Pseudoproline Dipeptides Pseudoprolindipeptide Dipeptides De Pseudoproline

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(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

[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

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

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

2 EP 2 344 516 B1

wherein R1 and R2 are as above, with serine or threonine into the dipeptide of formula

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

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

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(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

30 wherein R1 and R2 are as above is converted with serine or threonine into the dipeptide of formula

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 hydrochloride 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. [0023] Usually the coupling takes place by adding a solution of the activated ester obtained from the activation reaction

4 EP 2 344 516 B1

to an aqueous suspension of serine or threonine and the inorganic base. [0024] Suitable inorganic bases are alkali carbonates such as lithium-, sodium- or potassium-carbonates or hydroxides or mixtures thereof. [0025] Preferred are lithium carbonate and /or lithium hydroxide, more preferred mixtures of lithium carbonate and 5 lithium hydroxide. [0026] The inorganic base is as a rule applied stoechiometrically related to serine or threonine. [0027] The ratio serine or threonine to amino acid derivative of formula II is usually selected in the range of 1.5 to 4.0 to 1, preferably 2.0 to 3.0 to 1. [0028] The pH of the reaction mixture is expediently maintained in a range of 7.5 to 9.5. 10 [0029] After completion of the conversion the reaction mixture is acidified with a mineral acid. Suitable mineral acids are aqueous sulfuric acid or aqueous HCl, preferably aqueous sulfuric acid. [0030] The dipeptide of formula III can be isolated following methods known to the skilled in the art. In a preferred embodiment of the invention the dipeptide of formula III is directly, without its isolation, used in process step b).

15 Step b)

[0031] Step b) requires effecting the ring closure of the dipeptide of formula III with a compound of formula

wherein R3, R4, R9a and R9b are as above, in the presence of an acidic catalyst. 25 [0032] Preferably the ring closure is effected with 2,2-dimethoxypropane. Ideally the compounds of formula IV are used in an amount of 6.0 to 16.0 equivalents, preferably 7.0 to 12.0 equivalents in relation to the di-peptide obtained in step b). [0033] In a preferred embodiment the 2,2-dimethoxypropane is continuously added to the reaction mixture while in parallel the methanol generated is continuously distilled of. 30 [0034] The reaction temperature is usually maintained in the range of 15°C to 35°C, preferably between 20°C and 30°C. [0035] 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 0.30 equivalents, preferably 0.08 to 0.15 equivalents in relation to the dipeptide of formula III obtained in step b). 35 [0036] The organic solvent ideally applied for the conversion in step b) is substantially freed of water. Suitable solvents are toluene or tetrahydrofuran or mixtures thereof. [0037] The work up of the reaction mixture and the isolation of the target product of formula I can takes place applying a procedure comprising

40 a) extracting the reaction mixture with water, while maintaining a pH in the range of 7.0 to 9.0, preferably in the range of 7.5 to 8.5. b) extracting the water phase with an organic, water immiscible solvent, while maintaining a pH in the range of 5.5 to 6.0, preferably in the range of 5.5 to 5.7. c) isolating the target product of formula I from the organic phase and optionally by 45 d) crystallizing the target product of formula I in an organic solvent.

[0038] The adjustment of the pH in step a) of the work up procedure can happen with a common aqueous buffer e.g. with an aqueous sodium bicarbonate solution, while the pH in step b) can be adjusted by using an aqueous solution of a mineral acid e.g. with aqueous sulfuric acid. 50 [0039] The organic, water immiscible solvent is preferably toluene. [0040] Isolation in step c) usually happens by partly evaporation of the organic solvent, where after the target product may further be purified by crystallization in a suitable organic solvent such as in a mixture of toluene, isopropanol and heptane. [0041] The following examples illustrate the invention. 55

5 EP 2 344 516 B1

Examples

Synthesis of (S,S)-3-[3-tert.-butoxy-2-(9H-fluoren-9-yl-methoxycarbonylamino)-propionyl]-2,2-dimethyl-oxazolidine- 4-carboxylic acid 5 [0042]

Example 1:

20 [0043] A solution of 16.1 g N-hydroxysuccinimide and 40.0 g Fmoc-L-Ser(tBu)-OH in 200 mL of THF was added at 20°C within 30 to 60 minutes to a suspension of 26.0 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 80 mL DMF and 80 mL THF. The resulting mixture was stirred at ambient temperature for 4 hours and then added within 30 to 45 minutes to a pre- cooled (-5°C) suspension of 8.75 g lithium hydroxide monohydrate, 6.1 g lithium carbonate and 33.2 g L-Serine in 240 g of water. The resulting mixture was allowed to warm to room temperature within 30 minutes 25 and then stirred at this temperature for another hour. The mixture was then cooled to -5°C and the pH was adjusted from 8.5 to 2.0-2.5 with approx. 150 g of sulfuric acid (20% in water). The biphasic mixture was allowed to warm to room temperature and the lower aqueous layer was then separated. The aqueous layer was extracted with 200 mL of toluene. The combined organic layers were diluted with 150 mL of toluene and then washed with 5 3150 mL of water. From the organic layer water was removed by azeotropic distillation with toluene and THF. The water free (<0.05%) toluene/THF 30 solution (approx. 500 mL) was treated with 1.00 g methanesulfonic acid. To the mixture was added within 6 to 10 hours a solution of 100 g of 2,2- dimethoxypropane in 660 mL toluene. During the entire dosing volatiles were distilled off under reduced pressure (80-30 mbar) and at a temperature of 20 to 28°C, keeping the reaction volume (at approximately 600 mL) constant. After complete addition, the mixture was concentrated to a final volume of approx. 500 mL and then treated with 1.35 g triethylamine. Water (50 mL) was added and the layers were separated. The organic layer was treated with 35 250 g of sodium bicarbonate (5% in water). The biphasic mixture (pH ~7.5) was heated to 35-40°C and stirred at this temperature for 30 to 45 minutes. The layers were separated and the organic layer was extracted with 3 370 g of sodium bicarbonate (5% in water). The combined product containing aqueous layers were treated at 35-40°C with 360 mL of toluene and the pH was adjusted to 5.5 by the drop wise addition of approximately 50 g of sulfuric acid (20% in water). The aqueous layer was separated and the organic layer washed with 23 50 g of water. The resulting organic layer was 40 cooled to ambient temperature and diluted with 100 mL of water. The pH was adjusted to 4 by the addition of a few drops of sulfuric acid (20% in water). The lower aqueous layer was removed and the organic layer washed with 2380 g of water. The organic layer was concentrated to dryness. The residue was treated with 400 mL of isopropanol and the resulting solution was concentrated to dryness. The residue was diluted with 90 mL isopropanol and 90 mL heptane and the mixture was heated to 50°C to achieve a clear solution. 400 mL of heptane were added within 3 to 4 hours. The 45 mixture was then cooled to -10°C within 13-16 hours and the resulting suspension stirred at this temperature for at least 4 hours. The crystals were filtered off, washed with 80 mL of pre-cooled heptane and dried at 40-50°C / <30 mbar to afford 31.6 g (60%) of ( S,S)-3-[3-tert.-butoxy-2-(9H-fluoren-9-yl-methoxycarbonylamino)-propionyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid as colorless crystals with a HPLC assay of 99.0 % (m/m).

50 Example 2:

[0044] A solution of 16.1 g N-hydroxysuccinimide and 40.0 g Fmoc-L-Ser(tBu)-OH in 200 mL of THF was added at 20°C within 30 to 60 minutes to a suspension of 26.0 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 80 mL DMF and 80 mL THF. The resulting mixture was stirred at ambient temperature for 4 hours and then added 55 within 30 to 60 minutes to a pre- cooled (-5°C) suspension of 8.75 g lithium hydroxide monohydrate, 6.1 g lithium carbonate and 33.2 g L-Serine in 270 g of water. The resulting mixture was allowed to warm to room temperature within 30 minutes and then stirred at this temperature for another hour. The mixture was then cooled to -5°C and the pH was adjusted to 2.5 with 137 g of sulfuric acid (20% in water). The biphasic mixture was allowed to warm to room temperature and the

6 EP 2 344 516 B1

lower aqueous layer was then separated. The aqueous layer was extracted with 210 mL of toluene. The combined organic layers were diluted with 100 mL of toluene and then washed with 53130 mL of water. From the organic layer water was removed by azeotropic distillation with toluene and THF. The resulting toluene/THF solution (approx. 550 mL) was then treated with 1.00 g methanesulfonic acid. To the mixture was then added within 8 to 10 hours a solution 5 of 134 g of 2,2-dimethoxypropane in 1040 mL of toluene. During the entire dosing volatiles were distilled off under reduced pressure (80-30 mbar) and at a temperature of 25 to 32°C, keeping the reaction volume (at approximately 600 mL) constant. After complete addition, the mixture was concentrated to a final volume of approx. 500 mL. The reaction mixture was treated with 250 g of sodium bicarbonate (5% in water). The biphasic mixture was heated to 35-40°C and stirred at this temperature for 15 minutes. The layers were separated and the organic layer was extracted with 100 g of 10 sodium bicarbonate (5% in water). The combined product containing aqueous layers were washed with toluene (150 mL). The aqueous layer was treated at 35-40°C with 300 mL of toluene and the pH was adjusted to 5.7 by the drop wise addition of approximately 45 g of sulfuric acid (20% in water). The aqueous layer was then separated and the organic layer washed with 33 80 g of water. The resulting product containing organic layer was treated with 80 mL of water. The pH was adjusted to 4 by the addition of a few drops of sulfuric acid (20% in water). The lower aqueous layer was removed 15 and the organic layer washed with 2 380 g of water. The organic layer was concentrated to a residual volume of approximately 170 mL. The mixture was heated to 55-60°C and isopropanol (15 mL) was added. The resulting clear solution was treated at 55-60°C within 2-4 hours with 300 mL of heptane. The resulting suspension was cooled to 0°C within 10 hours and stirred at this temperature for 3 hours. The crystals were filtered off, washed with 100 mL of pre- cooled heptane and dried at 50°C / <30 mbar to afford 33.6 g (63%) of (S,S)-3-[3-tert.-butoxy-2-(9H-fluoren-9-yl-methoxycarbonylami- 20 no)-propionyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid as colorless crystals with a HPLC assay of 99.4 % (m/m).

Claims

25 1. Process for the manufacture of a compound of formula

35 wherein R1 is a side chain of an alpha amino acid, R 2 is an amino protecting group and R 3 and R4 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

40 a) converting an amino acid derivative of the formula

wherein R1 and R2 are as above, with serine or threonine into the dipeptide of formula

thereby using a water soluble carbodiimide as activating agent

7 EP 2 344 516 B1

and b) effecting the ring closure of the dipeptide of formula III with a compound of formula

3 4 3 wherein R and R are independently selected from hydrogen or C1-4-alkyl, with the proviso that not both R 10 4 9a 9b and R are hydrogen and R and R independently is C1-4-alkyl, in the presence of an acidic catalyst; and further characterized in that 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; b) extracting the water phase with an organic, water immiscible solvent, while maintaining a pH in the range 15 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.

2. Process of claim 1, characterized in that R1 is a side chain selected from valine, leucine, isoleucine, methionine, 20 phenylalanine, asparagine, glutamine, glutamic acid, histidine, lysine, arginine, aspartic acid, alanine, serine, threonine, tyrosine, tryptophan, cysteine, glycine and aminoisobutyric acid.

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

25 4. Process of claims 1 to 3, characterized in that the water soluble carbodiimide is EDC or a salt thereof.

5. Process of claim 4, characterized in that EDC or a salt thereof is applied together with HOSu.

6. Process of claims 1 to 5, characterized in that the ratio serine or threonine to amino acid derivative of formula II 30 is selected in the range of 1.5 to 4.0 to 1.

7. Process of claims 1 to 6, characterized in that the conversion in step a) is performed in the presence of an inorganic base.

35 8. Process of claim 7, characterized in that the inorganic base is selected from an alkali carbonate or an alkali hydroxide and from mixtures thereof.

9. Process of claims 1 to 8, characterized in that the conversion in step a) is performed at a pH in the range of 7.5 to 9.5.

40 10. Process of claims 1 to 9 characterized in that the conversion in step a) is performed at a temperature in the range of -10°C to 25°C.

11. Process of claims 1 to 10, characterized in that the reaction mixture, after the conversion in step a) is acidified with a mineral acid. 45 12. Process of claims 1 to 11, characterized in that the dipeptide of formula III is directly, without being isolated, used in process step b).

13. Process of claims 1 to 12, characterized in that the compound of formula IV used for the ring closure in step b) is 50 2, 2-dimethoxypropane.

14. Process of claim 13, characterized in that 2,2-dimethoxypropane is continuously added to the reaction mixture while in parallel the methanol generated is continuously distilled of.

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

16. Process of claims 1 to 15, characterized in that the ring closure in step b) is effected in the presence of toluene

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or tetrahydrofuran or of mixtures thereof.

17. Process of claims 1 to 16, characterized in that the ring closure in step b) is performed at a temperature in the range of 15°C to 35°C. 5 18. Process of claim 1, characterized in that the organic, water immiscible solvent is toluene.

19. Process of claim 18, characterized in that the target product is crystallized from a mixture of toluene, isopropanol and heptane. 10

Patentansprüche

1. Verfahren zur Herstellung einer Verbindung der Formel 15

wobei R1 eine Seitenkette einer alpha-Aminosäure ist, R2 eine Aminoschutzgruppe ist und R3 und R4 unabhängig 3 4 aus Wasserstoff oder C 1-4-Alkyl ausgewählt sind, mit der Maßgabe, dass R und R nicht beide Wasserstoffe sind, R5 Wasserstoff oder Methyl ist, umfassend 30 a) Umwandeln eines Aminosäurederivats der Formel

wobei R1 und R2 wie oben sind, mit Serin oder Threonin, in ein Dipeptid der Formel

unter Verwendung eines wasserlöslichen Carbodiimids als Aktivierungsmittel und 55 b) Durchführen des Ringschlusses des Dipeptids der Formel III mit einer Verbindung der Formel

9 EP 2 344 516 B1

3 4 3 wobei R und R unabhängig aus Wasserstoff oder C 1-4-Alkyl ausgewählt sind, mit der Maßgabe, dass R und 10 4 9a 9b R nicht beide Wasserstoffe sind und R und R unabhängig C1-4-Alkyl sind, in Gegenwart eines sauren Katalysators;

und weiterhin dadurch gekennzeichnet, dass die Zielverbindung der Formel I durch ein Aufarbeitungsverfahren erhalten wird, welches umfasst: 15 a) Extrahieren des Reaktionsgemisches mit Wasser unter Aufrechterhaltung eines pH im Bereich von 7, 0 bis 9,0; b) Extrahieren der Wasserphase mit einem organischen, nicht mit Wasser mischbaren Lösungsmittel unter Aufrechterhaltung eines pH im Bereich von 5,5 bis 6,0; c) Gewinnen der Zielverbindung der Formel I aus der organischen Phase, und gegebenenfalls durch 20 d) Kristallisieren der Zielverbindung der Formel I in einem organischen Lösungsmittel.

2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass R1 eine Seitenkette ausgewählt aus Valin, Leucin, Isoleucin, Methionin, Phenylalanin, Asparagin, Glutamin, Glutaminsäure, Histidin, Lysin, Arginin, Asparaginsäure, Alanin, Serin, Threonin, Tyrosin, Tryptophan, Cystein, Glycin und Aminoisobuttersäure ist. 25 3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass R2 aus Fmoc, Z, Moz, Troc, Teoc und Voc ausgewählt ist.

4. Verfahren nach den Ansprüchen 1 bis 3,dadurch gekennzeichnet, dass das wasserlösliche Carbodiimid EDC 30 oder ein Salz davon ist.

5. Verfahren nach Anspruch 4,dadurch gekennzeichnet, dass EDC oder ein Salz davon zusammen mit HOSu verwendet wird.

35 6. Verfahren nach den Ansprüchen 1 bis 5, dadurch gekennzeichnet, dass das Verhältnis Serin oder Threonin zu Aminosäurederivat der Formel II im Bereich von 1,5 bis 4,0 zu 1 gewählt ist.

7. Verfahren nach den Ansprüchen 1 bis 6, dadurch gekennzeichnet, dass die Umwandlung in Schritt a) in Gegenwart einer anorganischen Base durchgeführt wird. 40 8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, dass die anorganische Base aus einem Alkalicarbonat oder einem Alkalihydroxid und Gemischen davon, ausgewählt ist.

9. Verfahren nach den Ansprüchen 1 bis 8, dadurch gekennzeichnet, dass die Umwandlung in Schritt a) bei einem 45 pH im Bereich von 7,5 bis 9,5 durchgeführt wird.

10. Verfahren nach den Ansprüchen 1 bis 9, dadurch gekennzeichnet, dass die Umwandlung in Schritt a) bei einer Temperatur im Bereich von -10°C bis 25°C durchgeführt wird.

50 11. Verfahren nach den Ansprüchen 1 bis 10, dadurch gekennzeichnet, dass das Reaktionsgemisch nach der Um- wandlung in Schritt a) mit einer Mineralsäure angesäuert wird.

12. Verfahren nach den Ansprüchen 1 bis 11, dadurch gekennzeichnet, dass das Dipeptid der Formel III ohne isoliert zu werden direkt im Verfahrensschritt b) verwendet wird. 55 13. Verfahren nach den Ansprüchen 1 bis 12, dadurch gekennzeichnet, dass die Verbindung der Formel IV, welche für den Ringschluss in Schritt b) verwendet wird, 2,2- Dimethoxypropan ist.

10 EP 2 344 516 B1

14. Verfahren nach Anspruch 13, dadurch gekennzeichnet, dass 2,2-Dimethoxypropan dem Reaktionsgemisch kontinuierlich zugegeben wird, wobei parallel dazu das gebildete Methanol kontinuierlich abdestilliert wird.

15. Verfahren nach den Ansprüchen 1 bis 14, dadurch gekennzeichnet, dass dersaure Katalysator für den Ringschluss 5 in Schritt b) aus Methansulfonsäure, (+) Campher- 10-sulfonsäure, p-Toluolsulfonsäure, Pyridinium-p-toluolsulfonat ausgewählt ist.

16. Verfahren nach den Ansprüchen 1 bis 15, dadurchgekennzeichnet, dass der Ringschlussin Schrittb) in Gegenwart von Toluol oder Tetrahydrofuran oder Gemischen davon, durchgeführt wird. 10 17. Verfahren nach den Ansprüchen 1 bis 16, dadurch gekennzeichnet, dass der Ringschluss in Schritt b) bei einer Temperatur im Bereich von 15°C bis 35°C durchgeführt wird.

18. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass das organische, nicht mit Wasser mischbare Lö- 15 sungsmittel Toluol ist.

19. Verfahren nach Anspruch 18, dadurch gekennzeichnet, dass das Zielprodukt aus einem Gemisch von Toluol, Isopropanol und Heptan kristallisiert wird.

20 Revendications

1. Procédé pour la production d’un composé de formule 25

dans laquelle R1 représente une chaîne latérale d’un alpha-aminoacide, R2 représente un groupe protecteur de la 35 3 4 fonction amino et R et R sont choisis indépendamment entre un atome d’hydrogène et un groupe alkyle en C 1 à 3 4 5 C4, sous réserve que R et R ne représentent pas tous deux un atome d’hydrogène, R représente un atome d’hydrogène ou un groupe méthyle, comprenant

a) la conversion d’un dérivé d’aminoacide de formule 40

dans laquelle R1 et R2 répondent aux définitions précitées, avec la sérine ou la thréonine en le dipeptide de formule

en utilisant à cette fin un carbodiimide hydrosoluble comme activateur et

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b) la conduite de la cyclisation du dipeptide de formule III avec un composé de formule

3 4 dans laquelle R et R sont choisis indépendamment entre un atome d’hydrogène et un groupe alkyle en C 1 à 3 4 9a 9b C4, sous réserve que R et R ne représentent pas tous deux un atome d’hydrogène, et R et R représentent 10 indépendamment un groupe alkyle en C1 à C4, en présence d’un catalyseur acide ; caractérisé en outre en ce que le composé cible de formule I est obtenu par un mode opératoire de traitement comprenant

a) l’extraction du mélange réactionnel avec de l’eau, tout en maintenant un pH dans l’intervalle de 7,0 à 9,0 ; 15 b) l’extraction de la phase aqueuse avec un solvant organique non miscible à l’eau, tout en maintenant un pH dans l’intervalle de 5,5 à 6,0 ; c) l’obtention du produit cible de formule I à partir de la phase organique et, facultativement d) la cristallisation du produit cible de formule I dans un solvant organique.

20 2. Procédé suivant la revendication 1, caractérisé en ce que R1 représente une chaîne latérale choisie entre la valine, la leucine, l’isoleucine, la méthionine, la phénylalanine, l’asparagine, la glutamine, l’acide glutamique, l’histidine, la lysine, l’arginine, l’acide aspartique, l’alanine, la sérine, la thréonine, la tyrosine, le tryptophane, la cystéine, la glycin e et l’acide amino-isobutyrique.

25 3. Procédé suivant la revendication 1 ou 2,caractérisé en ce que R2 est choisi entre des groupes Fmoc, Z, Moz, Troc, Teoc et Voc.

4. Procédé suivant les revendications 1 à 3, caractérisé en ce que le carbodiimide hydrosoluble est le EDC ou un de ses sels. 30 5. Procédé suivant la revendication 4, caractérisé en ce que le EDC ou un de ses sels est appliqué conjointement avec du HOSu.

6. Procédé suivant les revendications 1 à 5, caractérisé en ce que le rapport de la sérine ou de la thréonine au dérivé 35 d’aminoacide de formule II est choisi dans l’intervalle de 1,5 à 4,0 à 1.

7. Procédé suivant les revendications 1 à 6,caractérisé en ce que la conversion dans l’étape a) est effectuée en présence d’une base inorganique.

40 8. Procédé suivant la revendication 7, caractérisé en ce que la base inorganique est choisie entre un carbonate de métal alcalin, un hydroxyde de métal alcalin et leurs mélanges.

9. Procédé suivant les revendications 1 à 8, caractérisé en ce que la conversion dans l’étape a) est effectuée à un pH dans l’intervalle de 7,5 à 9,5. 45 10. Procédé suivant les revendications 1 à 9, caractérisé en ce que la conversion dans l’étape a) est effectuée à une température dans l’intervalle de -10°C à 25°C.

11. Procédé suivant les revendications 1 à 10, caractérisé en ce que le mélange réactionnel, après la conversion dans 50 l’étape (a), est acidifié avec un acide minéral.

12. Procédé suivant les revendications 1 à 11, caractérisé en ce que le dipeptide de formule III est utilisé directement, sans être isolé, dans l’étape b) du procédé.

55 13. Procédé suivant les revendications 1 à 12, caractérisé en ce que le composé de formule IV utilisé pour la cyclisation dans l’étape b) est le 2,2-diméthoxypropane.

14. Procédé suivant la revendication 13, caractérisé en ce que le 2,2- diméthoxypropane est ajouté de manière continue

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au mélange réactionnel tout en éliminant continuellement en parallèle, par distillation, le méthanol engendré.

15. Procédé suivant les revendications 1 à 14, caractérisé en ce que le catalyseur acide pour la cyclisation dans l’étape b) est choisi entre l’acide méthanesulfonique, l’acide (+)campho-10-sulfonique, l’acide p-toluènesulfonique et le p- 5 toluènesulfonate de pyridinium.

16. Procédé suivant les revendications 1 à 15, caractérisé en ce que la cyclisation dans l’étape b) est effectuée en présence de toluène, de tétrahydrofurane ou de leurs mélanges.

10 17. Procédé suivant les revendications 1 à 16, caractérisé en ce que la cyclisation dans l’étape b) est effectuée à une température dans l’intervalle de 15°C à 35°C.

18. Procédé suivant la revendication 1, caractérisé en ce que le solvant organique non miscible à l’eau est le toluène.

15 19. Procédé suivant la revendication 18, caractérisé en ce que le produit cible est cristallisé à partir d’un mélange de toluène, d’isopropanol et d’heptane.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• WO 2008000641 A [0003]

Non-patent literature cited in the description

• JACS, 1996, vol. 118, 9218-9227 [0002] • GREEN T. Protective Groups in Organic Synthesis. • GREEN T. Protective Groups in Organic Synthesis. John Wiley and Sons, Inc, 1991, 10-142 [0013] John Wiley and Sons, Inc, 1991, 309-385 [0012]