(19) TZZ ¥_6B_T (11) EP 2 344 516 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 1/02 (2006.01) C07K 5/06 (2006.01) 20.03.2013 Bulletin 2013/12 C07K 5/062 (2006.01) C07D 263/06 (2006.01) (21) Application number: 09783513.6 (86) International application number: PCT/EP2009/062568 (22) Date of filing: 29.09.2009 (87) International publication number: WO 2010/040660 (15.04.2010 Gazette 2010/15) (54) Pseudoproline dipeptides Pseudoprolindipeptide Dipeptides de pseudoproline (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A-00/46239 WO-A-2008/000641 HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR • TORSTEN WÖHR ET AL: "Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection (30) Priority: 07.10.2008 EP 08165968 Technique in Peptide Synthesis" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, (43) Date of publication of application: AMERICAN CHEMICAL SOCIETY, 20.07.2011 Bulletin 2011/29 WASHINGTON,DC. US, vol. 118, no.39, 2 October 1996 (1996-10-02), pages 9218-9227, (73) Proprietor: F. Hoffmann-La Roche AG XP002447320 ISSN: 0002-7863 cited in the 4070 Basel (CH) application • WOHR T ET AL: "Pseudo-Prolines in Peptide (72) Inventor: HILDBRAND, Stefan Synthesis: Direct Insertion of Serine and CH-4460 Gelterkinden (CH) Threonine Derived Oxazolidines in Dipeptides" TETRAHEDRON LETTERS, ELSEVIER, (74) Representative: Rauber, Beat AMSTERDAM, vol. 36, no. 22, 29 May 1995 F.Hoffmann-La Roche AG (1995-05-29), pages 3847-3848, XP004027998 Patent Department ISSN: 0040-4039 675 / 3. OG Grenzacherstrasse 4070 Basel 2 (CH) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 344 516 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 344 516 B1 Description [0001] The invention relates to a novel process for the manufacture of a compound of the formula 5 10 [0002] The pseudo proline dipeptides 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, 15 118, 9218-9227]. The presence ofΨ Pro within a peptide sequence results in the disruption ofβ-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". [0003] A synthetic approach to pseudoproline dipeptides is published in PCT Publication WO 2008/000641. Access 20 to the compound of formula I is accomplished via an ammonium salt intermediate of formula 25 30 wherein R1, R2, R5,R6, R7 and R8 are defined in the PCT Publication mentioned above. [0004] One major disadvantage of the approach known in the art is the need to purify the dipeptide by isolation of its ammonium salt intermediate, which prior to the ring closure has to be liberated to the dipeptide. Accordingly this synthesis turned out not to be suitable for the application on a technical scale. [0005] Object of the present invention is to provide a short and technically feasible synthesis of the pseudo proline 35 dipeptides of formula I which allows for obtaining the product with a high yield. [0006] The object has been achieved with the process as outlined below. The process for the manufacture of a compound of formula 40 45 wherein R1 is a side chain of an alpha amino acid, R2 is an amino protecting group and R3 and R are independently 3 4 5 selected from hydrogen or C1-4 alkyl with the proviso that not both R and R are hydrogen R is hydrogen or methyl comprising 50 a) converting an amino acid derivative of the formula 55 2 EP 2 344 516 B1 wherein R1 and R2 are as above, with serine or threonine into the dipeptide of formula 5 10 thereby using a water soluble carbodiimide as activating agent and b) effecting the ring closure of the dipeptide of formula III with a compound of formula 15 20 3 4 wherein R and R are independently selected from hydrogen or C1-4-alkyl, with the 3 4 9a 9b proviso that not both R and R are hydrogen and R and R independently is C1-4-alkyl, in the presence of an acidic catalyst, wherein the target compound of formula I is obtained by a work up procedure comprising a) extracting the reaction mixture with water, while maintaining a pH in the range of 7.0 to 9.0; 25 b) extracting the water phase with an organic, water immiscible solvent, while maintaining a pH in the range of 5.5 to 6.0; c) obtaining the target product of formula I from the organic phase and optionally by d) crystallizing the target product of formula I in an organic solvent. 30 [0007] 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. [0008] The term "C 1-4- 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. 35 [0009] The term "side chain of an amino acid" used for the substituent R 1 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, aminoisobutyri c acid and proline. [0010] It is understood that in side chains of amino acids which carry a hydroxy group the hydroxy group is optionally 40 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. [0011] 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. In a more preferred embodiment R1 stands for a side chain of serine or threonine. 45 [0012] 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 as defined under R2 should withstand under acidic conditions. Preferably Fmoc, Z, Moz, Troc, Teoc or Voc more preferably Fmoc is used. [0013] The term "hydroxy protecting group" refers to any substituents conventionally used to hinder the reactivity of 50 the hydroxy group. Suitable hydroxy protecting groups are described in Green T., "Protective Groups in Organic Syn- thesis", 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. [0014] The meaning of the abbreviations used in the description and the claims is as outlined in the table below: 55 Fmoc 9-Fluorenylmethoxycarbonyl Z Benzyloxycarbonyl 3 EP 2 344 516 B1 (continued) Fmoc 9-Fluorenylmethoxycarbonyl tBu t-butyl 5 Moz p-Methoxybenzyloxycarbonyl Troc 2,2,2-Trichloroethoxycarbonyl Teoc 2-(Trimethylsilyl)ethoxycarbonyl 10 Voc Vinyloxycarbonyl TBDMS t-Butyldimethylsilyl ether TBDPS t-Butyldiphenylsilyl ether HOBt 1-Hydroxybenzotriazole 15 HOSu N-Hydroxysuccinimide EAC 1-Ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (iodide) EDC (3-Dimethylamino-propyl)-ethyl-carbodiimide (hydrochloride) 20 Step a) [0015] In the first step a) the amino acid derivative of formula 25 30 wherein R1 and R2 are as above is converted with serine or threonine into the dipeptide of formula 35 40 thereby using a water soluble carbodiimide as activating agent. [0016] 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. 45 [0017] Suitable water soluble carbodiimide activating agents are EDC or EAC or salts thereof, preferably the hydro- chloride salt of EDC. [0018] As a rule the water soluble carbodiimide activating agent is applied together with a further activating agent selected from HOSu or HOBt. [0019] Preferred activating agent is EDC.HCl/HOSu. 50 [0020] The EDC is usually applied in an amount of 1.0 to 1.5 equivalents and the HOSu is usually applied in an amount of 1.0 to 1.5 equivalents related to one equivalent of the amino acid derivative of formula II. [0021] 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 tetrahydrofuran and / or N, N-dimethylformamide at a temperature of -10°C to 25°C. 55 [0022] The coupling with serine or threonine, preferably with L-serine or L-threonine, can then be performed at a temperature of -10°C to 25°C in the presence of an inorganic base.