protecting group for use in the above sequence is t-butoxycarbonyl. Procedures for substituting said group on a given amino group are well known. In general they comprise acylating the appropriate amino compound with the corresponding carbonyl chloride or anhydride in a reaction inert solvent, e.g. water, methylene chloride or tetrahydrofuran, in the presence of a base (acid acceptor) e.g., sodium or potassium hydroxide when water is solvent; and, when an organic solvent is used, in the presence of a tertiary amine such as a

triethylamine or pyridine. When an aqueous solvent system is used the pH of the reaction is typically held at about pH 8-10, and preferably at pH 9. The protected amino groups are converted to the unprotected amino groups by procedures known to those skilled in the art as appropriate to the particular group employed. The t-butoxycarbonyl group is, for example, readily removed by treatment with

dichloromethane saturated with hydrogen chloride gas.

Various hydroxy-protecting groups are also known and are described in the literative sources already cited above. A preferred hydroxy protecting group is t-butyldimethylsilyl. This is introduced as previously described and is readily removed by treatment with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature.

Activation of carboxy groups as a means of

expediting a given acylation reaction is also

methodology known to those skilled in the art.

Especially useful in the herein described reaction sequence are the use of anhydrides and activated esters, particularly those esters derived from N-hydroxyphthalimide, N-hydroxysuccinimide or 1-hydroxybenzotriazole, all of which are used in peptide syntheses.

A dehydrative coupling agent is used to form the activated ester. Representative of such coupling agents are 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide, N,N^,dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, ethoxyacetylene, diphenylketene and N-ethyl-5-phenylisoxazoline-3'-sulfonate. The reaction conditions for using such coupling agents are well described in the literature. In general they comprise the use of a reaction inert solvent and temperatures ranging from ambient to 100°C. The above-mentioned carbodiimide reagents are favoured since they permit use of ambient reaction temperature and afford

satisfactory yields of the desired esters.

Upon completion of the coupling reactions leading to the final products, the various protecting groups can be removed by the appropriate techniques previously discussed, and the compounds of the formula (I)

isolated and purified using conventional procedures such as recrystallisation or column chromatography.

The above sequences can be adapted as appropriate to be performed with any of the variants claimed for R to R⁸, X and Het by appropriate selection of starting materials.

Thus, according to a further aspect, the invention includes a process for preparing a compound of the formula (I) which comprises removing the protecting group from a compound of the formula:

wherein X¹ is a selectively removable hydroxy-protecting group, and isolating the compound of formula (I) and optionally forming a pharmaceutically

acceptable salt thereof.

The preferred protecting group for X¹ is t-butyldimethylsilyl; this is removed by treatment with tetra-n-butylammonium fluoride in an organic solvent, preferably tetrahydrofuran.

The novel intermediates of formulae (VIII), (IX), and (X), also form part of this invention.

Examples of pharmaceutically acceptable salts of the compounds (I) are acid-addition salts, e.g. sulfates, bisulfates, phosphates, lactates, mesylates, fumarates, citrates, succinates and gluconates.

In the treatment of patients having a retrovirus infection, especially HIV, the compounds (I) will be administered by any suitable route, e.g. by the oral, parenteral (e.g. subcutaneous, intravenous,

intramuscular, or intradermal), rectal, nasal, topical (including buccal and sublingual) or vaginal routes. The formulations, which will contain an antiviral agent of the invention together with one or more

pharmaceutically acceptable carriers and optionally other therapeutic agents, can be prepared according to conventional techniques well known in pharmacy. Oral dosage forms include in particular syrups, tablets and capsules which may contain flavouring agents in

addition to an inert carrier. Tablets can be made by conventional compression or moulding techniques by compressing a powder of the appropriate ingredients, e.g. the antiviral agent in conjunction with a binder, diluent, lubricant and surface-active agent. Rectal formulations will be in suppository form, and vaginal formulations as, for example, tampons, creams or foams. Parenteral formulations will be in sterile form, e.g. as vials for injection containing aqueous or non-aqueous diluents, buffers and antioxidants so that the formulation will be isotonic with blood. In general, the appropriate dose of the anti-retroviral agents of the formula (I) will be from 1-50 mg/kg/day, preferably 1-25 mg/kg/day given in up to six divided doses per day. There may be of course instances where higher or lower dosages are merited according to the age, weight, degree of illness and response of the patient, and appropriate therapy will be as determined by the medical practitioner.

The compounds of formula (I) may be used in combination with other drugs, some of which may

potentiate their activity. Such drugs include the following:-

(a) Reverse transcriptase inhibitors such as AZT, ddI, ddC, foscarnet, TIBO compounds, dipyrido- diazepinones or 6-substituted acyclopyrimidine (HEPT) derivatives;

(b) gp120-CD4 blockers such as dextran sulphate and soluble CD4, including its combination with toxic agents such as pseudomonas toxin;

(c) tat antagonists, such as D-penicillamine;

(d) other retrovirals protease inhibitors, such as

Ro 31-8959; and

(e) biological response modifiers including

interferons, interleukins or colony stimulating factors, e.g. GM-CSF.

The compounds of the invention were evaluated for antiviral activity by dissolving the test compound in 50 μl of DMSO and diluting in RPMI 1640, a complex salts solution with a pH of 7.2, to 1 mg/ml. Testing was performed at 0.001, 0.01, 0.1, 1 and 10 μg/ml against HIV 1 (strain IIIB) in a human T-cell line (H9). Untreated control infections were initiated at the same time.

Seven days post infection, tissue culture

supernatants were titrated for the presence of

infectious virus on C8166 cells (human T-cell line). On days 3, 5, and 7 cultures were examined

for appearance of syncytia. Control infections, untreated with drug, show typical viral cytopathic effects, including formation of syncytia and cell death. The IC₁₀₀ quoted is the lowest test

concentration affording complete protection to the culture. Using this test method, the compounds had an IC₁₀₀ values in the range 0.1 to 10.0 μg/ml.

The preparation of certain starting materials and of the compounds of formula (I) will now be more particularly illustrated by reference to the following experimental Examples. The purity of compounds was routinely monitored by thin layer chomatography using Merck Kieselgel 60 F₂₅₄ plates. 1H-Nuclear magnetic reasonance spectra were recorded using either a Nicolet QE-300 or a Bruker AC-300 spectrometer and were in all cases consistent with the proposed structures.

Chemical shifts are given in parts-per-million

downfield from tetramethylsilane using conventional abbreviations for designation of major peaks: s, singlet; d, doublet; t, triplet; m, multiplet and b, broad. Optical rotations were read at a concentration of 0.1% in methanol at 25°C unless otherwise stated. All temperatures are in degrees Celsius.

PREPARATION OF STARTING MATERIALS AND INTERMEDIATES

PREPARATION 1

(S) -5-[ (S) -1-t-Butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone)

a) Ethyl (4S,5S)- and (4R,5S)-5-t-butoxycarbonylamino- 4-hydroxy-6-phenylhex-2-ynoate

A solution of diisopropylamine (6.4 ml) in dry tetrahydrofuran (25 ml) was stirred under nitrogen at -25°C and a 1.6 molar solution of n-butyllithium in hexane (24.4 ml) was added over 5 minutes, keeping the temperature below -20°. After a further 15 minutes at -20° the solution was cooled to -70° and ethyl

propiolate (3.8 g) was added dropwise over 10 minutes, keeping the temperature below -65°. The resulting yellow suspension was stirred at -70° for a further 20 minutes and then treated dropwise, over 10 minutes, with a solution of N-t-butoxycarbonyl-L-phenylalaninal (6.5 g, see J. R. Luly et al, J. Org. Chem., 1987, 52, 1487) in dry tetrahydrofuran (15 ml), again keeping the temperature below -65°. The clear yellow solution was stirred at -70° for 2 hours then treated with acetic acid (4 ml). The cooling bath was removed and the mixture was allowed to warm to -30° at which point water (100 ml) and ethyl acetate (100 ml) were added with vigorous stirring. Separation of the organic layer, followed by successive washing with 1 molar hydrochloric acid (50 ml), saturated aqueous sodium bicarbonate (50 ml) and saturated brine (50 ml), gave the crude product as an oil after drying (Na₂SO₄) and evaporation of the solvent. The oil was purified by silica gel chromatography using ethyl acetate-hexane (1:4) as the eluent. Evaporation of the productcontaining fractions gave an oil which solidified on standing overnight. Recrystallisation from ether-hexane afforded the title compounds as an approximately 2:1 mixture (4S,5S:4R,5S) of diastereomers, (4.3 g), m.p. 98-99°. Found: C,65.62; H,7.42; N,4.33. C₁₉H₂₅NO₅ requires C,65.70; H,7.20; N,4.03%.

N.M.R. (CDCl₃) δ = 1.30-1.39 (m, 3H); 1.43 (s, 9H);

2.90-3.11 (m, 2H); 3.37-3.38 and 4.16-4.19 (2x brm, 1H); 3.93-4.04 (m, 1H); 4.22-4.33 (m, 2H); 4.51-4.56 (m, 1H); 4.77-4.79 and 4.87-4.90 (2 x brm, 1H); 7.24-7.35 (m, 5H). b) Ethyl (4S,5S)- and (4R,5S)-5-t-butoxycarbonylamino-4-hydroxy-6-phenylhexanoate

The above product (1.17 g) was dissolved in ethanol (50 mg) and 5% Pd-BaSO catalyst was added. The mixture was then hydrogenated at 50 psi (344.7 kPa) for 2 hours. Filtration, followed by evaporation of the solvent under vacuum, gave the title compounds (approximately 2:1 mixture of diastereomers) as a white solid, (1.18 g), m.p. 125-126°. Found: C,64.91;

H,8.40; N,3.98, C₁₉H₂₉NO₅ requires C,64.95; H,8.26; N,3.98%.

N.M.R. (CDCl₃) δ = 1.24-1.33 (m, 3H); 1.39 and 1.42 (2 x s, 9H); 1.72-1.95 (m, 2H); 2.38-2.62 (m,2H); 2.77-2.98 (m,2H); 3.02-3.04 and 3.40-3.42 (2 x m, 1H exch. D₂O); 3.59-3.91 (m, 2H); 4.08-4.22 (m, 2H); 4.58-4.61 and 4.86-4.89 (2 x m, 1H); 7.22-7.37 (m, 5H). c) (S)-5-[(S)-1-t-Butoxycarbonylamino-2-phenylethyl]¬gamma-butyrolactone

The gamma-hydroxyester from (b) above was

dissolved in 2.5% acetic acid-toluene (35 ml) and the solution was heated at reflux for 2 hours. After cooling and evaporation to dryness under vacuum, the residue was purified by silica gel chromatography, eluting with diethyl ether-hexane (40:60), to afford the title compound (0.4 g), m.p. 98-99°. Found:

C, 66.77; H,7.78; N,4.38. C₁₇H₂₃NO₄ requires: C, 66.88; H,7.54; N,4.59%. m/e = 306 (MH ).

N.M.R. (CDCl₃) δ = 1.42 (s, 9H); 2.11-2.19 (m, 2H); 2.51-2.58 (m, 2H); 2.87-3.02 (m, 2H); 4.00-4.07 (m, 1H) ; 4 . 47-4. 52 (m, 1H) ; 4. 63 (d , J = 10 , NH) ; 7 . 26-7 . 36

(m, 5H) .

[α] ²⁵ _D -22 . 6 ° (c = 1 , MeOH) .

I. R. (KBr) 1775 , 1690 , 1525 cm^-1.

PREPARATION 2

(R)-2-Benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoic acid a) (R)-3-Benzyl-(S)-5-[ (S)-1-t-butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone.

A cold (-10°) solution of hexamethyldisilazane (7.9 ml) in tetrahydrofuran (15 ml) was treated over 3 minutes with 1.6M n-butyllithium in hexane (23 ml), keeping the temperature below 0° . After a further 5 minutes at 0º the solution was cooled to -70° and a solution of (S)-5-[(S)-1-t-butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone (5 g) in tetrahydrofuran (38 ml) was added, keeping the temperature below -65°. The solution was stirred at -70° for 15 minutes before benzyl bromide (1.95 ml) in tetrahydrofuran (12.5 ml) was added over 1 minute and the solution stirred at -70° for an additional 10 minutes before being treated with acetic acid (6.5 ml) and the cooling bath removed.

Water (50 ml) and ethyl acetate (50 ml) were added and the mixture allowed to warm to room temperature.

Separation of the organic layer, followed by drying (MgSO₄) and evaporation of the solvent under vacuum gave the crude product as an oil. Chromatography on silica-gel, eluting with diethyl ether-hexane (50:50), gave the title compound as a clear oil (3.52 g). Found: C, 73.22; H, 7.50; N, 3.50. C₂₄H₂₉NO₄ requires C,

72.91; H, 7.34; N, 3.54%.

[α]_D ²⁵ -14° (c = 0.1%, MeOH)

N.M.R. (CDCl₃) δ = 1.37(s,9H); 1.95-2.30 (m, 2H); 2.79-3.20(m,5H); 3.92-4.01(m, 1H); 4.21-4.25 (m, 1H); 4.52- 4 . 56 (m , 1H) ; 7 . 21-7 . 36 (m, 10H) . b) (R)-2-Benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoic acid.

A suspension of the product from (a) (17.63 g) in dioxan (120 ml) and water (60 ml) was treated with sodium hydroxide (1N, 53.5 ml) at room temperature. The reaction was stirred for 3 hours before being acidified to pH 5 by the addition of acetic acid. After standing for a further 30 minutes the precipitate was filtered off and washed with water. This solid was dissolved in ethyl acetate, dried (MgSO₄) and evaporated under vacuum to a white solid which was triturated with hexane, filtered and dried to give the intermediate hydroxy acid (17.85 g). A solution of this hydroxy acid in N,N-dimethylformamide was treated with imidazole (29.38 g) and t-butyldimethylsilyl chloride (32.53 g) at room temperature. After stirring for 18 hours, the solvent was evaporated under vacuum, the residue treated with ice/water, 10% citric acid to pH 4, and extracted using 2 × 400 ml portions of ethyl acetate. The combined extracts were dried (MgSO₄) and evaporated under vacuum to a pale oil (29.2 g). A solution of this oil in tetrahydrofuran (240 ml) was treated with acetic acid (240 ml) and water (80 ml) at room temperature. After stirring for 2 hours at room temperature and 18 hours at 4°C the solution was evaporated under vacuum and the residue partitioned between water (400 ml) and ethyl acetate (400 ml). The separated organic layer was washed with water (2 × 400 ml), saturated brine (100 ml), dried (MgSO₄) and evaporated under vacuum to a pale oil. Chromatography on silica-gel, eluting with diethyl ether-hexane (70:30) gave the title compound as a white glass (22.4 g).

m/e 528 (MH) ⁺ N.M.R. (DMSO-d₆) δ = 0.10(s,6H) ; 0.95(s,9H);

1.30(m,10H); 1.35(m,1H) ; 1.95(m,1H); 2.40(m,1H) ;

2.72(m,2H); 2.85(m,1H); 3.60(m,1H) ; 3.75 (m,1H);

6.88(d,1H) ; 7.22(m,10H).

PREPARATION 3

5-Bromomethylisoquinoline

A solution of an isomeric mixture of 5- and 7-bromoisoquinoline (5:7, 40:60; 1.0 g; see Glyde and Taylor, J. Chem. Soc. Perkin Trans 2, 1975, 1783), in dry tetrahydrofuran (15 ml) was treated with 1.6M n-butyllithium in hexane (3.3 ml) at -70°C. The reaction was maintained at this temperature for 30 minutes and then a solution of dry dimethylformamide (0.74 ml) in dry tetrahydrofuran (5 ml) added. After a further 15 minutes, the reaction was quenched with ethanol (5 ml) and allowed to warm to room temperature. Saturated ammonium chloride solution (10 ml) and diethyl ether (15 ml) were then added sequentially and the organic phase separated, washed with saturated brine, dried (MgSO₄) and evaporated under vacuum. Purification by chromatography on silica gel eluting with hexane-ethyl acetate (50:50) and isolation of the higher running aldehyde gave 5-formylisoquinoline as an unstable yellow solid (0.05 g). Rf 0.3 (hexane-ethyl acetate 50:50).

A solution of this product (0.88 g) in dry methanol (20 ml) was treated with sodium borohydride (0.53 g) at 5°C and the resulting mixture allowed to warm to room temperature and maintained for 1 hour at this temperature. The solution was diluted with diethyl ether (20 ml) and water (10 ml) then added. The two phases were separated and the aqueous phases extracted with diethyl ether (20 ml). The two organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried (MgSO₄), and

evaporated under vacuum to give 5-hydroxymethyl- isoquinoline as a pale yellow coloured powder, (0.37 g), m.p. 71-72°C. Found: C,75.06; H,5.72; N,8.66.

C₁₀H₉NO requires C,75.45; H,5.70; N,8.80%.

A solution of the above product (1.11 g) in glacial acetic acid (15 ml) was treated with 49% aqueous hydrobromic acid (30 ml) and the resulting mixture heated to reflux for 2 hours. The reaction was then concentrated under vacuum and the residue

suspended in methylene chloride and basified with saturated aqueous sodium bicarbonate. The two phases were separated and the aqueous phase extracted with methylene chloride. The combined organic phases were washed with saturated aqueous sodium bicarbonate solution, dried (MgSO₄) for 0.5 hour and evaporated under vacuum at room temperature to give the title product as a colourless solid. The solid was

azeotroped with toluene and then used directly (1.28 g).

N.M.R. (CDCl₃) δ = 4.90(S,2H); 7.50(t,1H); 7.70(d,1H); 7.90(m,2H); 8.65(d,1H); 9.25(S,1H).

PREPARATION 4

7-Bromomethylisoguinoline

7-Formylisoquinoline was obtained from the initial step of Preparation 3 as the lower running product and isolated as a yellow solid (0.08 g). Rf 0.25 (hexane-ethyl acetate 50:50). Reaction with sodium borohydride as described above gave 7-hydroxymethylisoquinoline, m.p. 129-130°C.

A solution of the hydrochloride salt of the above product (0.05 g) in thionyl bromide (0.5 ml) was heated to 60°C and maintained for 45 minutes. The reaction was then ice-cooled and excess water carefully added, followed by diethyl ether (15 ml). Concentrated aqueous ammonia was then added to pH 9 and the ether phase separated, washed with water and dried (MgSO₄) . A solution of hydrogen chloride in isoproyl alcohol (0.06 ml, 5.9 N) was then added and the resulting cloudy suspension evaporated under vacuum at room temperature. The residue was azeotroped with toluene to give the product hydrochloride as a colourless solid (0.04 g).

m/e (MH)⁺ 222

N.M.R. (DMSO-d₆) δ = 5.05(s,2H); 8.10(d,1H);

8.25(d,1H); 8.30(d,1H); 8.45(s,1H); 8.65(d,1H);

9.70(s,1H).

PREPARATIONS 5-13

The following compounds of formula (IV) were prepared by the procedure described in Preparation 2 but using the appropriate substituted benzyl bromide or bromomethylisoquinoline to alkylate the gamma-butyrolactone in step (a) followed by ring opening and reaction with t-butyldimethylsilylchloride as described in step (b).

PREPARATION 14

1-(N-t-Butoxycarbonyl)-3-methanesulphonyloxy-azetidine a) Azetidine-3-ol hydrochloride (2.10 g) was stirred in methylene chloride (40 ml) and diisopropylethylamme (2.59 g) was added followed by di-t-butyldicarbonate (4.36 g). The mixture was stirred at room temperature for 4 hours and the solvent was evaporated under vacuum. The residue was dissolved in ethyl acetate (200 ml) and washed with 1.5H hydrochloric acid (50 ml), saturated sodium bicarbonate solution (25 ml) and brine (25 ml). The organic layer was dried (MgSO₄), filtered and was evaporate under vacuum.

Chromatography on silica gel, eluting with ethyl acetate-hexane (50 : 50) , gave 1- (N-t-butoxycarbonyl) -3-hydroxy-azetidine as a white solid (2.57 g), m.p. 51-53°C. Found: C,55.29; H,8.70; N,7.98. C₈H₁₅NO₃ requires C,55.47; H,8.73; N,8.09%.

b) A solution of the above product (1.0 g) in

methylene chloride (35 ml) was treated with

methanesulphonyl chloride (0.75 ml) and pyridine (1.5 ml) and the mixture stirred for 3 days at room

temperature. The solution was diluted with methylene chloride (75 ml), washed with aqueous citric acid solution (5%, 100 ml), saturated aqueous sodium

bicarbonate (100 ml), dried (MgSO₄) and evaporated under vacuum to give the title product as a colourless oil (1.4 g). Found: C,43.00; H,6.80; N,5.50. C₉H₁₇NO₅S requires C,43.03; H,6.77; N,5.58%. m/e 269 (MNH₄)⁺.

N.M.R. (DMSO-d₆) δ = 1.37(s,H); 3.24(s,3H); 3.88-3.96(m,2H); 4.17-4.28 (m,2H); 5.25(m,1H).

PREPARATION 15

1-(N-Benzyloxycarbonyl)-(R)-3-methanesulphonyl-oxypyrrolidine

The title compound was prepared from 1-(N-benzyloxycarbonyl)-(R)-3-hydroxypyrrolidine (J. Med. Chem., 1992, 35, 1764), using the procedure described in Preparation 14 above, except that

triethylamine was used as base instead of pyridine, to give the product as an oil. Found: C,51.60; H,5.80; N,4.30. C₁₃H₁₇NO₅S 1/6 H₂O requires C,51.63; H,5.78;

N,4.63%. m/e MH⁺ 300.

N.M.R. (CDCl₃) δ = 2.15(m,1H); 2.55(m,1H); 3.0(s,3H); 3.45-3.80(m,4H); 5.10(s,2H); 5.25(m,1H); 7.30(m,5H).

PREPARATION 16

1-(N-Benzyloxycarbonyl)-(S)-3-(paratoluene)sulphonyl-oxypyrrolidine

To a solution of 1-(N-benzyloxycarbonyl)-(R)-3-hydroxypyrrolidine (7.90 g) in dry tetrahydrofuran (100 ml) under nitrogen was added triphenylphosphine (13.58 g). The resulting solution was cooled to -30°C and methyl tosylate (10.06 g) added, followed by

deithylazodicarboxylate (10.41 g) over a 0.5 hour period. After a further 1 hour, the reaction was allowed to warm to room temperature and maintained for

65 hours. The reaction was evaporated under vacuum, the residue dissolved in dichloromethane, washed with water, dried (MgSO₄) and evaporated under vacuum to an oil. Purification by chromatography on silica gel eluting with methylene chloride-methanol (98:2 to

96:4), followed by a second chromatography eluting with hexane-ethyl acetate (80:20 to 50:50) gave the product as a golden coloured oil (10.22 g). Found: C,60.59;

H,5.68; N,3.67. C₁₉H₂₁NO₅S requires C,60.78; H,5.64;

N, 3.73%.

[α]_D ²⁵ 9° (c = 0.1%, MeOH)

N.M.R. (CDCl₃) δ = 1.85-2.25(m,2H); 2.45(s,3H); 3.40- 3.65(m,4H); 4.95-5.15(m,3H); 7.20-7.35 (m, 7H);

7.75(d,2H). PREPARATION 17

1-(N-t-Butoxycarbonyl)-4-methanesulphonyloxypiperidine

The title compound was prepared from 1-(N-t-butoxycarbonyl)-4-hydroxypiperidine, following the procedure described in Preparation 14, m.p. 85-86°C. Found: C,47.2; H,7.66; N,4.91. C₁₁H₂₁NO₅S requires

C,47.3; H,7.58; N,5.02%.

N.M.R. (CDCl₃) δ = 1.5(s,9H); 1.85(m,2H); 2.0(m,2H); 3.08(s,3H); 3.35(m,2H); 3.75(m,2H); 4.9(m,1H).

PREPARATION 18

1-(N-t-Butoxycarbonyl)-3-(imidazol-1-yl)azetidine

A solution of imidazole (0.41 g) in N,N-dimethylformamide (30 ml) was treated with sodium hydride (60%, 0.24 g) and the mixture stirred at room temperature for 1 hour. 1-(N-t-Butoxycarbonyl)-3-methanesulphonyloxy-azetidine (Preparation 14). (1.4 g) was added and the mixture was heated at 75°C for 3 days. The solvent was then removed under vacuum and the residue dissolved in ethyl acetate (50 ml), washed with water (2 × 50 ml), dried (MgSO₄) and evaporated under vacuum to give a colourless oil. Purification by chromatography on silica gel eluting with methylene chloride-methanol-concentrated aqueous ammonia

(93:7:1) gave the title compound as an oil (0.72 g). m/e 224 (MH)⁺

N.M.R. (DMSO-d₆) δ =1.40 (s, 9H); 3.95-4.06 (m, 2H); 4.26-4.35 (m, 2H); 5.12 (m, 1H); 6.96 (s, 1H); 7.43 (s, 1H); 7.79 (s, 1H).

PREPARATIONS 19-25

The following compounds of formula (V) were prepared following the procedure described in

Preparation 18 above but using the appropriate starting material from Preparations 14 to 17 and reacting with the appropriate substituted or unsubstituted imidazole or triazole.

PREPARATION 26

(a) 1-(N-t-Butoxycarbonyl)-4-(4-methylimidazol-1-yl)piperidine

Reaction of 4-methylimidazole with 1- (N-t-butoxycarbonyl) -4-methanesulphonyloxypiperidine gave two regioisomeric products, which were separated by

chromatography on silica-gel, eluting with

dichloromethane : methanol : concentrated aqueous ammonia (96:3.5:0.5). Major isomer, Rf 0.47. m/e 265.9 (MH) ⁺

N.M.R. (DMSO-d₆) δ = 1.43 (s, 9H); 1.7 (dq, 2H); 1.93 (bd, 2H); 2.07 (s, 3H); 2.83 (m, 2H); 4.05 (m, 3H);

6.95 (s, 1H); 7.55 (s, 1H).

(b) 1-(N-t-Butoxycarbonyl)-4-(5-methylimidazol-1-yl)_piperidine

Minor isomer, Rf 0.52. m/e 265.9 (MH) ⁺

N.M.R. (DMSO-d₆) δ = 1.43 (s, 9H); 1.7 (dq, 2H); 1.9 (bd, 2H); 2.05 (s, 3H); 2.88 (m, 2H); 4.05 (m, 3H); 6.6 (s, 1H); 7.65 (s, 1H).

PREPARATION 27

1-(N-t-Butoxycarbonyl)-4-(imidazol-2-yl)-(1,2,5,6-tetrahydropyridine

(a) 1-(Diethoxymethyl)imidazole (6.8 g) was stirred in dry tetrahydrofuran (50 ml) under nitrogen at -40 ºC. n-Butyllithium (25 ml, 1.6N in hexane) was added at such a rate that the temperature remained below -35ºC. 1- (N-t-Butoxycarbonyl)-4-keto-piperidine (2.65 g) in dry tetrahydrofuran (10 ml) was added dropwise over 10 minutes, keeping the temperature below -40 °C, and the resulting mixture stirred at -40°C for 2 hours. The reaction mixture was stirred with hydrochloric acid (50 ml, 0.1N) for 15 minutes, ethyl acetate (50 ml) was then added and the resulting mixture stirred for 5 minutes. The organic layer was separated and the aqueous layer extracted with ethyl acetate (1 × 50 ml). The combined organic extracts were washed with

saturated sodium bicarbonate solution (1 × 50 ml), then with saturated sodium chloride solution. The organic layer was then dried (MgSO₄) and evaporated to a yellow oil, 8.0 g. Chromatography on silica-gel, eluting with ethyl acetate: methanol: concentrated aqueous ammonia (90:10:1), yielded 1-(N-t-butoxycarbonyl)-4-hydroxy-4-imidazol-2-ylpiperidine a cream solid, 2.2 g. m/e 268.0 (MH)⁺.

(b) The above product (267 mg) was stirred with diisopropylamine (350 ml) in dry dimethylformamide (2 ml) at 0ºC. Methanesulphonyl chloride (155 ml) was added in one portion, and the reaction mixture stirred for 2 hours at 0ºC. Further portions of

diisopropylamine (350 ml) and methanesulphonyl chloride (155 ml) were then added and the reaction mixture stirred for 16 hours at room temperature. The resulting mixture was diluted with water (4 ml), adjusted to pH 9 with 1M sodium hydroxide solution, and extracted with ethyl acetate (3 × 10 ml). The combined organic extract was dried (MgSO₄), and evaporated to a gum, 260 mg.

Chromatography on silica-gel, eluting with

dichloromethane: methanol: concentrated aqueous ammonia (95:5:1) gave the title product as a yellow gum, 144 mg. m/e 250.1 (MH)⁺.

N.M.R. (CDCl₃) δ = 1.46 (ε, 9H); 2.6 (bs, 2H); 3.53 (t, 2H); 4.0 (bs, 2H); 6.3 (m, 1H); 7.0 (s, 2H); 9.4 (bs, 1H).

PREPARATION 28

1-(N-t-Butoxycarbonyl)-4-imidazol-2-ylpiperidine

The product from Intermediate Preparation (27) (0.55 g) was dissolved in ethanol (30 ml) and hydrogentated at 30 p.s.i. (2.0 bar) with palladium on carbon catalyst (200 mg, 10%). Filtration of catalyst and removal of solvent gave a foam, 0.55 g. m/e 252.1 (MH)⁺. N.M. R. (CDCI₃) δ = 1. 4 (s , 9H) ; 1. 68 (qd, 2H) ; 1. 95 (bd, 2H) ; 2 .75 (bt , 2H) ; 2 . 92 (bt, 1H) ; 4 . 1 (bd, 2H) ; 6. 9 (s , 2H) ; 8 . 77 (bs , 1H) .

PREPARATION 29

1-(N-t-Butoxycarbonyl)-4-(imidazol-1-yl)methyl-1,2,5,6-tetrahvdropyridine

(a) A 60% oil dispersion of sodium hydride (4 g) in dry dimethylsulphoxide (100 ml) was washed free of oil with hexane and then heated at 70°C with stirring for 1 hour. Dry tetrahydrofuran (100 ml) was added and the reaction cooled to -20°C. A solution of

trimethylsulphonium iodide (20.4 g) in

dimethylsulphoxide (80 ml) was then added, followed by 1-(N-t-butoxycarbonyl)-4-ketopiperidine (19.9 g) in dry tetrahydrofuran (100 ml) and the reaction stirred for 0.5 hour at -10°C and then 1 hour at room temperature. Water (500 ml) was then added and the mixture extracted with ethyl acetate (3 × 250 ml). The combined extracts were then washed with brine, dried (MgSO₄) and

evaporated under vacuum. Purification by chromatography on silica gel eluting with cyclohexane-ether-isopropyl alcohol (60:40:1) gave 1-(N-t-butoxycarbonyl)-piperidine-4-spiro-2'-oxirane as a colourless solid, (19.6 g), m.p. 65-66°C. Found C, 62.11; H, 9.06; N, 6.55. C₁₁H₁₉NO₃ requires C, 61.97; H, 8.92; N, 6.57%.

(b) A stirred solution of imidazole (1.91 g) in dry acetonitrile (30 ml) under nitrogen was treated with an 80% oil dispersion of sodium hydride (0.84 g) and the resulting mixture heated to 60°C until solution

occurred. After 15 minutes the product from step (a) (2.0 g) was added and the reaction maintained for 5 hours. After allowing the reaction to stand at room temperature overnight, the solvent was evaporated under vacuum and the oily residue partitioned between methylene chloride (40 ml) and water (20 ml). The organic phase was separated and washed with water, dried (MgSO₄) and evaporated under vacuum. Purification by chromatography on silica gel, eluting with methylene chloride-methanol-concentrated 880 aqueous ammonia (95:4:1) gave 1-(N-t-butoxycarbonyl)-4-hydroxy-4-(imidazol-1-yl)methylpiperidine as a colourless powder (2.13 g). Found C, 58.35; H, 8.30; N, 14.52. C₁₃H₂₃N₃O₃ ¹/₁₀ CH₂Cl₂ requires C, 58.43; H, 8.07; N, 14.50%.

m/e 282 (MH)⁺.

(c) A stirred solution of the product from step (b) (2.0 g) and triethylamine (5.44 ml) in dry methylene chloride (80 ml) at 0 to 5°C was treated with a

solution of methanesulphonyl chloride (2.20 ml) in dry methylene chloride (10 ml) and the resulting mixture allowed to warm to room temperature and maintained for 14 hours. The reaction mixture was then washed with water, dried (MgSO₄) and evaporated under vacuum.

Purification of the residue by chromatography on silica gel eluting with methylene chloride-methanol-concentrated 880 aqueous ammonia (96:4:0 to 95:4:1) gave the product as a golden coloured oil (1.36 g).

Found C, 59.93; H, 7.60; N, 14.79. C₁₃H₂₁N₃O₂ ¹/₄ CH₂Cl₂ requires C, 60.14; H, 7.61; N, 14.77%.

m/e (MH)⁺ 264

N.M.R. (CDCl₃) δ = 1.45 (s, 9H); 1.97 (m, 2H); 3.50 (t, 2H); 3.90 (s, 2H); 4.50 (s, 2H); 5.50 (s, 1H); 6.90 (s, 1H); 7.10 (s, 1H); 7.55 (s, 1H).

PREPARATION 30

1-(N-t-Butoxycarbonyl)-4-(imidazol-1-yl)methyl-piperidine

A solution of the product from Preparation 29 (1.33 g) in absolute ethanol (25 ml ) was hydrogenated with stirring over 10% palladium on charcoal (0.3 g) at 50 p.s.i. (3.5 bar), room temperature, for 4 hours. The reaction mixture was then filtered and evaporated under vacuum, azeotroping with methylene chloride.

Purification by chromatography on silica gel eluting with methylene chloride-methanol (96:4) gave the product as a colourless oil (1.09 g). Found C, 63.08; H, 8.60; N, 15.50. C₁₃H₂₃N₃O₂1/4 CH₂Cl₂ requires C, 63.36; H, 8.74; N, 15.84%. m/e (MH) ⁺ 266

N.M.R. (CDCI₃) δ = 1.15 (m, 2H); 1.45 (s, 9H); 1.58 (m, 2H); 1.85 (m, 1H); 2.65 (t, 2H); 3.80 (d, 2H); 4.10 (m, 2H); 6.90 (s, 1H); 7.10 (s, 1H); 7.45 (s, 1H).

PREPARATION 31

1-(N-t-Butoxycarbonyl)-4-(imidazol-1-yl)-1,2,5,6-tetrahydropyridine

Imidazole (8.20 g) was stirred in dry methylene

chloride (30 ml) at -10°C and thionyl chloride (4.8 ml) in dry methylene chloride (30 ml) added. The resulting slurry was allowed to warm to room temperature and after 2 hours a solution of 1-(N-t-butoxycarbonyl)-4-keto-piperidine (6.0 g) in dry methylene chloride (50 ml) was added dropwise. The reaction mixture was stirred overnight and then evaporated under vacuum.

Potassium carbonate (6.0 g) in water (30 ml) was added to the oily residue and the product extracted with methylene chloride (2 × 60 ml). The combined extracts were washed with water (40 ml), dried (MgSO₄) and evaporated under vacuum. Purification by chromatography on silica gel eluting with ethyl acetate-methanol

(100:0 to 90:10), gave the product as an oil (1.0 g). N.M.R. (CDCI₃) δ = 1.50 (s, 9H); 2.55 (s, 2H); 3.70 (t, 2H); 4.05 (s, 2H); 5.80 (s, 1H); 7.10 (s, 2H); 7.65 (s, 1H).

PREPARATION 32

1-(N-Benzyloxycarbonyl)-4-(1 , 2 ,4-triazol-4-yl)piperidine To a solution of 1-(N-benzyloxycarbonyl)-4-ketopiperidine (5.0 g) in methanol (25 ml) was added ammonium acetate (16.5 g) and sodium cyanoborohydride (0.94 g) and the mixture was stirred at room

temperature for 24 hours. Solvent was then removed under reduced pressure and the residue partitioned between ethyl acetate and 1M sodium hydroxide solution. The ethyl acetate layer was separated, dried over magnesium sulphate, and evaporated under reduced pressure to give a yellow oil. Chromatography of this residue on silica-gel, eluting with methylene chloride-methanol-concentrated aqueous ammonia (95:5:1) gave 4-amino-1-(N-benzyloxycarbonyl)-piperidine as a yellow oil. A solution of this product (1.5 g) in toluene (20 ml) was treated with dimethylformamide azine (1.0 g) and p-toluenesulphonic acid (0.1 g), and the mixture was heated under reflux for 24 hours. Solvent was then removed under reduced pressure and the residue was chromatographed on silica-gel, eluting with methylene chloride-methanol-concentrated aqueous ammonia

(93:7:1), to give the title compound as a colourless oil. m/e (MH⁺) 287

N.M.R. (DMSO-d₆) δ = 1.82 (m, 2H); 2.01 (m, 2H); 2.83-3.09 (m, 2H); 4.12 (m, 2H); 4.40 (m, 1H); 5.11 (s, 2H); 7.28-7.45 (m, 5H); 8.65 (s, 2H).

PREPARATION 33

1-(N-t-Butoxycarbonyl-(S)-valyl)-3-(imidazol-1-yl)azetidine

A solution of 1-(N-t-butoxycarbonyl)-3-(imidazol-1-yl)azetidine (from Preparation 18) (0.72 g) in

methylene chloride (30 ml) was saturated with hydrogen chloride at 0°C and kept at this temperature for a further 1 hour. The solvent was removed under vacuum to give the amine hydrochloride which was dissolved in N,N-dimethylformamide (25 ml) and the solution was treated at room temperature with N-t-butoxycarbonyl-(S)-valine N-hydroxysuccinimide ester (1.01 g) and N,N-diisopropylethylamine (1.7 ml). The reaction mixture was stirred for 18 hours at room temperature and the solvent was then removed under vacuum. Purification of the residue by chromatography on silica gel, eluting with methylene chloride-methanol-concentrated ammonia (97:7:1) gave the title compound as a colourless foam (0.79 g). m/e 323 (MH) ⁺

N.M.R. (DMSO-d₆) δ = 0.89 (m, 6H); 1.38 (s, 9H); 1.90 (m, 1H): 3.61-3.78 (m, 1H); 3.92-4.10 (m, 1H); 4.24-4.80 (m, 3H); 5.19 (m, 1H); 6.98 (s, 1H); 7.06 (dd, 1H); 7.38 (s, 1H); 7.77 (s 1H).

PREPARATIONS 34-43

The following compounds of formula (VII) where R⁴ is (S)-isopropyl and R⁷ and R⁸ are hydrogen were

prepared following the procedure of Preparation 33 using the appropriate intermediate from Preparations 18 to 32 and coupling to N-t-butoxycarbonyl-(S)-valine N-hydroxysuccinimide ester.

PREPARATIONS 44-48

The following compounds of formula (VII) where R⁴ is sec-butyl and m is O were prepared following the production of Preparation 33 using N-t-butoxycarbonyl- (S)-isoleucine N-hydroxy-succinimide ester in the coupling step.

PREPARATION 49

1-(N-t-Butoxycarbonyl-(S)-isoleucyl)-4-ketopiperidine

The title compound was prepared using the same procedure as described for Preparation 33 but using 4-ketopiperidine hydrochloride hydrate instead of 3- (imidazol-1-yl)azetidine hydrochloride and (S)-isoleucine N-hydroxysuccinimide ester, m/e 313 (MH)⁺ [α]_D ²⁵ -16° (c = 0.34%, MeOH)

N.M.R. (CDCl₃) δ = 0.9 (m, 6H); 1.2 (m, 1H); 1.4 (s,

9H); 1.6 (m, 1H); 1.75 (m, 1H); 2.5 (m, 4H); 3.7 (m,

2H); 4.15 (m, 2H); 4.55 (m, 1H); 5.2 (d, 2H).

PREPARATION 50

N-( (R)-2-Benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoyl)-(S)-valine

The title compound was prepared by the method described in S. J. deSolms et al . , J. Med Chem., 1991, 34, 2852.

PREPARATION 51

1-Isocyano-3-methyl-1- (p-toluenesulphonyl)-but-1-ene The title compound was prepared from p-toluenesulphonylmethylisocyanide and isobutyraldehyde by the method of Van Leusen, Schaart and Van Leusen Recueil, 98, No. 5, 258 (1979). i.r. (Nujol) 2100cm^-1 m/e 267 (M+ NH₃) ⁺

N.M.R. (CDCl₃) δ = 1.15 (d, 6H); 2.5 (s, 3H); 2.84 (m, 1H); 6.88 (d, 1H); 7.4 (d, 2H); 7.85 (d, 2H).

PREPARATION 52

3-Oxetanyloxycarbonyloxysuccinimide

Oxetan-3-ol (2.0 g) and N,N-diisopropylethylamine (7.76 g) were dissolved in methylene chloride (50 ml) and the solution was added dropwise to a solution of bis-trichloromethyl carbonate (2.69 g) in methylene chloride (100 ml) maintained at -20 °C over a period of 15 minutes under an atmosphere of nitrogen. The

solution was then stirred for a further 15 minutes at -20ºC and N-hydroxy-succinimide (3.45 g) was added in one portion. The solution was allowed to warm to room temperature over a period of 2 hours and then washed with water (50 ml), saturated aqueous sodium

bicarbonate (50 ml) and brine (25 ml). The organic layer was then dried (MgSO4), filtered and the solvent was removed under vacuum to give the title compound as a light brown oil, (4.65 g). N.M.R. (CDCl₃) δ = 2.83 (s, 4H); 4.75 (m, 2H); 4.90 (m, 2H); 5.58 (m, 1H).

EXAMPLE 1

1-[N-( (R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy- 6-phenylhexanoyl)- (S )-valyl]-3-(imidazol-1-yl)azetidine

a) 1-[N-( (R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-t-butyldimethylsilyloxy-6-phenylhexanoyl)- (S)-valyl]-3-(imidazol-1-yl)azetidine

A solution of 1-(N-t-butyloxycarbonyl-(S)-valyl)-3-(imidazol-1-yl)azetidine ( from Preparation 33, 0.79 g) in methylene chloride (50 ml) was saturated with hydrogen chloride at 0° and kept at this temperature for a further 1 hour. The solvent was evaporated under vacuum to give the amine as a colourless solid. A solution of this product in dimethylformamide (20 ml) was added to an active ester solution previously prepared by stirring together (R)-2-benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy)-6-phenylhexanoic acid (from Preparation 2, 1.32 g), 1-hydroxy-benzotriazole (0.36 g), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (0.58 g) and N,N-diisopropylethylamine (1.75 ml) in dimethylformamide (75 ml) for 20 minutes. After stirring for a further 24 hours, the solvent was removed under vacuum and the residue partitioned between ethyl acetate and water. The ethyl acetate layer was separated, dried (MgSO₄) and removed under vacuum to yield a colourless oil. Purification by chromatography on silica-gel eluting with ethyl acetate-methanol-concentrated aqueous ammonia (90:10:1) gave the title compound as a colourless foam (1.1 g). Found: C,66.04; H,8.26;

N,9.61. C₄,H₆₁N₅O_S Si. 1/2 H₂O requires C,66.39; H,8.36; N,9.44%.

m/e 733 (MH) ⁺

[α]_D ²⁵ -6° (c = 0.29%, MeOH)

N.M.R. (DMSO-d₆) δ = 0.12(m,6H); 0.90 (m, 15H); 1.25- 1.29(2 x s,9H); 1.96(m,1H); 2.32-2.77 (m, 4H); 2.89(m,1H); 3.48-3.74 (m,2H); 3.88-4.06 (m, 3H) ; 4.18- 4.66(m,4H); 5.12(m,1H) ; 6.77(t,1H); 6.98(s,1H) ; 7.07- 7.32(m,10H); 7.40-7.82(2 x s,1H) ; 7.76-7.96(2 x s,1H);

8.0-8.20(2 x d,1H) .

b) 1-[N-((R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)- (S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazol- 1-yl)azetidine

The product from step (a) (1.19 g) was dissolved in tetrahydrofuran and treated with a 1M solution of tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature. After 48 hours the solvent was removed under vacuum, the product taken up in ethyl acetate, washed with saturated aqueous sodium

bicarbonate and water, dried over MgSO₄ and the solvent evaporated under vacuum. Purification by

chromatography on silica-gel eluting with methylene chloride-methanol-concentrated aqueous ammonia (93:7:1) followed by recrystallisation from ethyl acetate/hexane gave the product as a colourless solid, (0.52 g), m.p.

124-126°. Found: C,67.99; H,8.01; N,11.06. C₃₅H₄₇N₅O₅ requires C,68.05; H,7.67; N,11.34%. m/e 618 (MH) ⁺

[α]_D ²⁵ -3° (c = 0.14%, MeOH)

N.M.R. (DMSO-d₆) δ = 0.80(m,6H); 1.21-1.47 (m, 1H);

1.27(s,9H) 1.58-1.71 (m,1H); 1.81-1.98 (m,1H); 2.44-2.62(m,2H) 2.68-2.92(m,3H); 3.37-3.61 (m, 2H); 3.85-4.02(m,2H) 4.13-4.36(m,2H); 4.41-4.56 (m,2H);

5.18(m,1H) 6.43(d,1H); 6.98(S,1H); 7.05-7.30 (m,10H);

7.26-7.77(2 x s,1H); 7.38-7.82(2 x s,1H); 7.88-7.98(2 x d,1H). EXAMPLES 2-10

The following compounds were prepared following the general procedure described in Example 1 but using the appropriate protected carboxylic acid intermediate of formula (IV) and the appropriate amine of formula (VIII) in the coupling step (a) followed by removal of the t-butyldimethylsilyl protecting group as described in step (b).

EXAMPLE 11

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)- 2-(4-(trifluoromethoxy)benzyl)hexanoyl)- (S)-valyl]-(S)-3-(imidazol-1-yl)pyrrolidine

The procedure of Example 1 was followed using 1- (N-t-butoxycarbonyl-(S)-valyl)-3(S)-imidazol-1-yl)pyrrolidine (Preparation 36) in the coupling step followed by removal of the t-butyldimethylsilyl group as described in Example 1(b) to give the title product. m.p. 111-112°C. Found: C, 61.90; H, 6.85; N, 9.61.

C₃₇H₄₈F₃N₅O₆ requires C, 62.08; H, 6.76; N, 9.78%.

m/e (MH)⁺ 716

[α]_D ²⁵ 2° (c = 0.1%, MeOH) [α]² ₅₃₆₅+ 14° (c = 0.1%, MeOH)

N.M.R. (DMSO-d6) δ = 0.65-0.90 (m, 6H); 1.15-1.35 (m,

10H); 1.60 (m, 1H); 1.85 (m, 1H); 2.05-2.90 (m, 7H);

3.20-4.10 (m, 6H); 4.20 (m, 1H); 4.55 (m, 1H); 4.85 (m, 1H); 6.40 (d, 1H); 6.90-6.95 (2 x s, 1H); 7.10-7.30 (m,

10H); 7.70-7.80 (2 x s, 1H); 7.90-8.0 (2 x d, 1H).

EXAMPLE 12

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)- 2-(4-(trifluoromethoxy)benzyl)hexanoyl)- (S)-valyl]-(R)-3-(imidazol-1-yl)pyrrolidine

The above procedure was followed but starting with

1-(N-t-butoxycarbonyl-(S)-valyl-3(R)-imidazol-1-yl)pyrroldine to give the title product, m.p. 109°C.

Found: C, 61.44; H, 7.05; N, 9.71. C₃₇H_4gF₃N₅O₆ ²/₅ H₂O requires C, 61.47; H, 6.80; N, 9.69%. m/e (MH) ⁺ 716

[α]_D ² ₅ -23° (c = 0.1%, MeOH)

N.M.R. (DMSO-d6) δ = 0.70-0.85 (m, 6H); 1.15-1.40 (m,

10H); 1.60 (m, 1H); 1.85 (m, 1H); 2.00-2.90 (m, 7H);

3.25-3.70 (m, 5H); 3.95 (m, 1H); 4.20 (m, 1H); 4.45- 4.65 (m, 1H); 4.75-4.95 (2 x m, 1H); 6.40 (m, 1H); 6.90

(s, 1H); 7.05-7.25 (m, 10H); 7.70-7.75 (2 x s, 1H);

7.90-8.05 (2 x d, 1H). EXAMPLE 13

1-[N-( (R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine

a) 1-[N-( (R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy-6-phenylhexanoyl)-(S)-valyl-4-(imidazol-1-yl)piperidine

The title compound was prepared from 1-(N-t-butoxycarbonyl-(S)-valyl-4-imidazol-1-ylpiperidine and (S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy-(R)-2-benzyl-6-phenylhexanoic acid using the same procedure as described in Example 1 step (a).

Purification by chromatography on silica-gel eluting with ethyl acetate-methanol-concentrated aqueous ammonia (90:10:1) gave the product as a colourless foam (0.71 g). Found: C,67.32; H,8.44; N,9.22. C₄₃H₆₅N₅O₅ Si. 1/4 H₂O requires C, 67.54; H,8.56; N,9.15%.

m/e 760 (MH)⁺

N.M.R. (DMSO-d₆) δ = 0.12(m,6H); 0.91(m,15H); 1.13-1.30(m,2H); 1.26(s,9H); 1.47-1.76 (m, 2H); 1.89-2.08(m,4H); 2.36-2.97 (m, 6H); 3.50-3.75 (m, 2H); 3.99-4.61(m,4H); 6.79(m,1H); 6.90(d,1H); 7.07-7.29 (m, 11H); 7.68-7.73(2 x s,1H); 7.93-7.99(2 x d,1H).

b) 1-[N-(R)-2-Benzyl- (S)-5-(t-butoxycarbonylamino)- (S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine

The title compound was prepared from the product of step a) above by the same procedure as described for Example 1 step b). Purification by chromatography on silica-gel eluting with methylene chloride-methanol-concentrated aqueous ammonia followed by

recrystallisation from ethyl acetate/hexane gave the product as a colourless solid, m.p. 121-123° (0.48 g). Found: C,68.38; H,7.96; N,10.59. C₃₇H₅₁N₅O₅. 1/4 H₂O requires C,68.30; H,7.92; N, 10.77%.

m/e 646 (MH)⁺ [α]²⁵ -10° (c = 0.1, MeOH)

N.M.R. (DMSO-d₆) δ = 0.77 (m, 6H); 1.19-1.37 (m, 1H);

1.28(s,9H); 1.48-1.72(m,3H); 1.82-2.04 (m, 3H) ; 2.42-2.94(m,7H); 3.38-3.60 (m,2H)); 3.96-4.15 (m, 1H);

4.28(m,1H); 4.37-4.58 (m, 3H); 6.43(d,1H); 6.88(s,1H); 7.04-7.29 (m,11H); 7.65-7.69(2 x s,1H); 7.84-7.88(2 x d,1H).

Examples 14-37

The following compounds were prepared following the procedure described in Example 13 but using the appropriate protected carboxylic acid intermediate of formula (IV) and the appropriate valine or isoleucine derivative of formula (VIII) in the coupling step (a) followed by removal of the t-butyldimethylsilyl protecting group as described in step (b).

EXAMPLE 31

1-[N-( (R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(1,2,4-triazol-4-yl)piperidine

The benzyloxycarbonyl protecting group was removed from 1-(N-benzyloxycarbonyl)-4-(1,2,4-triazol-4-yl)-piperidine (Preparation 32) by catalytic hydrogenation and the resulting amine product coupled to N-((R)-2-benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy)-6-phenylhexanoyl)-(S)-valine following the procedure described for Example 1 step (a), to give 1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoyl)-(S)-valyl]-4-(1,2,4-triazol-4-yl)piperidine, m/e 761 (MH⁺) .

The above product was treated with tetra-n-butylammonium fluoride following the procedure of

Example 1 step (b) to give the title product. Found:

C,64.90; H,7.93; N,12.48. C₃₆H₅₀N₆O₅. H₂O requires

C,65.06; H,7.83; N,12.65%. m/e 647 (MH) ⁺

[α]_D ² ₅ -9° (c = 0.12 MeOH)

N.M.R. (DMSO-d₆) δ = 0.70-0.92 (m, 6H); 1.15-1.41(m, 10H);

1.46-1.76(m,3H); 1.81-2.16 (m, 3H); 2.53-2.96 (m,7H);

3.38-3.64 (m,2H); 3.95-4.15 (m, 1H); 4.27-4.61(m,4H);

6.36-6.44(m,1H); 7.01-7.12 (m, 10H); 7.78-7.91 (m, 1H);

8.54(s,1H); 8.60(s,1H).

EXAMPLE 32

1-[N-( (S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)- 2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl]-4-fimidazol-1-yl)1,2,5,6-tetrahydropyridine; tartrate

a) Reaction of (S)-5-t-butoxycarbonylamino-(S)-4-tbutyldimethylsilyloxy)-(R)-2-(4-trifluoromethoxy¬benzyl)-6 phenylhexanoic acid and the amine prepared by deprotection of 1-(N-t-butoxycarbonyl)-4-imidazol-2- yl(1,2,5,6-tetrahydropyridine) following the procedure of Example 1(a) gave 1-[N-((S)-5-t-butyloxycarbonyl¬amino)-(S)-4-t-butyldimethylsilyloxy)-6-phenyl-(R)-2- (4-trifluoromethoxybenzyl)hexanoyl-(S)-valyl]-4- (imidazol-1-yl)-1,2,5,6-tetrahydropyridine. Found:

C,62.19; H,7.39; N,8.38. C₃₃H₆₂F₃N₅O₆Si 1/10 CH₂Cl₂ requires C,62.27; H,7.37; N,8.23%.

m/e 842 (M)⁺

[α]_D ² ₅ -10° (c = 0.1%, MeOH)

N.M.R. (CDCl₃) δ = 0.10 (m,6H); 0.80 (d,6H);

0.90(s,9H); 1.35(s,9H); 1.20-1.95 (m,5H); 2.35- 2.55(m,3H); 2.70(m,2H); 3.41-4.10 (m, 6H) ; 4.55(m,1H);

4.65(d,1H); 5.60-5.80(2 x s, 1H); 6.25(d,1H); 6.90- 7.30(m,11H); 7.65(s,1H).

b) Deprotection by reaction with tetra-n-butylammonium fluoride following the procedure of

Example 1(b) gave the title product, free base. This was dissolved in absolute ethanol and treated with a solution of 1-tartaric acid (0.14 g) in absolute ethanol. Addition of ether gave a precipitate which was filtered and dried to give the 1-tartrate salt as a colourless solid, m.p. 92-152°C (0.61 g). Found: C,

57.40; H, 6.64; N, 7.51. C₃₈H₃₈F₃N₅O₆. CH,₆O₆ V₃ H₂O requires

C, 57.11; H, 6.23; N, 7.93%.

m/e (MH)⁺ 728

[α]_D ² ₅ +6.7° (c = 0.1%, MeOH)

[α]² ₅₃₆₅- 6.7° (c = 0.1%, MeOH)

N.M.R. (DMSO-d6) δ = 0.75 (m, 6H); 1.25 (s, 9H); 0.95- 1.40 (m, 1H); 1.50 (m, 1H); 1.90 (m, 1H); 2.35-2.95 (m,

7H); 3.0-3.85 (m, 4H); 4.0 (m, 1H); 4.10 (m, 1H); 4.25

(s, 2H); 4.40-4.60 (m, 2H); 6.0 (m, 1H); 6.40 (d, 1H);

7.0 (s, 1H); 7.10-7.30 (m, 9H); 7.50 (s, 1H); 7.95 (m,

2H) . EXAMPLE 33

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hvdroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-( S)-isoleucyl]-4-(5-isopropylimidazol-1-yl)piperidine a) Deprotection of 1-(N-t-butoxycarbonyl-(S)-isoleucyl)-4-ketopiperidine (Preparation 49) followed by reaction with (S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoic acid (Preparation 10) gave 1-[N-((S)-5-(t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl-(S)-isoleucyl]-4-ketopiperidine. Found: C,62.50; H,8.00; N,5.20.

C₄₂H₆₂F₃₃O₇Si requires C, 62 . 60 ; H, 7 . 80 ; N, 5. 20% .

m/e 806. 5 (MH) ⁺ . [α]_D ²⁵ -5. 30 (c = 0 . 1% , MeOH) . b) The above product (2.0 g) and aqueous ammonia (specific gravity 0.88), in ethanol (25 ml) were hydrogenated with palladium on carbon (5 100 mg) at 30 p.s.i. (2.0 bar) for 4 hours. Filtration of catalyst and evaporation of solvent gave a colourless foam, 2.0 g, which was chromatographed on silica-gel, eluting with dichloromethane:methanol: concentrated aqueous ammonia (90:10:1), to give, after concentration of the appropriate fractions 1-[N-((S)-5-t-butoxycarbonyl-amino-(S)-4-t-butyldimethylsilyloxy-6-phenyl-(R)-2-(4-trifluoromethoxy)benzyl)hexanoyl)-(S)-isoleucyl]-4-aminopiperidine as a colourless glass, 1.53 g.

c) The product from step b) above (404 mg) and 1-isocyano-3-methyl-1-(p-toluenesulphonyl)-but-1-ene, (from Preparation 51, 150 mg) were stirred together in methanol (15 ml) with diisopropylethylamme (100 mg) for 16 hours. The solvent was removed at 40°C and the residue purified by chromatography on silica-gel, eluting with dichloromethane:methanol:concentrated aqueous ammonia (98:2:0.4), to give 1-[N-((S)-5-(t- butoxycarbonylamino)-(S)-4-t-butyldimethylsilyoxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl]-4-(5-isopropylimidazolyl-1-yl)piperidine as a colourless glass (380 mg). m/e 900 (MH⁺) .

d) The product from step c) above was deprotected by treatment with tetra-n-butylammonium fluoride following the procedure of Example (1b) to give the title

product.

Found : C, 63.5; H, 7.70; N, 8.70. C₄₂H₅₈F₃N₅O₆. ¹/₂H₂O requires. C, 63.5; H, 7.48; N, 8.81%.

m/e 785.9 (MH)⁺

N.M.R. (DMSO-d₆) δ = 0.8 (m, 6H); 1.05 (m, 1H); 1.2 (m, 6H); 1.3 (s, 9H); 1.3-2.0 (m, 7H); 2.5-3.0 (m, 6.5H);

3.2 (bt, 0.5H); 3.3 (m, 1H); 3.58 (m, 1H); 4.13 (m,

3H); 4.4-4.65 (m, 4H); 6.43 (m, 1H); 6.63 (s, 1H); 7.1- 7.3 (m, 9H); 7.47, 7.6 (s,s, 1H); 7.95 (bt, 1H).

EXAMPLE 34

1-[N-((S)-4-Hydroxy-(S)-5-(oxetan-3-yloxycarbonyl-amino)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)-hexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine

The product from Example 16 (4.0 g) was dissolved in methylene chloride (40 ml) and cooled in an ice bath. Trifluoroacetic acid (10 ml) was added dropwise over a period of 5 minutes and the solution stirred at 0°C for 1.5 hours. The solvent was evaporated under vacuum and the residue taken up in ethyl acetate (250 ml) and washed with 1M sodium hydroxide (50 ml) and brine (50 ml). The organic solution was dried (MgSO₄), filtered and the solvent was evaporated under vacuum. Chromatography on silica gel, eluting with ethyl acetate-methanol-concentrated aqueous ammonia (90:10:1) gave 1-[N-(S)-5-amino-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl]-4- (imidazol-1-yl)piperidine as a white solid (2.98 g). m/e 630 (MH)⁺ N.M.R. (DMSO-d₆) δ = 0.78(m,6H); 1.34-1.75 (m, 4H); 1.81- 2.07(m,3H); 2.37-2.93 (m,8H); 3.05-3.29 (m, 3H); 4.05- 4.56(m,5H); 6.88(s,1H); 7.10-7.27 (m, 10H); 7.67(d,1H);

7.95(m,1H);

b) The product from step a) (0.818 g) was dissolved in methylene chloride (30 ml). 3-Oxetanylcarbonyloxy-succinimide (0.344 g) was added and the solution stirred at room temperature for one hour. The solution was then washed with 0.5M sodium hydroxide (15 ml) and brine (15 ml), dried (MgSO₄), filtered and evaporated under vacuum. The resulting solid was recrystallised from ethyl acetate to give the title compound as a white solid, m.p. 201-203 °C. Found: C,60.32; H,6.22;

N,9.15. C₃₇H₄₆F₃N₅O₇ 1/2 H₂O requires C,60.15; H,6.41;

N,9.48%.

m/e 730 (MH)⁺

[α]_D ² ₅ +3° (c = 0.1%, MeOH), [α]² ₅₃₆₅+ 32° (c = 0.1, MeOH)

N.M.R. (DMSO-d₆) δ = 0.77 (m, 6H); 1.22-2.05 (m,7H); 2.50- 2.93 (m,8H); 3.42-3.60(m,2H); 4.08(d,1H); 4.22- 4.73 (m,7H); 5.12(m,1H); 6.89(s,1H); 7.11-7.28 (m,11H);

7.67(d,1H); 7.86(d,1H) .

EXAMPLE 35

1-[N-(S)-4-Hydroxy-(S)-5-(oxetan-3-yloxycarbonylamino)-6-phenyl -(R)-2-(3-phenylprop-2-enyl)hexanoyl)-(S)-isoleucyl]-4-(imidazol-1-yl)piperidine

A solution of the product from Example 18 (0.45 g) in methylene chloride (20 ml) was treated with anhydrous trifluoroacetic acid (4 ml) at 0-5ºC for 5 hours. The reaction was then concentrated under vacuum and the residual gum azeotroped with toluene (x3) and dried. A solution of the crude amine product and diisopropylethylamme (0.82 ml) in methylene chloride (25 ml) was then cooled to 5ºc and a solution of 3-oxetanyloxycarbonyloxysuccinimide (0.22 g) in methylene chloride (5 ml) added dropwise. The reaction was allowed to warm to room temperature, maintained for 17 hours and then washed with water, dried (MgSO₄), and evaporated under vacuum. Purification by chromatography on silica gel eluting with methylene chloride-methanol-concentrated aqueous ammonia (95:4:1) gave product as a colourless foam, m.p. 157-160°C (0.26 g).

Found : C, 66.45; H, 7.49; N, 10.22. C₃₈H₄₉N₅O₆ ³/₄H₂O requires C, 66.60; H, 7.43; N, 10.22%.

[α]_D ² ₅ +32° (c = 0.1%, MeOH)

N.M.R. (DMSO-d₆) δ = 0.75 (m, 6H); 1.25-2.95 (m, 12H);

3.05-3.65 (m, 4H); 3.95-4.75 (m, 10H); 5.05 (m, 1H);

6.10 (m, 1H); 6.30 (d, 1H); 6.80-6.85 (2 x s, 1H);

6.95-7.35 (m, 11H); 7.55-7.65 (2 x s, 1H); 7.95 (d, 1H).

EXAMPLE 36

1-[N-(S)-4-Hydroxy-(S)-5-(isopropyloxycarbonylamino)-6-phenyl-(R)-2-(3-phenylprop-2-enyl)hexanoyl)-(S)-isoleucyl]-4-(imidazol-1-yl)piperidine; tartrate

The title compound was prepared by the procedure described above for Example 35, except that

isopropylchloroformate was used for reaction with the amine intermediate. Purification by chromatography on silica gel eluting with methylene chloride-methanol-concentrated aqueous ammonia (97:2:1 to 95:4:1) gave the product as a colourless powder, which was

recrystallised from ethyl acetate-hexane. The free base was dissolved in ethanol and treated with a solution of 1-tartaric acid. Addition of diethyl ether gave the tartrate salt as a colourless powder, m.p. 156-157°C.

Found : C , 61. 60 ; H, 6 . 87 ; N , 8 . 38 . C₃₈H₅₁N₅O₅ C₄H₆O₆ ¹/₂ H₂O requires C, 61.75; H, 7.16; N, 8.57%.

m/e 658 (MH)⁺

[α]_D ² ₅ +26° (c = 0.1%, MeOH) N.M.R. (DMSO-d₆) δ = 0.75 (m, 6H) ; 1. 0 (m, 6H) ; 1. 25-3 . 65 (m, 14H) ; 3 . 95-4 . 70 (m, 10H) ; 6 . 10 (m, 1H) ; 6. 30 (d, 1H) ; 6. 65 (d, 1H) ; 6. 85-6. 90 (2 x s , 1H) ; 7 . 05-7 . (m, 11H) ; 7 . 65-7 . 75 (2 x s , 1H) ; 7 . 90 (d , 1H) .

EXAMPLE 37

1-[N-(S)-5-(t-Butoxycarbonylamino)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)-4-((S)-valyloxy)hexanoyl)-(S)-valyl]-4-(imidazol-1-yl)piperidine; tartrate

a) N-Benzyloxycarbonyl-L-valine (1.03 g) and

dicyclohexylcarbodiimide (0.51 g) were dissolved in dichloromethane (25 ml) and the mixture was stirred for 3 hours. The precipitated dicyclohexylurea was removed by filtration and the filtrate evaporated to a white foam. This white foam was commoned with the product of Example 16 in N,N-dimethylformamide (20 ml) and 4-dimethylaminopyridine (0.025 g. added. After stirring at room temperature fcr five days the fixture was partitioned between ethyl acetate and water. Drying of the organic extract (MgSO₄) and evaporation of the solvent followed by silica-gel chromatography, eluting with ethyl acetate:methanol (0-10%) gave 1-[N-(3)-4-(N-benzyloxycarbonyl-(S)-valyloxy)-(S)-5-(t-butoxy¬carbonylamino)-6-phenyl-(R)-2-(4-(trifluoromethoxy)-benzyl)hexanoyl-(S)-valyl]-4-(imidazol-1-yl)piperidine as a white foam. Found: C,62.63; H,6.73; N,8.37. C₅₁H₆₅ F₃N₆O₉. H₂O requires C,62.43; H,6.88; N,8.56%.

m/e 963 (MH)⁺

b) The product from step a) above (0.95 g) was dissolved in absolute ethanol (50 ml), and the solution was treated with 10% palladium on charcoal (0.1 g) and hydrogenated at 60p.s.i., (4.1 bar) at room temperature for 4 hours. After removal of the catalyst the filtrate was evaporated to dryness. Purification by silica-gel chromatography eluting with dichloromethane-methanol-concentrated aqueous ammonia (97:3:0.5) gave the product as a white foam. This foam was dissolved in ethyl acetate (6 ml) and a solution of tartaric acid (0.089 g) in 10% methanol ethyl acetate (10 ml) was added. Evaporation of the solvent and trituration with diethyl ether gave the product as a white glass (0.48 g), m.p. 122°C. Found C, 56.74; H, 6.78; N, 8.56.

C₄₃H₅₉F₃N₆O₇:C₄H₆O₆:H₂O requires C, 56.61; H, 6.77; N, 8.43%.

N.M.R. (DMSO-d₆) δ = 0.8-0.95 (m, 12H); 1.2 (s, 11H); 1.35-1.77 (m, 2H); 1.8-2.15 (m, 4H); 2.4-3.2 (m, 8H); 3.51 (m, 1H); 3.8 (m, 1H); 4.1 (m, 1H); 4.3 (m, 1H); 4.4-4.65 (m, 2H); 4.81 (m, 1H); 6.83 (s, 1H); 7.0-7.32 (m, 12H); 7.66 (d, 1H); 7.73-8.0 (m, 1H).

Claims

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof or bioprecursor therefor, wherein-:

R¹ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, aryl,

heterocyclyl or CONR⁹R¹⁰;

R² is C₁-C₆ alkyl, C₃-C₈ cycloalkyl (C₁-C₄) alkyl, aryl (C₁-C₄)alkyl or heterocyclyl(C₁-C₄) alkyl;

R³ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₈

cycloalkyl (C₁-C₄) alkyl, aryl (C₁-C₄) alkyl, aryl (C₂-C₄)-alkenyl, heteroeyelyl(C₁-C₄) alkyl or heterocyclyl (C₂-C₄)-alkenyl;

R⁴ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl,

aryl or heterocyclyl;

carbocyclic ring;

X is a 4-10 membered mono or bicyclic heterocyclic group containing carbon ring atoms and one ring

nitrogen atom through which the group is attached to the adjacent carbonyl group; the group may be saturated or partially unsaturated and, in addition to the -(CR⁷R⁸)_m-Het substituent, it may be substituted by up to 4 further substituents each independently chosen from F, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, OR¹¹ or NR⁹R¹⁰;

R¹¹ is H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl;

n and m are each independently 0, 1 or 2;

2. A compound as claimed in claim 1 having the stereochemistry:

wherein R¹ to R⁸, n, m, X and Het are as previously defined in claim 1.

3. A compound as claimed in claim 1 or claim 2 wherein R¹ is t-butyl, isopropyl, or oxetanyl and n is 0.

4. A compound as claimed in claim 3 wherein R² is benzyl.

5. A compound as claimed in claim 3 or claim 4 wherein R⁴ is isopropyl or sec-butyl.

6. A compound is claimed in any one of claims 3 to 5 wherein R³ is benzyl optionally substituted in the phenyl ring by methyl, fluoro, chloro, iodo, CF₃, or OCF₃, or R³ is 3-phenylpropyl or 3-phenyl-prop-2-enyl.

7. A compound as claimed in any one of claims 3 to 6 wherein each of R⁵, R⁶, R⁷ and R⁸ is H.

8. A compound as claimed in any one of claims 3 to 7 wherein m is 0 or 1.

9. A compound as claimed in claim 1 wherein said compound is:

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)- isoleucyl]-4-(imidazol-1-yl)piperidine

10. A process for preparing a compound of the formula (I) as claimed in claim 1 which comprises removing the protecting groups from a compound of the formula:

wherein X¹ is a selectively removable hydroxyprotecting group and R¹ to R⁸, X and Het are as defined in claim 1, and isolating the compound of formula (I) and optionally forming a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising a compound of the formula (I) or (la) or a pharmaceutically acceptable salt thereof or bioprecursor therefor as claimed in any one of claims 1 to 9, together with a pharmaceutically acceptable diluent or carrier.

12. A compound of the formula (I), or (la) or a pharmaceutically acceptable salt thereof or

bioprecursor therefor, as claimed in any of claims 1 to 9 for use in medicine, in particular for use in the treatment or prophylaxis of human retroviral

infections.

13. The use of a compound of the formula (I) or a (la) as claimed in any one of claims 1 to 9, or a

pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment or phrophylaxis of human retroviral infections.

14. A method of treating human retroviral infections which comprises administering an effective amount of a compound of the formula (I) or (la) as claimed in any one of claims 1 to 9.

PROCESS CLAIMS

15. A process for producing a compound having the formula:

or a pharmaceutically acceptable salt thereof,

wherein-:

R¹ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, aryl,

heterocyclyl or CONR⁹R¹⁰;

R² is C₁-C₆ alkyl, C₃-C₈ cycloalkyl (C₁-C₄) alkyl, aryl (C₁-C₄) alkyl or heterocyclyl (C₁-C₄) alkyl;

R³ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₈

cycloalkyl (C₁-C₄) alkyl, aryl (C₁-C₄) alkyl, aryl(C₂-C₄)- alkenyl, heterocyclyl (C₁-C₄) alkyl or heterocyclyl (C₂-C₄)- alkenyl;

R⁴ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl,

aryl or heterocyclyl;

carbocyclic ring;

nitrogen atom through which the group is attached to the adjacent carbonyl group; the group may be saturated or partially unsaturated and, in addition to the - (CR⁷R⁸)_m-Het substituent, it may be substituted by up to 4 further substituents each independently chosen from F, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, OR¹¹ or NR⁹R¹⁰;

each of R⁹ and R¹⁰ is independently H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl, or R⁹ and R¹⁰ may be joined together to form, with the nitrogen to which they are attached, a 4 to 8 membered nitrogen-containing heterocyclic group,

R¹¹ is H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl;

n and m are each independently 0, 1 or 2;

wherein any alkyl or cycloalkyl group included in the aforementioned definitions may optionally be fully or partially substituted by fluorine;

which comprises removing the protecting group from a compound of the formula:

wherein X¹ is a selectively removable hydroxy-protecting group and R¹ to R⁸, X and Het are as defined in claim 1, and isolating the compound of formula (I) and optionally forming a pharmaceutically acceptable salt thereof.

16. A process as claimed in claim 1 wherein the selectively removable hydroxy protecting group X¹ is t-butyldimethylsilyl and it is removed by reaction with tetra-n-butylammonium floride in an organic solvent.

17. A process as claimed in claim 1 wherein the compound of formula (I) has the stereochemistry-:

wherein R¹ to R⁸, n, m, X and Het are as previously defined in claim 1.

18. A process as claimed in claim 1, claim 2 or claim 3 wherein R¹ is t-butyl, isopropyl, or oxetanyl and n is 0.

19. A process as claimed in claim 4 wherein R² is benzyl.

20. A process as claimed in claim 4 or 5 wherein R⁴ is isopropyl or sec-butyl.

21. A process is claimed in any one of claims 4 to 6 wherein R³ is benzyl optionally substituted in the phenyl ring by methyl, fluoro, chloro, iodo, CF₃, or OCF₃, or R³ is 3-phenylpropyl or 3-phenyl-prop-2-enyl.

22. A process as claimed in any one of claims 4 to 7 wherein each of R⁵, R⁶, R⁷ and R⁸ is H.

23. A process as claimed in any one of claims 4 to 8 wherein m is 0 or 1.

24. A process as claimed in claim 1 wherein said compound of formula I produced is-:

1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4- hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazol-1- yl)azetidine,

1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4- hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazol-1- yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6- phenyl-(R)-2-(3-phenylprop-2-en-1-yl)hexanoyl)-(S)- valyl]-4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6- phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)- valyl]-4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(R)-2-(4- chlorobenzyl)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)- valyl]-3-(imidazol-1-yl)azetidine or

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6- phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)- isoleucyl]-4-(imidazol-1-yl)piperidine.