WO2008000641A1

WO2008000641A1 - Pseudo proline dipeptides

Info

Publication number: WO2008000641A1
Application number: PCT/EP2007/055988
Authority: WO
Inventors: Thomas Ammann; Stephan Goetzoe; Bernd Thern; Sandra Welz; Klaus-Juergen Wolter
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2006-06-28
Filing date: 2007-06-18
Publication date: 2008-01-03
Also published as: TWI383995B; BRPI0713065B1; AU2007263805A1; BRPI0713065A2; CN101472939B; EP2038295B1; DE602007012436D1; TW200808831A; JP2009541412A; ATE497966T1; AU2007263805B2; EP2038295A1; MX2008015723A; CA2654914C; IL195657A; CA2654914A1; CN101472939A; KR101431282B1; KR20090021361A; JP5292289B2

Abstract

Disclosed is a three step process for the manufacture of pseudo proline dipeptides of the formula (I) wherein R1 is a side chain of an alpha amino acid , R2 is an amino protecting group and R3 and R4 are independently selected from hydrogen or C1-4-alkyl, R5 is hydrogen or methyl starting from an amino acid derivative of the formula (II) wherein R1 and R2 are as above. Pseudo proline dipeptides can be used as reversible protecting groups for Ser, Thr and Cys and thus are versatile tools in peptide chemistry.

Description

PSEUDO PROLINE DIPEPTIDES

The invention relates to a novel process for the manufacture of a compound of the formula

The pseudo proline dipetides of formula I can be used as reversible protecting groups for Ser, Thr, and Cys and prove to be versatile tools for overcoming some intrinsic problems in the field of peptide chemistry [JACS 1996, 118, 9218-9227]. The presence of ΨPro within a peptide sequence results in the disruption of j8-sheet structures considered as a source of intermolecular aggregation. The resulting increased solvation and coupling kinetics in peptide assembly such as Fmoc solid phase peptide synthesis facilitates chain elongation especially for peptides containing "difficult sequences".

Object of the present invention is to provide a short and technically feasible synthesis of the pseudo proline dipetides of formula I which allows for obtaining the product with a high yield and without any chromatographic purification step.

The object has been achieved with the process as outlined below. The process for the manufacture of a compound of formula R⁴

wherein R¹ is a side chain of an alpha amino acid , R² is an amino protecting group and R and R are independently selected from hydrogen or Ci 4-alkyl, R is hydrogen or methyl, comprises

a) converting an amino acid derivative of the formula

wherein R and R are as above, with serine or threonine and crystallizing the resulting dipeptide as ammonium salt of formula

wherein R , R and R are as above and

R⁶, R⁷ and R⁸ are independently selected from hydrogen, Ci 4-alkyl or C3 7- cycloalkyl, with the proviso that not all of R , R and R are hydrogen, in a subsequent step

b) releasing the free acid from the ammonium salt of formula III in the presence of an acid and removing the amine by extraction and

c) effecting the ring closure with a compound selected from

wherein R³ and R⁴ are independently selected from hydrogen or Ci ₄-alkyl, with the proviso that not both R and R are hydrogen, R ^a and R independently is Ci 4-alkyl, R has the meaning of Ci ₄-alkyl, Ci ₄-alkanoyl or aryl and R¹¹ is hydrogen or Ci ₃-alkyl, in the presence of an acidic catalyst.

It is further understood that the serine or threonine can be used either in its L- or in their D -configuration, as racemate or in various mixtures of their isomers. Preferably the L-configuration is used. The term "Ci 4- alkyl" refers to a branched or straight- chain monovalent saturated aliphatic hydrocarbon radical of one to four carbon atoms. This term is further exemplified by radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl and t-butyl.

The term "C_{3 7}-cycloalkyl" refers to a cycloalkyl group containing from 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

The term "aryl" relates to the phenyl or naphthyl group, preferably the phenyl group, which can optionally be mono- or multiply-substituted by halogen, hydroxy, CN, CF3, NO2, NH2, N(H,alkyl), N( alkyl) 2, carboxy, aminocarbonyl, alkyl, alkoxy, aryl and/or aryloxy. Preferred aryl group is phenyl.

The term "alkanoyl" relates to a Ci 4-alkyl carbonyl group, such as to acetyl, n-propanoyl, isopropanoyl, n-butanoyl, s-butanoyl and t-butanoyl, preferably acetyl.

The term "side chain of an amino acid" used for the R particularly refers to side chains of the alpha amino acids selected from valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamine, glutamic acid, histidine, lysine, arginine, aspartic acid, alanine, serine, threonine, tyrosine, tryptophan, cysteine, glycine, aminoisobutyric acid and proline.

It is understood that in side chains of amino acids which carry a hydroxy group the hydroxy group is optionally protected by a hydroxy protecting group as defined below. In side chains that carry additional amino groups the amino group is optionally protected by an amino protecting group as defined below.

R¹ preferably stands for a side chain of valine, leucine, isoleucine, phenylalanine, asparagine, glutamine, glutamic acid, lysine, aspartic acid, alanine, serine, threonine, tyrosine and tryptophan. More preferred are serine and threonine.

The term "amino protecting group" refers to any substituents conventionally used to hinder the reactivity of the amino group. Suitable amino protecting groups are described in Green T., "Protective Groups in Organic Synthesis", Chapter 7, John Wiley and Sons, Inc., 1991, 309-385. Suitable amino protecting groups are Fmoc, Cbz, Moz, Boc, Troc, Teoc or Voc. Preferred amino protecting group is Fmoc.

The term "hydroxy protecting group" refers to any substituents conventionally used to hinder the reactivity of the hydroxy group. Suitable hydroxy protecting groups are described in Green T., "Protective Groups in Organic Synthesis", Chapter 1 , John Wiley and Sons, Inc., 1991, 10-142. Suitable hydroxy protecting groups are t-butyl, benzyl, TBDMS or TBDPS. Preferred hydroxy protecting group is t-butyl. - A -

The meaning of the abbreviations used in the description and the claims is as outlined in the table below:

Step a)

In the first step a) an amino acid derivative of the formula

wherein R and R are as above is reacted with serine or threonine and the resulting dipeptide is crystallized as ammonium salt of formula

wherein R¹, R², R⁵, R⁶, R⁷ and R⁸ are as above.

The amino acid derivatives of formula II are as a rule commercially available compounds. Suitable amino acid derivatives of formula II according to the preferences given for R¹ and R² are Fmoc-L-Ser(tBu)-OH, or Fmoc-L-Thr(tBu)-OH.

Prior to the coupling with serine or threonine amino acid derivative of formula II is expediently activated with an activating reagent.

Suitable activating reagents can be selected from DIC/HOSu,

DIC/Pentafluorphenol, DIC/HOBt, DCC/HOSu, DCC/Pentafluorophenol, DCC/HOBt, EDC(xHCl)/HOSu or HBTU/HOBt. Preferred coupling agent is DIC/HOSu. The DIC is usually used in an amount of 1.0 to 1.4 equivalents and the HOSu is usually used in an amount of 1.0 to 1.8 equivalents related to one equivalent of the amino acid derivative of formula I.

As a rule the activation reaction is performed in the presence of a suitable organic solvent, such as ethylacetate, N, N-dimethylformamide, acetone or tetrahydrofuran, preferably ethylacetate at a temperature of -5°C to 25°C.

The coupling with serine or threonine, preferably with L-serine or L-threonine, can then be performed at a temperature of 10⁰C to 30⁰C in the presence of an organic solvent, such as in ethylacetate, acetone or tetrahydrofuran or mixtures thereof with water. Preferred solvent is a mixture of acetone and water.

The ratio serine or threonine to amino acid derivative of formula II is usually selected in the range of 1.5 to 3.0 to 1, preferably 2.0 to 1. The pH of the reaction mixture is expediently set at a value of 7.5 to 9.0.

The formation of the ammonium salt of formula III happens by adding to the dipeptide previously formed an amine of formula

^R\

N V

R^7/ V wherein R⁶, R⁷ and R⁸ are independently selected from hydrogen, Ci ₄-alkyl or

C3 7-cycloalkyl, with the proviso that not all of R , R and R are hydrogen.

Suitable amines of formula V are those wherein R⁶, R⁷ and R⁸ are independently selected from hydrogen, ethyl or cyclohexyl, with the proviso that not all R , R and R are hydrogen. Cyclohexylamine, dicyclohexylamine and triethylamine are the preferred amines; dicyclohexylamine is the most preferred amine of formula V used. The crystallization is commonly effected in suitable organic solvents such as in lower alcohols like methanol, ethanol, n-propanol or i-propanol or in ethylacetate or tetrahydrofuran. Preferred solvent is ethanol.

The ammonium salts of formula III have previously not been described and thus are a further embodiment of the present invention.

Preferred ammonium salts are the dicyclohexylammonium salts of formula III wherein R¹ and R² are as described above, R⁵ is hydrogen or methyl, R⁶ is hydrogen and R and R are cyclohexyl. More preferred are compounds of formula III wherein:

a) R¹ stands for the L-serine side chain with OtBu protection, R² is Fmoc, R⁵ is H,

R is hydrogen and R and R are cyclohexyl.

b) R¹ stands for the L-serine side chain with OtBu protection, R² is Fmoc, R⁵ is methyl, R is hydrogen and R and R are cyclohexyl.

c) R¹ stands for the L-threonine side chain with OtBu protection, R² is Fmoc, R⁵ i is H, R is hydrogen and R and R are cyclohexyl.

d) R¹ stands for the L-threonine side chain with OtBu protection, R² is Fmoc, R⁵ i is methyl, R is hydrogen and R and R are cyclohexyl.

Step b)

In the subsequent step b) the free acid of the di-peptide is released in the presence of an acid and the protonated amine of formula V is removed by extraction. Particularly the free acid of the ammonium salt of formula III is released in the presence of a mineral acid, taken up in an organic solvent while the amine is removed by extraction with water and/ or an aqueous solution of a mineral salt.

Suitable mineral acids are aqueous sulfuric acid or aqueous HCl, preferably aqueous sulfuric acid. Suitable organic solvent for taking up the free acid can be selected from ethylacetate, t-butyl methyl ether or methylenchloride. t-Butyl methyl ether has been found to be the preferred solvent.

The organic phase containing the free acid is as a rule washed several times with 5 water and/ or an aqueous solution of a mineral salt, like sodium chloride in order to completely remove the amine.

Step c)

In step c) the ring closure of the free acid of the di-peptide obtained in step b) is effected with a compound selected from

wherein R and R are independently selected from hydrogen or Ci ₄-alkyl, with the proviso that not both R³ and R⁴ are hydrogen, R^9a and R^9b independently is Ci 4-alkyl, R has the meaning of Ci ₄-alkyl, Ci ₄-alkanoyl or aryl and R is hydrogen or Ci ₃-alkyl, in the presence of an acidic catalyst.

15 Preferably the ring closure is effected with compounds of the formula IVa and IVc, more preferably the compounds are selected from 2, 2-dimethoxypropan, 2- methoxypropen or 2-acetoxypropen, whereby 2, 2-dimethoxypropan is the most preferred compound.

Ideally the compounds of formula IV are used in an amount of 6.0 to 16.0

20 equivalents, preferably 7.0 to 10.0 equivalents in relation to the di-peptide obtained in step b).

Suitable acidic catalysts are selected from methane sulfonic acid, (+) camphor- 10- sulfonic acid, p-toluenesulfonic acid or pyridinium p-toluenesulfonate, while methane sulfonic acid is preferred. The acidic catalyst is usually applied in an amount of 0.05 to 25 0.30 equivalents, preferably 0.10 to 0.20 equivalents in relation to the di-peptide obtained in step b).

The ring closure is effected in the presence of an organic solvent, such as in tetrahydrofuran, methylenechloride or toluene, preferably in tetrahydrofuran at reflux temperature.

30 Isolation and work up of the target product can be performed by using methods which are known to the skilled in the art. The following examples illustrate the invention without limiting it.

Examples

Example 1

A 1000 mL double jacketed glass reactor equipped with a mechanical stirrer, a Pt-

100 thermometer, reflux condenser, a dropping funnel and a nitrogen inlet was charged with 25 g (64.9 mmol) of Fmoc-L-Ser(tBu)-OH (1), 9.66 g (83.1 mmol) of N- hydroxysuccinimide and 180 mL of ethyl acetate. The resulting suspension was cooled to 0⁰C. A solution of 10.49 g (83.1 mmol) of diisopropyl carbodiimide in 20 mL of ethyl acetate was added within 15 minutes. The resulting mixture was stirred at 0⁰C for 2 h and then for another hour at room temperature and sampled. The solvent was completely removed under reduced pressure (ca. 220 mbar) at a jacket temperature of maximal 50⁰C. The residue was treated with 250 mL of acetone at an internal temperature of 35°C to 40⁰C, cooled to 20⁰C and treated with 13.5 mL of water. The pH was set with 1.0 mL of 1 M HCl to pH 2 - 3 and the resulting mixture was stirred for 12 h at 20⁰C and sampled. The suspension was then cooled to -5°C to 0⁰C and stirred for 1 h at this temperature. The precipitate was filtered off and the reactor and filter was rinsed with 50 mL of cold acetone (0⁰C). The clear and colorless filtrate was added at 20⁰C within 60 minutes to a solution of 13.57 g (127.8 mmol) of L-serine and of 13.63 g (257 mmol) of sodium carbonate in 122.5 ml of water. The resulting mixture was stirred for 1 h at 20⁰C and sampled. The pH was set with 28 g of HCl (37%) to pH 2 - 3 and the organic solvent was removed under reduced pressure (< 250 mbar) at a jacket temperature of maximal 50⁰C. The resulting suspension was treated at 35°C to 40⁰C with 125 mL of ethyl acetate and the resulting clear biphasic solution was cooled to 20⁰C. The phases were separated and the organic phase was twice extracted with totally 250 mL of ethyl acetate. The combined organic layers were three times washed with totally 225 mL of aqueous NaCl (10% w/w). The resulting organic solution was concentrated and the solvent almost completely removed under reduced pressure at a jacket temperature of maximal 50⁰C. The residue was dissolved in 250 mL of ethanol where after a part of the solvent (75 mL) was removed again under reduced pressure (ca. 170 mbar) at a jacket temperature of maximal 50⁰C. The resulting solution was treated with 462.5 mL of ethanol and cooled to 20⁰C. About 20% (ca. 29.5 mL) of a solution of 11.83 g (63.9 mmol) of dicyclohexylamine in 118 mL of ethanol was added. The mixture was seeded whereupon the product started to precipitate. The suspension was stirred for 1 h at RT and subsequently, the rest of the dicyclohexyl amine solution was slowly added within at least 2 h. The dropping funnel was rinsed with 25 mL of ethanol. The internal temperature was lowered to 0⁰C within 4 h where after the suspension was stirred over night at this temperature. The precipitate was filtered with suction, the filter cake was washed with 117.5 mL of cold ethanol (0⁰C) and dried under vacuum (50⁰C, 20 mbar) to afford 35.7 g (yield 82% starting from (S) -3- tert-Butoxy-2-(9H-fluoren-9-ylmethoxycarbonyl-amino) -propionic acid, 96.8%(w/w) purity based on HPLC) of (S,S)-2- [3-tert-Butoxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)-propionyl-amino] -3-hydroxy-propionic acid dicyclohexyl - ammonium salt (3) as a colorless solid.

The HPLC analysis was performed using an external standard of pure (S,S)-2- [3- tert-Butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionyl-amino] -3-hydroxy- propionic acid dicyclohexyl-ammonium salt (3). Conditions for HPLC: Column XBridge C18 (Waters), 4.6 x 150 mm, 3.5μm;UV detection 206 nm; solutions for gradient: water (A), 2OmM KH₂PO₄-buffer, pH 2.5 (B), acetonitrile (C); flow 1.0 mL/min; 20⁰C.

Gradient:

Retention Times:

(S,S)-2- [3-tert-Butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionyl- amino] -3-hydroxy-propionic acid dicyclohexyl-ammonium salt (3) 8.4 min

Fmoc-L-Ser(tBu)-OH ( 1) 11.6 min

This HPLC-method results in a value for the assay of the free acid of (3). From this value, the assay of the corresponding dicyclohexylammonium salt is calculated, assuming a stoichiometric ratio of 1 : 1 of free acid and dicyclohexyl ammonium.

A GC analysis using an internal standard of dodecane is used to measure the content of dicyclohexyl amine. Conditions for GC: Column fused silica, 100% polydimethylsiloxane, 1 μm, L= 15 m, ID=O.25 mm; carrier gas hydrogen, pressure: 53 kPa, Hn. velocity: 73 cm/s, split-ratio: 1:100.

Temperature program:

Retention Times:

Dodecane 4.10 min

Dicyclohexylamine 4.90 min

Example 2

A 500 mL double jacketed glass reactor equipped with a mechanical stirrer, a Pt-100 thermometer, reflux condenser, a dropping funnel with cotton filter, and a nitrogen inlet was charged with 25.0 g (37.0 mmol) of (S,S)-2-[3-tert-Butoxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)-propionylamino] -3-hydroxy-propionic acid dicyclohexylammonium salt (3), 100 mL of tert-butyl methyl ether and a solution of 4.70 g of sulfuric acid (96%) in 44.3 mL of water. The mixture was stirred for 90 minutes at room temperature. The aqueous phase was separated and the organic phase was twice washed with a total of 76 ml of aqueous sodium chloride (0.5%-w/w) and again with 38 mL of water. The organic solvent was completely removed under reduced pressure (500 - 100 mbar) and at a jacket temperature of 50⁰C. The foamy residue was dissolved in 100 mL of tetrahydrofuran and the solvent was again completely removed reduced pressure (500 - 100 mbar) and at a jacket temperature of 50⁰C. The residue was dissolved in 450 ml of tetrahydrofuran and the resulting clear solution was treated with 35.4 g (333 mmol) of 2,2-dimethoxy propane and 0.65 g (6.7 mmol) of methanesulfonic acid. The mixture was heated under reflux at a jacket temperature of 85°C while leading back the distillate over 73 g of molecular sieve (0.4 nm). After 16 h, the slightly yellowish solution was cooled to 20⁰C and sampled, and the mixture was treated with 0.828 g (8.14 mmol) of triethylamine and stirred for 10 minutes. The solvent was completely removed under reduced pressure (350 - 100 mbar) and at a jacket temperature of 50⁰C. The residue was treated with 100 mL of tert-butγl methyl ether and again completely concentrated under reduced pressure (350 - 100 mbar) and at a jacket temperature of 50⁰C. The residue was dissolved in 175 mL of tert-butγl methyl ether and cooled to 20⁰C to 25°C. The solution was treated with 87.5 mL of water and stirred for 10 minutes. The phases were separated and the organic phase was completely concentrated under reduced pressure (350 - 100 mbar) and at a jacket temperature of 50⁰C. The foamy residue was dissolved in 100 mL of tert-butγl methyl ether and completely concentrated under reduced pressure (350 - 100 mbar) and at a jacket temperature of 50⁰C. This step was twice repeated with a total of 200 mL of tert-butγl methyl ether. The residue was dissolved in 45.2 mL of tert-butγl methyl ether at 20⁰C to 25°C and treated with 22.6 mL of Isopropanol. At this temperature, the solution was treated with 175 mL of pentane, seeded, then kept stirring for at least 15 minutes, and again slowly treated with 200 mL of pentane within 1 h. The resulting solution stirred for 4 to 16 h and then cooled to 0⁰C within 1 - 2 h and again stirred for another 2 h at this temperature. The precipitate was filtered with suction, the filter cake was washed in two portions with a total of 60 ml of cold pentane (0⁰C) and dried under vacuum (50⁰C, 20 mbar) to afford 14.3 g (yield 75% starting from (S,S)-2-[3- tert-Butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-3-hydroxy- propionic acid dicyclohexyl-ammonium salt, 98.7%(w/w) purity based on HPLC) of (S,S)-3-[3-tert-Butoxy-2-(9H-fluoren-9-yl-methoxycarbonylamino)-propionyl]-2,2- dimethyl-oxazolidine-4-carboxylic acid (4) as a colorless solid.

The HPLC analysis was performed using an external standard of pure (S,S)-3-[3- tert-Butoxy-2-(9H-fluoren-9-yl-methoxycarbonylamino)-propionyl]-2,2-dimethyl- oxazolidine-4-carboxylic acid (4). Conditions for HPLC: Column XB ridge C18 (Waters), 4.6 x 150 mm, 3.5μm; UV detection 206 nm; solutions for gradient: water (A), 20 mM KH₂PO₄-buffer, pH 2.5 (B), acetonitrile (C); flow 1.0 mL/min; 20⁰C.

Gradient:

Retention Times:

(S,S) -3- [3-tert-Butoxy-2-(9H-fluoren-9-yl-niethoxycarbonylaniino)-propionyl] ■ 2,2-dimethyl-oxazolidine-4-carboxylic acid (4) 7.3 min (S,S) -2- [3-tert-Butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino) -propionyl- amino]-3-hydroxy-propionic acid dicyclohexyl-ammonium salt (3) 3.0 min

Fmoc-L-Ser(tBu)-OH (1) 5.6 min

Claims

1. Process for the manufacture of a compound of formula

wherein R is a side chain of an alpha amino acid , R is an amino protecting group and R³ and R⁴ are independently selected from hydrogen or Ci 4-alkyl, R⁵ is hydrogen or methyl comprising

a) converting an amino acid derivative of the formula

wherein R¹ and R² are as above, with serine or threonine and crystallizing the resulting dipeptide as ammonium salt of formula

wherein R , R and R are as above and

c) effecting the ring closure with a compound selected from

wherein R³ and R⁴ are independently selected from hydrogen or Ci ₄-alkyl, with the proviso that not both R and R are hydrogen, R ^a and R independently is Ci 4-alkyl, R¹⁰ has the meaning of Ci 4-alkyl, Ci 4-alkanoyl or aryl and R¹¹ is hydrogen or Ci 3-alkyl, in the presence of an acidic catalyst.

2. Process of claim 1, characterized in that R¹ is a side chain selected from valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamine, glutamic acid, histidine, lysine, arginine, aspartic acid, alanine, serine, threonine, tyrosine, tryptophan, cysteine, glycine, aminoisobutyric acid and proline.

3. Process of claim 1 or 2, characterized in that R² is selected from Fmoc, Cbz, Moz, Boc, Troc, Teoc and Voc.

4. Process of claims 1 to 3, characterized in that the amino acid derivative of formula II is activated prior to its coupling with serine or threonine with an activating reagent.

5. Process of claim 4, characterized in that the activating reagent is selected from DIC/HOSu, DIC/Pentafluorphenol, DIC/HOBt, DCC/HOSu, DCC/Pentafluorphenol,

DCC/HOBt, EDC (xHCl)/HOSu or HBTU/HOBt.

6. Process of claim 4 and 5, characterized in that the activating reagent is DIC/HOSu.

7. Process of claims 1 to 6, characterized in that the ratio serine or threonine to amino acid derivative of formula I is selected in the range of 1.5 to 3.0 to 1.

8. Process of claims 1 to 7, characterized in that the ammonium salt of formula III is formed by adding to the dipeptide an amine of formula

^R\

N V

R^7/ V

wherein R , R and R are independently selected from hydrogen, Ci 4 alkyl or C3 7-cycloalkyl, with the proviso that not all of R⁶, R⁷ and R⁸ are hydrogen.

9. Process of claim 8, characterized in that R , R and R are independently selected from hydrogen, ethyl or cyclohexyl, with the proviso that not all of R⁶, R⁷ and R⁸ are hydrogen.

10. Process of claims 8 to 9, characterized in that dicyclohexylamine is added.

11. Process of claims 8 to 10, characterized in that the crystallization takes place in an organic solvent selected from methanol, ethanol, n-propanol, i-propanol, ethylacetate or tetrahydrofuran.

12. Process of claims 1 to 11, characterized in that the free acid of the ammonium salt of formula III is released in the presence of a mineral acid, taken up in an organic solvent while the amine is removed by extraction with water and/ or an aqueous solution of a mineral salt.

13. Process of claims 1 to 12, characterized in that the ring closure is effected with 2, 2-dimethoxypropan, 2-methoxypropen or 2-acetoxypropen.

14. Process of claim 13, characterized in that the ring closure is effected with

2, 2-dimethoxypropan.

15. Process of claims 1 to 14, characterized the acidic catalyst for the ring closure is selected from methane sulfonic acid, (+) camphor- 10-sulfonic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate.

16. Process of claims 1 and 13 to 15, characterized in that the ring closure is effected in the presence of an organic solvent.

17. A compound of formula

wherein R is a side chain of an alpha amino acid and R is an amino protecting group and

R is hydrogen or methyl, R , R and R are independently selected from hydrogen,

Ci 4 alkyl or C3 7-cycloalkyl, with the proviso that not all of R ->6 , r R> 7 and R are hydrogen.

18. A compound of claim 17,

wherein R¹ and R² are as above, R⁶ is hydrogen and R⁷ and R⁸ are cyclohexyl.