WO2000047608A1

WO2000047608A1 - Process for the preparation of cyclic tetrapeptide derivatives

Info

Publication number: WO2000047608A1
Application number: PCT/GB1999/004401
Authority: WO
Inventors: Albert Andrzej Jaxa-Chamiec; Michael Kranz; Peter John Murray
Original assignee: Glaxo Group Limited
Priority date: 1999-02-12
Filing date: 1999-12-23
Publication date: 2000-08-17
Also published as: JP2002536457A; GB9903087D0; EP1150998A1; AU1879700A

Abstract

Provided is a process for the preparation of compounds of formula (II), or a protected derivative thereof, wherein each of B1, B2, B3 and B4 independently represent the skeleton of an α-amino acid, D1, D2 and D3 each independently represent carbonyl or methylene, with the proviso that only one of the groups selected from D1, D2, and D3 can be methylene, and T1, T2 and T3, which are optionally present, each independently represent hydrogen or an amino acid substituent or an activating group, an amine protecting group or a linker group, which process comprises cyclising a compound of formula (III) or a protected derivative thereof, wherein P is hydrogen or carboxyl protecting group or a linker group.

Description

Process For The Preparation Of Cyclic Tetrapeptide Derivatives.

The present invention relates to a process for the preparation of cyclic tetrapeptide derivatives.

Cyclic tetrapeptides are a well known class of compounds. There are nineteen naturally occurring cyclic tetrapeptides currently recognised, such as trapoxin A and B, chlamydocin, HC toxin I, II and III, bottromycin, apicidin, apicidin A, dihydrotentoxin, tentoxin and fenestin. However, viable synthetic routes to cyclic tetrapeptides and cyclic tetrapeptide derivatives have proved difficult to develop.

The central synthetic challenge which must be addressed in preparing cyclic tetrapeptides and synthetic derivatives thereof is the construction of the macrocyclic core. This is usually effected by ring closure of the corresponding linear tetrapeptide or tetrapeptide derivative. Such ring closure reactions are characterised by low yields, often due to the formation of the corresponding cyclooctapeptides via an intermolecular, head to tail dimerization process, even under high dilution conditions. Moreover, the success of the cyclisation process is very much dependent on the sequence and configuration of the amino acids present in the linear precursor.

It is known that the incorporation of an Λ/-substituted amino acid, such as proline or pipecolic acid, can allow an otherwise unfavourable cyclisation to proceed. Moreover, the use of a D-amino acid is also known to facilitate cyclisation especially when it occupies the Λ/-terminal position. All of the naturally occurring cyclic tetrapeptides include these types of amino acids in their composition.

The synthesis of cyclic tetrapeptides containing only amino acids of the L- configuration is known to be difficult and, by currently available methods, usually leads predominantly or exclusively to the formation of cyclic octamers via a competitive, head-to-tail dimerisation process. Significantly, only four or five of the naturally occurring cyclic tetrapeptides do not contain at least one amino acid of the D-configuration and all of these examples incorporate two proline residues. The preparation of a small number of cyclic tetrapeptides has been reported, in which all of the constituent α-amino acids have the L-configuration, and which exclude Λ/-substituted amino acids, see for example: K. B. Mathur, S. Rishi, M. M. Dhar, Ind. J. Chem., 1974, 12, 458. However the structural assignment of these compounds is based mainly on mass spectral evidence, and the authenticity of at least some of these claims has been vigorously contested by other workers in the area; see U. Schmidt, J. Langer, J. Peptide Res., 1997, 49, 67.

Although there has been some success in developing synthetic routes to specific cyclotetrapeptide macrocyclic cores, for example see: U. Schmidt, A. Libemecht, H. Griesser, F. Bartowiak, Angew. Chem., Int. Ed. Engl., 1984, 23, 318; application of the same conditions to form structurally related cyclotetrapeptides either work less effectively, see: J. E. Baldwin, R. M. Adlington, C. R. A. Godfrey, V. K. Patel, Tetrahedron, 1993, 49, 7837; or not at all, see: J. Taunton, J. L. Collins, S. L. Schreiber, J. Am. Chem. Soc, 1996, 118, 10412.

Thus, a generally applicable route to cyclic tetrapeptides and derivatives thereof has not yet been developed. Methodology that would deliver such molecules is essential if these structures are to be explored as a potential source of biologically active compounds.

We have now found a process for preparing cyclic tetrapeptide analogues by cyclising partially reduced, linear tetrapeptide derivatives.

Accordingly, in a first aspect, the present invention provides a process for the preparation of a compound of formula (II),

or a protected derivative thereof, wherein each of B^ B₂, B₃ and B₄ independently represent the skeleton of an α-amino acid, D_{1 τ} D₂ and D₃ each independently represent carbonyl or methylene, with the proviso that only one of the groups selected from D^ D₂, and D₃ can be methylene, and T_{1 t} T₂ and T₃, which are optionally present, each independently represent hydrogen or an amino acid substituent or an activating group, an amine protecting group or a linker group, which process comprises cyclising a compound of formula (III)

or a protected derivative thereof, wherein P is hydrogen or a carboxyl protecting group or a linker group.

We have discovered that the cyclisation of a partially reduced linear tetrapeptide proceeds more efficiently than the cyclisation of the corresponding fully oxidised tetrapeptide. The cyclisation of partially reduced linear tetrapeptides proceed in acceptable yield alleviating the need to tailor the reaction conditions to the specific linear tetrapeptide derivative to be cyclized. Accordingly, the present invention provides a general route to cyclic tetrapeptide reduced isosteres. This route may enable combinatorial chemistry techniques, which are unsuitable for the synthesis of fully oxidised tetrapeptides, to be applied to the synthesis of the corresponding partially reduced cyclic tetrapeptides.

Suitably, D₁ and one of D₂ or D₃ represent carbonyl and the other one of D₂ or D₃ represents methylene. Conveniently, D₃ is methylene and D^ and D₂ are carbonyl. Preferably D₂ is methylene and D. and D₃ are carbonyl.

Suitably, B_{1 (} B₂, B₃ and B₄ each independently represent the skeleton of any proteinogenic or non-proteinogenic α-amino acid. Conveniently, B B₂, B₃ and

B₄ each independently represent the skeleton of any proteinogenic α-amino acid. A preferred class of compounds of formula (II) is that wherein at least one of B_{1 t} B₂, B₃ and B₄ represents the skeleton of any non-proteinogenic α-amino acid. Compounds of formula (II) wherein one of B., B₂, B₃ and B₄ represents the skeleton of any non-proteinogenic α-amino acid are particluarly preferred.

Suitably B_{1 ?} B₂₎ B₃ and B₄ may each independently be of either L or D configuration. A convenient class of compounds of formula (II) are those wherein one of B.,, B₂, B₃ and B₄ is in the D configuration. A preferred class of compounds of formula (I) are those wherein B_{1 t} B₂, B₃ and B₄ are all of the L configuration.

The term amino acid substituent as used herein in the definition of T_{1 t} T₂ and T₃ is intended to cover any organic substituent which is bound to the α nitrogen atom of an amino acid used in the synthesis of a compound of formula (II) or (III), for example where the amino acid Λ/-methyl alanine is used in the synthesis of a compound of formula (II) or (III) the amino acid substituent is methyl.

The term α-amino acid as used herein is intended to encompass both proteinogenic and non-proteinogenic α-amino acids. Suitable α-amino acids for use according to the invention include alanine, 2-aminobutyric acid, α- aminoisobutyric acid, (S)-2-amino-8-oxo-decanoic acid, α-aminosuberic acid, arginine, asparagine, aspartic acid, 4-chlorophenylalanine, citrulline, β- cyclohexylalanine, cysteine, cystine, 3,4-dehydroproline, 3,5-diiodotyrosine, 2- fluorophenylalanine, 3-fiuorophenylalanine, 4-fluorophenylalanine, glutamic acid, glutamine, glycine, histidine, homocitrulline, homoserine, frat7s-4-hydroxyproline, β-hydroxyvaline, isoleucine, leucine, lysine, methionine, 4-nitrophenylalanine, norleucine, norvaline, ornithine, penicillamine, phenylalanine, phenylglycine, proline, pipecolic acid, sarcosine, serine, β-(2-thienyl)alanine, threonine, tryptophan, tyrosine, valine.

Suitably, B^ B₂, B₃ and B₄ each independently represent the skeleton of any α- amino acid having a molecular weight of less than 400 amu. Conveniently, B.,, B₂, B₃ and B₄ each independently represent the skeleton of any α-amino acid having a molecular weight of less than 350 amu. Another class of compounds of formula (II) is that wherein, B.,, B₂, B₃ and B₄ each independently represent the skeleton of any α-amino acid having a molecular weight of less than 250 amu. Compounds of formula (II) wherein B^ B₂, B₃ and B₄ each independently represent the skeleton of any α-amino acid having a molecular weight of less than 250 amu and wherein one of B.,, B₂, B₃ and B₄ represents the skeleton of any α-amino acids having a molecular weight of less than 150 amu are most preferred.

Reference to molecular weight means the molecular weight of the α-amino acid skeleton excluding protecting groups, activating groups, linker groups, solid resins and the like which may be bound to the α-amino acid.

Suitably, the T group attached to the nitrogen atom adjacent to the D group that is methylene is an activating group and/or an amine protecting group and/or a linker group and the remaining T groups are hydrogen or amino acid substituents. Conveniently, D₃ is methylene, T₃ is an activating group, amine protecting group or linker group and T, and T₂ are hydrogen or amino acid substituents. Preferably, D₂ is methylene, T, and T₃ are hydrogen or amino acid substituents and T₂ is an activating group, amine protecting group or linker group.

The term α-amino acid skeleton is used herein to define the part of an α-amino acid excluding the amine group or residue thereof and the carboxyl group or residue thereof. For example, the skeleton of the α-amino acid alanine is ethyl, the skeleton of the α-amino acid α-aminoisobutyric acid is 2-propyl, the skeleton of the α-amino acid isoleucine is 2-methylbutyl, the skeleton of the α-amino acid tryptophan is 3-ethylindole and the skeleton of the α-amino acid 2-amino-8-oxo- decanoic acid is 7-oxo-nonanyl. The α-amino acid skeleton may, together with the α nitrogen atom of the amino acid to which it is bound, form a heterocyclic ring, such as in, for example, proline and pipecolic acid.

The term activating group is used herein to describe a group capable of activating the nitrogen atom to which it is attached such that the nitrogen atom will undergo a coupling reaction with a suitable substrate. Preferably, the coupling reaction will be performed under Mitsunobu conditions, see O. Mitsunobu, Synthesis, 1981 , 1 , 1. Convenient activating groups include aromatic sulfonyl groups, alkyl sulfonyl groups and carbamate groups. Preferably, the activating group is selected from 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl and 2,4-dinitrobenzenesulfonyl. Most preferably, the activating group is 2- nitrobenzenesulfonyl.

The term linker group is used herein to mean the organic group or part of a group which binds the reacting substrate or reaction product, for example the compound of formula (II) or formula (III), to the polymeric support of a suitable resin. Suitable resins for connection to the C-terminus of the linear tetrapeptide derivative include 4-hydroxymethyl-3-methoxyphenoxybutyryl MBHA (HMPB- MBHA) resin, and 4-(2^'4^'-dimethoxyphenyl-hydroxymethyl)-phenoxy (Rink Acid) resin and modified Wang resins such as the chlorotrityl resin. Such resins are commercially available and may be used in a conventional manner. Suitable resins for connection to one of the α nitogen atoms of, for example, the compound of formula (II) or formula (III), include 4-(bromomethyl)phenoxyethyl polystyrene and 4-(bromomethyl)phenoxymethyl polystyrene. Such resins are commercially available and may be used in a conventional manner.

Conveniently, any one of T, to T₃ may be a group which is both an amine protecting group and a linker group, for example by using an amine protecting group bound to a resin, such reagents are commercially available and may be used in a conventional manner. Advantageously, any one of T, to T₃ may be a group which is an activating group and an amine protecting group and a linker group. For example see: P. J. Murray, C. Kay, L. Sandow, A. B. Holmes., Tetrahedron Lett., 1997, 38, 6941 ; wherein aromatic sulfonyl groups, previously disclosed herein as suitable activating groups, are described bound to solid supports. Preferably only one of T, to T₃ is an amine protecting group and/or activating group and/or linker group and the remaining T groups are hydrogen or amino acid substituents.

Compounds of formula (III) may be cleaved from the solid resin prior to cyclisation. Alternatively, the compound of formula (II) may be cleaved from the solid resin following cyclisation, for example when one of T, to T₃ is an activating group such as a 2-nitrobenzenesulfonyi bound to a solid support as described above, cleavage may occur at the sulphonamide moiety as described by T. Fukuyama, K.-C. Jow, M. Cheung, Tetrahedron Lett, 1995, 36, 6373. Advantageously, by judicious choice of solid resin and linker group, the compound of formual (II) may be cleaved from the solid resin concomitant with cyclisation. Optionally, the cleavage site may be located at a position on the linker group such that on cleavage the activating group remains attached to the compound of formula (II).

Conveniently, in compounds of formula (III) only one position is bound to a suitable resin. Thus, when P is a linker group, T_{1 f} T₂ and T₃ each independently represent hydrogen or an amino acid substituent or an activating group or an amine protecting group. Alternatively, when one of T,, T₂ and T₃ is a linker group, P is hydrogen or a carboxyl protecting group.

Typical amine and carboxyl protecting groups are well known to those skilled in the art and may be used in a conventional manner. See, for example: "Protective Groups in Organic Synthesis" by T. W. Greene and P. G. M. Wuts (John Wiley & Sons, 1991) or "Protecting Groups" by P. J. Kocienski (Georg Thieme Verlag, 1994). Examples of suitable amine protecting groups include acyl type protecting groups e.g. formyl, trifluoroacetyl, acetyl, urethane type protecting groups e.g. benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), f-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl and alkyl type protecting groups e.g. benzyl, trityl, chlorotrityl. Preferred α-amine protecting groups are those that may be cleaved under mild acidic conditions, such as Boc, chlorotrityl, and benzophenone imine. Examples of carboxyl protecting groups include esters such as fetf-butyl ester.

The cyclisation process may be effected under conventional peptide coupling conditions which are well known to those skilled in the art. For example see: "Peptide Chemistry: A Practical Textbook" M. Bodanszky, (2nd. rev. ed., Berlin and New York, Springer-Verlag, 1993), "Solid Phase Peptide Synthesis" J. M. Stewart, J. D. Young, (2nd ed., Rockford, Pierce Chemical Co., 1984).

The cyclisation process may be carried out in the presence of any suitable organic solvent. A medium comprising one or more aprotic solvents may be employed. Suitable solvents include chlorinated solvents such as dichloromethane or chloroform, amides for example DMF, Λ/-methylpyrrolidinone or dimethylacetamide and mixtures thereof, with dimethylformamide / dichloromethane mixtures being most preferred.

Cyclisation may be effected by any suitable reagent or combination of reagents. Suitable combinations of reagents include a coupling reagent such as benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate

(pyBOP), or benzotriazole-1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), or 2-(1 H-benzotriazole-1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophosphate (HBTU), or 2-(1H-benzotriazole-1-yl)- 1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (TBTU), dicyclohexylcarbodiimide (DCC) or 1 ,1'-carbonyl-diimidazole (CDI), and a base such as di- /sopropylethylamine (DIPEA) or triethylamine. Optionally, the cyclisation process may be performed in the presence of a coupling reagent, a base and N- hydroxybenzotriazole (HOBt). Preferably, the cyclisation process is carried out with pyBOP, HOBt and DIPEA.

The compound of formula (III), may be prepared by sequentially coupling a compound of formula (IV)

or a protected derivative thereof, wherein B₃ and B₄ represent the skeleton of an α-amino acid, P is hydrogen or a carboxyl protecting group or a linker group and T₃ represents an activating group, amine protecting group or linker group, with two suitably protected α-amino acids in a conventional manner.

Methods of coupling α-amino acids to form peptides or peptide mimetics are well known to those skilled in the art. For example see, "Peptide Chemistry: A Practical Textbook" M. Bodanszky, 2nd. rev. ed., Berlin and New York, Springer- Verlag, 1993, "Solid Phase Peptide Synthesis" J. M. Stewart, J. D. Young, 2nd ed., Rockford, Pierce Chemical Co., 1984. Conveniently, the synthesis of compounds of formula (III) and (IV), is effected on solid phase. Suitably, P is a linker group and T,, T₂ and T₃ each independently represent hydrogen or an amino acid substituent or an activating group or an amine protecting group.

The compound of formula (IV) may be prepared by any suitable method known to those skilled in the art. For example, compounds of formula (IV) may be prepared by reacting a compound of formula (V) with a compound of formula (VI) under Mitsunobu conditions,

(V) (VI)

wherein X is a hydroxy or halogen group, P is a carboxyl protecting group or a linker group, P^' is an amine protecting group, B₃ and B₄ represent the skeleton of an α-amino acid and T₃ is an activating group as described hereinbefore, see T. Fukuyama, K.-C. Jow, M. Cheung, Tetrahedron Lett, 1995, 36, 6373. By way of a further example, the compound of formula (IV) may be prepared by reacting a compound of formula (VII) with a compound of formula (VIII),

(^VM) (VIII)

wherein B₃ and B₄ represent the skeleton of an α-amino acid, P is a carboxyl protecting group or a linker group and P^' is an amine protecting group, the condensation reaction is followed by the reduction of the resulting imine, for example with sodium cyanoborohydride, and protection of the resulting secondary amine with a suitable amine protecting group T₃.

The synthesis of compounds of formula (III) illustrated above describes the preparation of a compound of formula (III) wherein D₃ is methylene. However, it will be apparent to those skilled in the art that the sequence of reactions may be readily varied such that the compound of formula (V) or (VII) may be reacted with a sequence of two or three amino acids to provide a compound of formula (III) wherein either of D₂ or D., is methylene.

In a further aspect, the present invention provides a compound of formula (II),

or a protected derivative thereof, wherein each of B^ B₂, B₃ and B₄ independently represent the skeleton of an α-amino acid, D.,, D₂ and D₃ each independently represent carbonyl or methylene, with the proviso that only one of the groups selected from D^ D₂, and D₃ can be methylene, and T_{1 f} T₂ and T₃, which are optionally present, each independently represent hydrogen or an organic substituent or an activating group, an amine protecting group or a linker group, with the proviso that the compound of formula (II) is not selected from 6- [(4-nitrophenyl)methyl]-1 ,4,7, 10-tetraazacyclododecane-2,5,8-trione, 6-[(4- nitrophenyl)methyl]-10-(trifluoroacetyl)-1 ,4,7, 10-tetraazacyclododecane-2,5,8- trione and 5-[(4-nitrophenyl)methyl]-3,6,9-trioxo-1 ,1dimethylethylester-1 ,4,7,10- tetraazacyclododecane-1-carboxylic acid.

Preferably, the compound of formula (II) is cyclic {Aoda-Trp-ψ-[CH₂N(ONS)]-lle- D-Pro}.

BIOLOGICAL DATA

Cyclic {Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro} was assessed for activity against Trypanosoma brucei, strain S427. The test was carried out against the bloodstream trypomastigote form (extracellular) grown in culture (in vitro). The medium used for growth was HMI- 18 supplemented with 10% heat inactivated foetal calf serum at 37°C in an 5% CO₂/ air mixture.

Parasites were incubated in medium containing drug for 72 hours. At the endpoint, the number of parasites in the control untreated culture was compared with the number in drug treated cultures and a % inhibition calculated. A standard pentamidine isethionate was included for comparison.

Cyclic {Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro} was found to have an ED₅₀ of 2.0 (μg ml ¹).

EXPERIMENTAL

Abbreviations

Amu Atomic mass units

Aoda (S)-2-Amino-8-oxo-decanoic acid Boc te/f-Butoxycarbonyl

BOP^® Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate

CBz Benzyloxycarbonyl

CDI 1 ,1'-Carbonyl-diimidazole DCC Dicyclohexylcarbodiimide

Dde 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl

DEAD Diethylazodicarboxylate

DIPEA Di-/^'sσpropylethylamine

DMF Λ/_;Λ/-Dimethylformamide ES Electrospray ionisation

Fmoc 9-Fluorenylmethoxycarbonyl

HBTU 2-(1 H-Benzotriazole-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate

HOBt Λ/-Hydroxybenzotriazole HPLC High Performance Liquid Chromatography LCMS Liquid Chromatography-Mass Spectrometry

Nal Napthylalanine

ONS 2-Nitrophenylsulfonyl pyBOP^® Benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate RT Room temperature

Rt Retention time (min)

TBTU 2-(1 H-Benzotriazole-1-yl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate TFA Trifluoroacetic acid

TS Thermospray filament ionisation

HPLC Analytical Methods

HPLC Eluents: A = 0.1% v/v of TFA in H₂O; Solvent B = 0.05% v/v of TFA in

MeCN / H₂O (95:5).

Method A

Instrument: Hewlett Packard 1050 Column: Supelcosil ABZ+PLUS, 330 mm x 4.6 mm, 3 μm particle size. Gradient: 10% to 95% solvent B in A at 1ml min^-1 over 10min. Detection Wavelength: As specified.

Method B Instrument: Hewlett Packard 1050

Column: Dynamax, C-18 reverse phase, 25 cm x 4.6 mm, 60 A particle size. Gradient: 10% to 95% solvent B in A at 1ml min ¹ over 15 min. Detection Wavelength: As specified.

HPLC Preparative Methods

Method C

Instrument: Gilson Autoprep

Column: Supelcosil ABZ+PLUS, 10cm x 21.2 mm, 5μm particle size. Gradient: 20% to 95% of solvent B in solvent A over 19 min, at 6 ml min^-1, held for 5 min. Detection: at 215 nm.

Method D

As for method B except for the gradient.

Gradient: 20% to 95% of solvent B in solvent A over 20 min, at 6 ml min^'1, held for 11 min.

LCMS Method

LCMS data was obtained on a Hewlett Packard HP1050 instrument using an ABZ+PLUS column (330 x 4.6 mm) with a supelcosil packing (3 μm particle size). The gradient used was 0 to 100% eluent B in 100 to 0% eluant A, over 3.5 min, at a flow rate of 1ml min^'1 [eluent A = 10 mmol NH₄OAc in H₂O containing 0.1% v/v HCO₂H; eluent B = a mixture of MeCN / H₂O (95:5) containing 0.05% v/v HCO₂H]. Mass spectra were obtained on a Platform Series II spectrometer using electrospray ionisation in +ve and - mode.

Mass Spectra

Low resolution mass spectra were obtained using a Hewlett Packard MS 5989B Engine mass spectrometer using thermospray ionisation in positive ion detection mode. Accurate mass measurements were performed on a VG Autospec magnetic sector instrument, using positive electrospray ionisation, in voltage scanning mode and employing PEG as internal calibration reference.

Dde-Tryptophanol

A solution of 2-acetyldimedone (9.8 g, 54.0 mmol) and L-tryptophanol (9.40 g, 49.0 mmol) in ethanol (250 ml) was heated at reflux, under nitrogen, for 4 h and then allowed to cool to RT. The volatiles were removed in vacuo to give an oily residue which was purified on silica gel, by column chromatography, eluting firstly with ethyl acetate / hexane (70:30), and then with ethyl acetate, to give a white gum. Trituration of this material with diethyl ether produced the title compound as a white powder.

HPLC: (Method A, 215 nm), Rt = 4.04 (100%); LCMS: Rt = 4.45, [M+H]⁺ = 316.

ONS-lle-OH

To a stirred solution of 2-nitrophenylsulfonyl chloride (5.69 g, 24.9 mmol) and H- lle-O'Bu.HCI (5.06 g, 23.0 mmol) in dichloromethane (200 ml) under nitrogen, at RT, was added dropwise DIPEA (11.8 ml, 67.9 mmol) and the reaction stirred at RT for 4.5 h. The mixture was concentrated in vacuo and the residue was taken up into ethyl acetate (100 ml) and was washed sequentially with 1M hydrochloric acid (50 ml), saturated sodium bicarbonate solution (50 ml) and brine (50 ml) and dried (MgSO₄). Concentration of the solution in vacuo afforded a green oil which crystallised on standing. Purification of the crude product by flash column chromatography, eluting with dichloromethane, gave a colourless oil which crystallised on standing. The solid was taken up into a mixture of trifluroracetic acid (30 ml) and dichloromethane (30ml) and stirred for 1 h at RT. Evaporation of the volatiles in vacuo gave the title compound, as a pale yellow liquid. HPLC: (Method A, 215 nm), Rt = 4.51 (100%); LCMS: Rt = 4.85, [M+H]⁺ = 317.

ONS-lle-D-Pro-O'Bu

To a stirred solution of ONS-lle-OH (5.55 g, 19.0 mmol) in a mixture of dichloromethane and DMF (20:1 , 350 ml) at RT, under nitrogen, was added TBTU (7.32 g, 22.8 mmol) and HOBt (3.08 g, 22.8 mmol). After 4 min DIPEA (16.5 ml, 95.0 mmol) was added and 4 min later a solution of H-Pro-O'Bu (3.95 g, 19.0 mmol) in a mixture of dichloromethane and DMF (20:1 , 350 ml) was added. After 18 h the mixture was concentred in vacuo to a yellow oil (30 g) and the residue was purified by flash column chromatography on silica gel, eluting with a mixture of ethyl acetate and hexane (75:25), to give the title compound, as a colourless viscous liquid.

HPLC: (Method A, 254 nm), Rt = 5.82 (100%); LCMS: Rt = 5.26, [M+H]⁺ = 470.

Dde-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-O^tBu

To a solution of ONS-lle-D-Pro-O'Bu (469 mg, 1.00 mmol), triphenylphosphine (1.05 g, 4.00 mmol) and Λ/-Dde-tryptophanol (630 mg, 2.00 mmol) in dry THF (40 ml) under nitrogen, at 0°C, was added dropwise DEAD (630μl, 4.00 mmol) over 15 min. The reaction mixture was stirred for 12 h whilst slowly warming to RT. Concentration of the solution in vacuo gave an orange oily residue which was purified by column chromatography on silica gel, eluting with a mixture of ethyl acetate and hexane (50:50), to afford a yellow oil which was purified further by HPLC (Method D) to give the title compound, as a colourless oil. HPLC; (Method A, 215 nm), Rt = 6.65 (100%); LCMS: Rt = 5.54, [M+H]⁺= 806.

H-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-O^tBu

To a solution of Dde-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-O^tBu (461 mg, 0.57 mmol) in methanol (5.0 ml) was added hydrazine hydrate, (5.0 ml of 55 wt % solution of hydrazine in water) at RT and the yellow solution was stirred for 2.5h. The mixture was then concentrated in vacuo to afford an equimolar mixture of the title compound and 3,6,6-trimethyl-1 ,5,6,7-tetrahydro-indazol-4-one as an impure yellow oil. HPLC: (Method A, 215nm), Rt = 4.84 (40%); LCMS: Rt = 4.58, [M+H]⁺ = 642.

Boc-Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-O^tBu

To a stirred solution of a portion of the material obtained above (194 mg, equivalent to 0.22 mmol of H-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-O^tBu) in a mixture of dichloromethane and DMF (20:1 , 10 ml) at RT, under nitrogen, was added TBTU (233 mg, 0.73 mmol) and HOBt (98 mg, 0.73 mmol). After 10 mins Boc- Aoda-OH (100 mg, 0.33 mmol) was added and 5 min afterwards, DIPEA (260 μl, 1.51 mmol) was added dropwise. After a further 3 h the mixture was concentrated in vacuo to yield a yellow oil. Purification by HPLC (Method D) afforded the title compound as a white foam. HPLC: (Method A, 215 nm), Rt = 7.02 (100%); LCMS: Rt = 5.72, [M+H]⁺ = 925.

H-Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-OH.TFA

To a solution of Boc-Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-O^tBu (160 mg, 0.17 mmol) in dichloromethane (5.0 ml) was added thioanisole (500 μl) and trifluoroacetic acid (5.0 ml) and the reaction mixture was stirred for 2 h at RT. Following concentration in vacuo the residue was purified by HPLC (Method C) to afford the title compound as a pale yellow foam. HPLC: (Method A, 230 nm), Rt = 4.45 (100%); LCMS: Rt = 4.56, [M+H]⁺ = 769.

Cyclic {Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro}

To a solution of H-Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro-OH (73 mg, 95 μmol) in a mixture of dichloromethane and DMF (20:1 , 350 ml) under nitrogen, was added TBTU (61 mg, 191 μmol) and HOBt (26 mg, 191 μmol). After 10 min, DIPEA (83 μl, 430 μmol) was added and the stirring was continued for 1 h. Concentration of the solution in vacuo gave a yellow residue which was purified by HPLC (Method D) to afford the title compound as a pale yellow powder. HPLC: (Method A, 215 nm), Rt = 6.07 (100%); LCMS: Rt = 5.28, [M+H]⁺ = 751.

Cyclic [Aoda-Trp-ψ-(CH₂NH)-lle-D-Pro] To a stirred solution of cyclic {Aoda-Trp-ψ-[CH₂N(ONS)]-lle-D-Pro} (40 mg, 53 μmol) and potassium carbonate (11 mg, 120 μmol) in DMF (0.50 ml) was added a solution of thiophenol (8.8 mg, 80 μmol, 1.5 eq) in DMF (82 μM, 100 μl). After 2 h, 16 h and 20 h additional portions of potassium carbonate (22 mg) were added and the reaction was stirred for a further 11 h. The mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate (5.0 ml) and water (1.0 ml). The organic phase was separated and extracted with 1M sodium carbonate solution (2 x 1 ml) and saturated brine (1 ml) and was concentrated in vacuo to a viscous oil. This was purified by HPLC (Method C) to afford the title compound as a white solid.

HPLC: (Method A, 215 nm), Rt = 4.00 (100%); LCMS: Rt = 4.51 , [M+H]⁺ = 566.

ONS-Phe-O'Bu

Triethylamine (1.97 ml, 14.1 mmol) was added to a stirred suspension of Phe- O'Bu HCI (2.00 g, 7.80 mmol) in dry dichloromethane (100 ml) at RT. After 10 min the mixture was cooled to 0°C; and a solution, of 2-nitrophenylsulfonyl chloride (4.67g, 21.1 mmol) in dichloromethane (50 ml) was added dropwise and the reaction mixture then stirred for 1 h at 0°C and for 24 h at RT. The solvent was removed in vacuo and the product purified by column chromatography on silica gel eluting with a mixture of hexanes and ethyl acetate (2:1) to yield a yellow oil (2.8g), which was crystallised from EtOAc; hexanes to give the title compound as a white solid; mp 87-88°C. HPLC: (Method A, 254 nm), Rt = 9.87 (100%); m/z (TS) 424 (M+NH₄)⁺.

Dde-Alaninol

To a solution of 2-acetyldimedone (2.50 g, 13.7 mmol) in EtOH (10 ml) was added (S)-alaninol (1.07 ml, 13.7 mmol) and the reaction mixture stirred for 60 h at RT. The solvent was removed by evaporation in vacuo and the residue taken up into EtOAc. Hexane was added dropwise to the solution until the mixture turned cloudy and a cream coloured was precipitate was produced. The solid was collected by filtration to give the title compound as an off white solid. HPLC: (Method B, 254 nm), Rt = 12.0 (100%); m/z (TS) 240 (M+H)⁺.

Dde-Ala^-[CH₂N(ONS)]-Phe-O⁴Bu To a stirred solution of Ph₃P (1.74 g, 6.62 mmol), Dde-alaninol (785 mg, 3.31 mmol), and ONS-Phe-O'Bu (1.30g, 3.31 mmol) in anhydrous THF (50 ml) was added DEAD (1.04 ml, 6.62 mmol) dropwise, at 0°C. The mixture was stirred at 0°C for 1 h and then RT for 18 h. The solvent was removed in vacuo, and the resulting orange oily residue was taken up into dichloromethane (200 ml) and extracted with water (3 x 100 ml). The organic layer was dried (MgSO₄) and the solvent was evaporated in vacuo to yield an orange oil. The crude product was purified by flash column chromatopgraphy, eluting with a mixture of hexanes and EtOAc ( 2:1), to give the title compound as a colourless viscous liquid. HPLC: (Method B, 254 nm), Rt = 9.87 (100%); m/z (TS) 628 (M+H)⁺.

H-Ala-ψ-tCH_jMONSJl-Phe-O'Bu

To a stirred solution of Dde-Ala-ψ-[CH₂N(ONS)]-Phe-O'Bu (700 mg, 1.06 mmol) in MeOH (3.1 ml) at RT was added hydrazine (1.32 ml of ~55% aqueous solution, 23.2 mol) in a single aliquot. After 3.5 min. the yellow solution was diluted with dichloromethane (30 ml) washed with water (3 x 10 ml), and the organic phase separated and dried (MgSO₄). Evaporation of the solvent in vacuo, and purification of the residue by column chromatography, eluting firstly with EtOAc and then with a mixture of dichloromethane: EtOH and cone. (0.88) ammonia (600:20:1), gave the title compound as a slightly impure (TLC), yellow liquid, which was used in the next step without further purification.

Fmoc-Phe-Ala-ψ-fCH.NfONSH-Phe-O u

To a stirred solution of H-Ala-ψ-[CH₂N(ONS)]-Phe-O^tBu (436 mg, 0.95 mmol) in a mixture of DMF and dichloromethane (5:2, 7.0 ml) was added Fmoc-Phe-OH (413 mg, 1.07 mmol), HOBt (144 mg, 1.06 mmol) and PyBOP (610 mg, 1.17 mmol), followed by DIPEA (371 μl, 2.13 mmol). After 1 h at RT the reaction mixture was diluted with dichloromethane (160 ml), was washed with water (3 x 50 ml) and the organic layer separated and dried (MgSO₄). The solvent was removed by evaporation in vacuo to yield a yellow oily residue which was purified by column chromatography, on silica gel, eluting with a mixture of hexanes and EtOAc (2:1), to give the title compound as a colourless, viscous liquid. HPLC: (Method A, 254 nm), Rt = 9.6 (100%); m/z (ES) 833 (M+H)\

Fmoc-Ala-Phe-Ala-ψ-tCH.NfONSH-Phe-O'Bu

To a solution of Fmoc-Phe-Ala-ψ-[CH₂N(ONS)]-Phe-O¹Bu (650 mg, 781 μmol) in dichloromethane (5.0 ml) was added, diethylamine (2.0 ml, 19 mmol,) and the mixture was stirred at RT for 5h. The volatiles were removed by evaporation in vacuo and the crude product was purified by column chromatography on silica gel, eluting with a mixture of dichloromethane, EtOH and cone. (0.88) ammonia (600:20:1). Evaporation of the solvents in vacuo gave H-Phe-Ala-ψ- [CH₂N(ONS)]-Phe-O'Bu as colourless viscous liquid. To a stirred solution of H- Phe-Ala-ψ-[CH₂N(ONS)]-Phe-O^tBu (415 mg, 68.0 mmol) in a mixture of DMF and dichloromethane (4:1 , 5.0 ml) at RT was added Fmoc-Ala-OH (233 mg, 75.0 mmol), HOBt (101 mg, 75.0 mmol) and PyBOP (389 mg, 75.0 mmol), followed by DIPEA (261 μl, 150 mmol). After 1 h the mixture was diluted with dichloromethane (100 ml), and was washed with water (3 x 50 ml), and was then dried (MgSO₄). The solvent was removed by evaporation in vacuo to yield a yellow oily residue, which was purified by column chromatography, on silica gel, eluting with a mixture of hexanes and EtOAc (2:1), to give the title compound as a white solid. HPLC: (Method A, 254 nm), Rt = 9.2 (100%); m/z (ES) 904 (M+H)⁺.

H-Ala-Phe-Ala-ψ-[CH₂N(ONS)]-Phe-OH.TFA

To a solution of Fmoc-Ala-Phe-Ala-ψ-[CH₂N(ONS)]-Phe-O^tBu (565 mg, 626 μmol) in dichloromethane (5.0 ml) was added, diethylamine (2.2 ml, 21 mmol,) and the mixture was stirred vigorously, at RT for 1.5h. The volatiles were removed by evaporation in vacuo. The crude product was purified by column chromatography on silica gel, eluting firstly with a mixture of hexanes and EtOAc (1 :1), followed by a mixture of dichloromethane, EtOH and cone (0.88) ammonia (600:20:1). Evaporation of the solvents in vacuo gave the amine as a white solid. This residue (483 μmol) was taken up into a mixture of TFA and water (5.0 ml, 95:5) and was stirred at RT for 1.5h. The volatiles were removed by evaporation in vacuo under high vacuum and the residue taken up in a mixture of acetonitrile and water (5 ml, 1 :1). The solution was lyophilized over 24h to give the title compound as a white powder. HPLC: (Method A, 254 nm), Rt = 4.74 (100%).

Cyclo {Ala-Phe-Ala-ψ-[CH₂N(ONS)]-Phe}

To a stirred solution of H-Ala-Phe-Ala-ψ-[CH₂N(ONS)]-Phe-OH TFA (43.0 mg, 58.1 μmol) in a mixture of dichloromethane and DMF (4:1 , 430 ml) at RT was added, HOBt (18.0 mg, 13.8 mmol,) and PyBOP (71.0 mg, 13.8 mmol), followed by DIPEA (48 μl, 27.5 mmol). After 48 h at RT the reaction mixture was diluted with dichloromethane (200 ml) and the solution was washed with water (3 x 150 ml) and then dried (MgSO₄). The solvent was evaporated in vacuo to give a yellow oily residue which was purified by preparative HPLC (Method D) to give the title compound as a white solid, mp 136-137°C.

HPLC: (Method A, 254 nm), Rt = 6.6 (100%); m/z (ES) 608(M+H)⁺. Found: (M+NH₄)⁺, 625.2435. C₃₀H₃₇N₆O₇S requires , 625.2444.

Claims

A process for the preparation of a compound of formula (II),

or a protected derivative thereof, wherein each of B_{1 τ} B₂, B₃ and B₄ independently represent the skeleton of an α-amino acid, D_{1 (} D₂ and D₃ each independently represent carbonyl or methylene, with the proviso that only one of the groups selected from D-, D₂, and D₃ can be methylene, and T_{1 f} T₂ and T₃, which are optionally present, each independently represent hydrogen or an amino acid substituent or an activating group, an amine protecting group or a linker group, which process comprises cyclising a compound of formula (III)

2. A process according to claim 1 wherein D₃ is methylene and D., and D₂ are carbonyl.

3. A process according to claim 1 wherein D₂ is methylene and D., and D₃ are carbonyl.

4. A process according to any of claims 1 to 3 wherein at least one of

B B₂, B₃ and B₄ independently represents the skeleton of any non- proteinogenic α-amino acid.

5. A process according to any of claims 1 to 3 wherein B_1τ B₂, B₃ and

B₄ each independently represent the skeleton of any proteinogenic α-amino acid.

6. A process according to any of claims 1 to 5 wherein T.,, T₂ and T₃ each independently represent hydrogen or an amino acid substituent or an activating group or an amine protecting group.

7. A process according to any of claims 1 to 5 wherein the T group attached to the nitrogen atom adjacent to the D group that is methylene is an activating group and/or an amine protecting group and/or a linker group and the remaining T groups are hydrogen or amino acid substituents.

8. A process according to any preceeding claim wherein B₁₍ B₂, B₃ and B₄ are all of the L configuration.

9. A compound of formula (II) or a protected derivative thereof, wherein each of B^ B₂, B₃ and B₄ independently represent the skeleton of an α-amino acid, D^ D₂ and D₃ each independently represent carbonyl or methylene, with the proviso that only one of the groups selected from D.,, D₂, and D₃ can be methylene, and T T₂ and T₃, which are optionally present, each independently represent hydrogen or an organic substituent or an activating group, an amine protecting group or a linker group, with the proviso that the compound of formula (II) is not selected from 6-[(4-nitrophenyl)methyl]-1 ,4,7,10- tetraazacyclododecane-2,5,8-trione, 6-[(4-nitrophenyl)methyl]-10- (trifluoroacetyl)-1 ,4,7,10-tetraazacyclododecane-2,5,8-trione and 5-[(4- nitrophenyl)methyl]-3,6,9-trioxo-1 , 1 dimethylethylester-1 ,4,7, 10- tetraazacyclododecane-1-carboxylic acid.