A PROCESS FOR THE PREPARATION OF H-TYR-D-ALA-PHE(F)-PHE-NH2

Abstract: The present invention discloses a new and improved process for the preparation of the tetrapeptide H-Tyr-D-Ala-Phe(F)-Phe-NH2 that is a peptide of formula (I), or a pharmaceutically acceptable salt thereof, as well as new intermediates in the preparation thereof. The novel process is a fragment synthesis and suitable for large-scale production.

Description: A PROCESS FOR THE PREPARATION OF H-TYR-D-ALA-PHE(F)-PHE-NH 2 FIELD OF THE INVENTION The present invention is directed to a new process for the preparation of a tetrapeptide, more specifically the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe- NH2, or a pharmaceutically acceptable salt thereof. In further aspects, the present invention also relates to new intermediates used in the process. BACKGROUND AND PRIOR ART WO 97/07129 discloses a process for producing inter alia the peptide H-Tyr-D-Ala- Phe(pF)-Phe-NH 2 . The peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 is also disclosed in WO 97/07130. Said peptide exhibits peripheral analgesic activity and selectivity for the μ- subtype of opioid receptors, and is particularly suitable in pain therapy. Furthermore, it is prepared using solid phase synthesis according to procedures well established in the art. The drawback with solid phase synthesis is that it is difficult to use in large-scale production, in addition to being expensive. WO99/47548 discloses a process for the preparation of the tetrapeptide H-Tyr-D-Ala- Phe(pF)-Phe- NHb using stepwise synthesis. The process of the present invention provides the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe- NH 2 in a simpler manufacturing process with, e.g. easier purification of the final product. Thus, the object of the present invention is to provide a novel process suitable for use in large-scale synthesis. A further object of the present invention is to provide a process containing as few reaction steps as possible. OUTLINE OF THE INVENTION The present invention provides a new process for large-scale preparation of the peptide H- Tyr-D-Ala-Phe(pF)-Phe-NH2, which is the peptide of formula (I) or a pharmaceutically acceptable salt thereof. The process according to the present invention for preparing the compound of formula (I) is a fragment synthesis (2+2). In a fragment synthesis a plurality of intermediate compounds, are prepared in parallel and then coupled together to give the key intermediate(s) or the final compound. This strategy should be compared to a traditional stepwise synthesis wherein a number of synthetic steps are performed sequential. The different approaches in a stepwise vs. fragment synthesis are schematically shown in Figure 1 below. B -→- C -→- D -→- E -→- F -→- G -→- product Stepwise synthesis G — *- product Fragment synthesis Figure 1. Stepwise vs. fragment synthesis The process of the present invention can be described as comprising the steps shown in Figure 2 below; 2 step 1 step 2 + Figure 2. Fragment synthesis according to the present invention Step 1 A coupling step wherein an activated tyrosine derivative (II), wherein A is an amino protecting group, and R is an activating agent residue group, and 2 R is H or a benzyl-like group; previously prepared by a pre-activation step or generated in situ, is reacted with the amino group of D-alanine, wherein the carboxyl group is protected as an ester, i.e. a compound of tthhee ffoorrmmuullaa DD--AAllaa--RR wwhheerreeiinn RR iiss tthhee eesstteerr ggrrooiup, e.g. OMe, in the presence of a solvent, providing a protected dipeptide derivative (IV) wherein A is an amino protecting group, R is an ester residue group, and 2 R is H or a benzyl-like group; Step 2 A deprotection step wherein a protected dipeptide derivative (IV) prepared in the previous step, is deprotected by treatment with aqueous base or acid to give the dipeptide derivative (V), wherein A is an amino protecting group, and 2 R is H or a benzyl-like group. However, if the activated tyrosine derivative is an activated ester or urethane protected N- carboxyanhydride (UNCA) of the structure (II') wherein A is an amino protecting group, and 2 R is H or a benzyl-like group then the carboxyl group of D-alanine needs no protection, i.e. can be a compound of the formula D-Ala-OH, and the coupling reaction thereby provides a protected dipeptide derivative V, which can be used directly in the step 3 without any further deprotection. The preparation and use of UNCA-derivatives is discussed by Fehrentz et al. (1995). "The use of N-urethane protected N-carboxyanhydrides (UNCAs) in amino acid and peptide synthesis." J. Pept. Sci., 1(2), 124-131 ; and by Fuller et al, (1996). "Urethane-protected a- aminoacid N-carboxanhydrides and peptide synthesis." Biopolymers (Peptide Science), 40(2), 183-205 which are incorporated herein by reference. Alternatively, the activated tyrosine derivative (II) can be reacted with the amino group of non-protected D-Ala, i.e. H-D-Ala-OH, providing dipeptide derivative (V) directly. Step 1' A coupling step wherein an activated /7-fluorophenylalanine derivative (VI), wherein A is an amino protecting group, and R is an activating agent residue group; previously prepared by a pre-activation step or generated in situ, is reacted with the amino group of phenylalanine, wherein the carboxyl group is protected as an ester or amide, i.e. a compound of the formula Phe-R (VII), wherein R is -NH2 or an ester residue group, e.g. OMe, in the presence of a solvent, providing a protected dipeptide derivative (VIII) wherein A is an amino protecting group, and R is -NH2 or an ester residue group; Step 2' A deprotection step wherein a protected dipeptide derivative (VIII) prepared in the previous step, is deprotected by either catalytic hydrogenation, base or acid treatment, depending on the amino protecting group used, to give the dipeptide derivative (IX), wherein R is -NH2 or an ester residue group; Step 3 A coupling step wherein an activated dipeptide derivative (V), wherein A is an amino protecting group, R is an activating agent residue group, and 2 R is H or a benzyl-like group previously prepared by a pre-activation step or generated in situ from compound (V), is reacted with the amino group of compound (IX) in the presence of a solvent, providing a protected tetrapeptide derivative (X) wherein A is an amino protecting group, R is -NH2 or an ester residue group, and 2 R is H or a benzyl-like group An additional transformation step is performed if the protected tetrapeptide derivative (X). prepared in the previous step, is an ester. Thus the ester compound (X) wherein R is an ester residue group, e.g. OMe, is reacted with an amine in an organic alcohol, preferably ammonia in methanol, providing the protected dipeptide derivative (X'), wherein A is an amino protecting group, R 1 is -NH 2 , and 2 R is H or a benzyl-like group Optionally, the additional step described above may be prepared on the protected dipeptide derivative (VIII), if R is an ester, whereby the ester compound (VIII) is reacted with an amine in an organic alcohol, preferably ammonia in methanol, providing the protected dipeptide derivative (VHP), wherein A is an amino protecting group, and R 1 is -NH 7 Step 4 A deprotection step wherein the protected tetrapeptide derivative (X) is deprotected either by catalytic hydrogenation, or treatment with acid or base, depending on the amino protecting group used, providing the final tetrapeptide (I), which optionally may be converted to a pharmaceutically acceptable salt thereof A person skilled in the art will appreciate suitable amino, carboxyl and tyrosine side-chain ex protecting groups, which may be used in the present invention The N -amino protecting group may be selected from any protecting group suitable in peptide synthesis, such as tert- butoxycarbonyl (Boc), 9-fluorenylmetoxycarbonyl (Fmoc) or benzyloxycarbonyl, often abbreviated Z-, just to mention three possible amino protecting groups However, benzyloxycarbonyl is particularly preferred to be used in the present invention since it is easily removed by catalytic hydrogenation, and contrary to the protecting group Boc, it does not require neutralization of the liberated amine C i-C^ alkyl esters and alkylaryl ester, such as benzyl, are preferred carboxyl protecting groups Methyl esters are particularly preferred carboxyl protecting groups Benzyl-like protecting groups are suitable tyrosine side-chain protecting groups to be used in the present invention Preferably no tyrosine side-chain protecting group is used Reference is made to J Meienhofer in The Peptides Vol 1 Eds E Gross & J Meienhofer, Academic Press Inc London 1979, pp 264-309 The peptides, Vol 1-9, E Gross & J Meienhofer Eds Academic Press Inc , London 1979-198~, Houben-Weyl Methoden der organischen Chemie, E Muller, ed , Vol 15 Part I-II Thieme, Stuttgart 1974, and M Bodanszky Principles of peptide Synthesis Springer Verlag, Berlin 1984 The pre-activation step preceding steps 1 , 1 ' and 3, or the in situ generation of the activated amino acid derivatives (II), (VI) and (V), is achieved by reacting an amino acid, wherein the amino function has been protected by a suitable protecting group, such as tert- butoxycarbonyl (Boc), 9-fluorenylmetoxycarbonyl (Fmoc) or benzyloxycarbonyl (Z), which are either commercially available or available by techniques known in the art, with an activating agent in the presence of a suitable amine and an organic solvent, to give the activated amino acid derivative. A schematic representation of a pre-activation step is shown below; (VI) aminoacid derivative activated aminoacid derivative wherein A is an amino protecting group, and R is an activating agent residue group; For the coupling step, in Steps 1, Land 3 described above, a variety of powerful solvents may be used, as long as the amino component is essentially soluble and available for immediate reaction with the activated peptide derivative. Examples of suitable solvents for the coupling step are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP), EtOAc, and mixtures thereof. As used herein, the term " benzyl-like group " denotes any substituted or un-substituted benzyl group that is hydrogenolyzed under similar reaction conditions as the benzyloxycarbonyl group. The term "pF' denotes a/?αra-fluoro substituent. The term "C 1-C alkyl ' denotes a cyclic or linear, straight or branched, substituted or unsubstituted alkyl group having from 1 to 6 carbon atoms Examples of said alkyl include but are not limited to methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert - butyl, cyclohexyl. and cyclopentyl The term "substituted" denotes a group that is substituted by one or more C|-C 6 alkyl, C C 6 alkoxy, halogen, amino, thiol, nitro, hydroxy, Cι-C 6 acyl or cyano groups Possible as well as preferred reagents and reaction conditions in each step are the following The pre-activation or in situ generation step Suitable activating agents may be selected from those that generates any of the commonly used activated ammo acid derivatives including, but not limited to, carbodπmides, activated esters, azide, or anhydrides Isobutylchloroformiate (iBuOCOCl) and 2-( lH- Benzotπazole-l-yl)- l , l,3,3-tetramethyluronιum tetrafluoroborate (TBTU) are the preferred activating agents together with UNCA-deπvatives The amount of activating agent is between 0 9- 1 2 molar equivalents, preferably 0 95- 1 05 equivalents From a practical point of view the amount of activating agent shall be as close to 1 0 as possible When isobutylchloroformiate (iBuOCOCl) is the activating agent, the activated peptide derivative will have the following structure, exemplified on D-alanine, The suitable amine may be selected from any tertiary amine However, NMM (N-methylmorphohne), di-isopropylethylamine and tπethylamine are preferred The amount of amine is between 0 9-2 0 molar equivalents, compared to the acid, and preferable between 0.95 to 1.5 molar equivalents. From a practical point of view the amount of suitable amine shall be at least equal to the molar amount of activating agent used. The organic solvent may be any organic solvent known to a person skilled in the art to be suitable in peptide chemistry. However, ethyl acetate, acetonitrile, acetone, tetrahydrofurane, DMF as well as mixtures thereof are preferred solvents in the pre- activation step. The coupling step; Steps 1, 1' and 3 The solvent used for the coupling step may be selected from a variety of solvents, as long as the amino component is essentially soluble and available for immediate reaction with the activated amino acid residue. Examples of suitable solvents for the coupling steps are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP), EtOAc, and mixtures thereof, of which acetone, EtOAc, NMP and DMF are preferred. Any temperature where the activated amino acid derivative is not degraded or the reaction rate is too slow may be used. The preferred range when isobutyl chloroformate is used as the activating agent is from 0°C to -20°C, and particularly preferred is from -5°C to -15°C. The preferred range when TBTU is used as the activating agent is around room temperature. The rate of addition is in both cases adjusted so that the preferred temperature is maintained in the reaction mixture. The deprotection step; Step V and 4 The catalyst used for hydrogenation may be selected from a great variety of catalysts as will be appreciated by a person skilled in the art. However 5% Pd on carbon is preferred. Any solvent that can dissolve at least some of the peptide is possible to use except ketones, such as acetone, or those solvents that poison the catalyst or react with the components of the reaction. A person skilled in the art will appreciate the choice of solvent. DMF and NMP are the preferred solvents. The deprotection step; Step 2 Hydrolysis of the ester residue group in compound (IV) can be achieved by any method known to the skilled person, e.g. aqueous acid, base treatment or hydro genolyzis, depending on the carboxyl protecting group used. Cj-Cό alkyl esters are preferred esters and treatment with aqueous base under standard conditions is the preferred method for ester hydrolysis. In a preferred embodiment of the present invention the protected amino acid, preferably oc using benzyloxycarbonyl- as N -amino protecting group, is activated as a mixed anhydride with isobutyloxycarbonylchloride, or a similar type of chloroformate. The method employed is based on the general method reviewed by J. Meienhofer in The Peptides, Vol. l, Eds: E. Gross & J. Meienhofer, Academic Press, Inc, London 1979, pp. 264-309. We have surprisingly found that the activation time can be extended to at least 30 min at a temperature about 0 - -15°C, contrary to the recommended 1-2 min at -15°C. We also found that strictly anhydrous conditions are not necessary as otherwise is recommended. This allows the present method to be used for large-scale production where the longer reaction times allow a safe and reproducible process to be carried out. The stereochemical integrity has been completely maintained and the chemical purity as well as yields have been typically over 90%. The generated mixed anhydride is coupled with the slow addition of the amino component (amino acid/ peptide amide or ester) at about 0 - -15°C and the reaction mixture is then allowed to reach 20-30°C in about 30-60 min. or longer before crystallization of the product is initiated directly from the reaction mixture. We have also surprisingly found that when using the present method, if appropriately selected solvent combinations are used, there is no need for a separate washing step prior to crystallization. DMF, acetonitrile, EtOAc and water are preferably used as solvents. A controlled crystallization achieves an excellent purification, shortens the filtering or centrifugation time during work up, as well as shortens the drying time, if dry intermediates are required. One important factor is to generate sufficiently large crystals with a relatively narrow size distribution not to block the filter medium or centrifugation cloth. It is very common for peptides in particular to generate gels or amorphous crystals that are almost impossible to filter. We have further surprisingly found that isobutylchloroformiate can be used in the key step in the fragment synthesis of the present invention, i.e. coupling step 3, without any substantial racemization of the D-alanine aminoacid fragment. INTERMEDIATES Another object of the present invention is to provide new intermediates that can be used in the preparation of compound of formula I. Accordingly, a further aspect of the present invention is a compound of the general formula X' wherein A is tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Z) or a substituted benzyloxycarbonyl derivative; R 1 is -NH 2 ,C r C 6 alkyl ester, benzyl ester, OH, 9-fluorenylmetyl ester or a substituted benzyl ester derivative: R 2 is H, a benzyl-like group, tert. butyl group, or 9-flourenylmefhyl group, as intermediates for use according to the present invention. DETAILED DESCRIPTION OF THE INVENTION The preparation of the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH, or a pharmaceutically acceptable salt thereof, will now be described in more detail by the following Examples, which should not be construed as limiting the invention. Furthermore, Scheme 1 below provides a detailed overview of the synthetic route followed for the preparation of the peptide of the formula (I) according to the present invention using an alanine derivative, wherein the carboxyl group is protected as an amid. The compound numbers referred to in the Examples below corresponds to the compound numbering in Scheme 1. Z-Phe(pF)-OH iBuOCOCl zv rmn. z-Phe(pF)-OCOOiBu (6) -10°C - +25°C EtOAc + DMF H-Phe(pF)-Phe-NH 2 40°C Z-Phe(pF)-Phe-NH 2 (9) Scheme 1 (8) Scheme 1, cont'd EXAMPLES Step l Preparation of Z-Tyr-D-Ala-OMe (Compound 4 in Scheme 1) Example 1 2,85g (22mmol) DEEA was added dropwise to an ice-cool solution of Z-Tyr-OH (2), H-D- Ala-OMe (3) and TBTU in 10ml of EtOAc (lOmmol each). The reaction mixture was left to assume room temperature after 25min at 4°C. The solvents were then evaporated and the residue partitioned between EtOAc and water. The organic phase was washed with 3 x 1M Na 2 CO 3 , 3x 1M KHSO 4 and brine before drying over Na 2 SO 4 . The product was crystallized by the addition of light petroleum ether to yield 3.2g (80%) and 99% pure. Step 2 Preparation of Z-Tyr-D-AIa-OH (Compound 5 in Scheme 1) Example 2 Z-Tyr-D-Ala-OMe ( l.Og, 2.46mmol, of purity 95%) was dissolved in dioxane (8mL). 1M NaOH (aq) (5.2mL) was then charged and the reaction left over night. The solvents were removed by vacuum distillation. The residue was dissolved in EtOAc (250mL) and extracted, first with brine (4x75mL) followed by 1M KHSO 4 (3x75mL). The organic layer was then dried over MgSO (anh d ) for several hours before filtration. The filtrate was evaporated to dryness by vacuum distillation. The residual oil was dissolved in Acetone EtOAc (2: 1, 5mL) and then isopropyl ether (30mL) was charged slowly in order to precipitate the product. The solid was filtered off and dried under reduced pressure at 30°C, yielding 0.8g (86%) of the product with a purity of 99% (HPLC). Example 3 Z-Tyr-OH (63,1 g, 200 mmol) and H-(D)-Ala-OMe x HCl (31,0 g, 222 mmol) were charged to a one liter reactor under nitrogen. Acetone (400 ml) was added and the slurry cooled to -20°C. Isobutylchloroformiat (30, 1 g, 220 mmol) was then added, quickly followed by N-Methylmorpholine (47,9 g, 472 mmol) while maintaining the temp at about -10°C. Upon completion of the NMM-charge the temp was allowed to reach 20°C, the precipitate filtered off and washed with Acetone (100ml). The product was slowly charged to NaOH (20 g, 500 mmol) in water (600 ml) at RT. HCl, 32% (60 ml) was added followed by evaporation of half the reaction volume at 70°C. Upon cooling and seeding the product crystallised. The product was isolated by filtration and dried in vacuum to yield 19.3 g Z- Tyr-(D)-Ala-OH (25%). Example 4 A 250 mL flask was charged with H-D-Alanine-OH (4,52 g; 50,2 mmol; 2 eq), potassium carbonate anhydrate (7,05 g; 50,2 mmol; 2 eq) and polyethylene glycol 200 (50 mL; 11,1 mL/g Alanine), which was stirred at ambient temperature. Another 250 mL flask was charged with Z-Tyrosine (8,00 g; 25,1 mmol) and EtOAc (100 mL; 12,6 mL/g Z- Tyrosine). After cooling (jacket temperature -17°C) to -9°C Isobutylchloroformate (3,55 mL: 30,1 mmol; 1,2 eq) was charged. N-Methylmorpholine (3,35 mL; 30,1 mmol; 1 ,2 eq) was charged keeping the temperature below -7°C. The cool mixture was charged to the first 250 mL flask containing the alanine via a peristaltic pump. The conversion was 90% by HPLC. The product was not isolated, but ready to use in the next step. Step 1' Preparation of Z-Phe(pF)-Phe-ΝH 2 (Compound 8 in Scheme 1) Example 5 Z-Phe(pF)-OH ( 1 eq.) is first dissolved in acetonitrile (MeCN)( 1.7L/mole) and cooled before addition of z ' -Butylchloroformiate ( 1.05 eq). The reaction is then controlled by the rate of addition, (about 20 minutes) 15 min actual, of N-Methylmorpholine ( 1.4eq). A reaction temperature between 0 and -15°C is recommended where the reaction occurs immediately upon addition of N-Methylmorphohne, yet prevents the mixed anhydride from decomposing to rapidly H-Phe-ΝH 2 x HCl ( 1 04 eq) is meanwhile dissolved in DMF (4 OL/mole), neutralized with s N-Methylmorpholine (1 04eq) and cooled to about -10°C This slurry is upon completion of the activation added at a rate that maintains the temperature around -10°C for about 15 minutes After completion of the coupling the product was crystallized from the reaction mixture by slow addition of 50% Ethanol/water (3 6L/mole) After 30 min a total of 2 85L/mole water lo in three portions were charged with about 25 min wait between each addition and at temperature of about 20°C The crystals can after about 17 hours be filtered or centπfuged and washed with 50% Ethanol/water followed by several portions of acetonitrile before drying under vacuum at 20-60°C Yield 90% and 99 9% purity 15 Step 2' Preparation of H-Phe(pF)-Phe-ΝH 2 (compound 9 in Scheme 1) Example 6 Z-Phe(pF)-Phe-NH prepared in the previous step is mixed with DMF (3 5L/mole) and a 0 Pd/C catalyst (5% Pd) is added 5%, by weight and the resulting mixture hydrogenated for more than 0 5 hours at 25-30°C and about 3bar H 2 The reaction mixture is then filtered and cooled to about -15°C before the next step 99 6% purity in solution and >99% conversion of starting material 5 Step 3 Preparation of Z-Tyr-D-Ala- PhefoFVPhe-NH? (compound 10 in Scheme 1) Example 7 The two building blocks Z-Tyr-D-Ala-OH ( 1 29mmol, 1 00 eq) and H-Phe( F)-Phe-NH : x o HCl (1 29mmol, 1 OOeq) were added to a round bottom flask containing solvent (DMF, 5mL) and the coupling reagent TBTU (1.28mmol, 0.99eq). The base (DIEA, 2.58mmol, 2. OOeq) was charged to this slurry. The reaction was monitored by HPLC and was terminated after 4h. The solvent was then evaporated. 22mL of a solvent mixture (EtOAc/Acetone MeOH 5: 10: 1) was charged to 2.4g of the remaining oil to yield the product as a solid, which was filtered and washed with acetone (3x5mL). The moist crystals were dried under reduced pressure at 30°C over night. The overall yield was 0.74g (80%) corrected for the purity 97% (HPLC). Example 8 Z-Tyr-(D)-Ala-OH (15.0 g, 39 mmol) and H-Phe(pF)-Phe-NH2xHCl (12.8 g, 35 mmol) were mixed with aceton (450 ml) in a one liter reactor. The slurry temperature was reduced to -10°C prior to addition of isobutylchloroformate (4.55 ml, 35 mmol). NMM (8.45 ml, 77 mmol) was slowly charged to maintain the temp at about -10°C. Upon completed addition the temp was increased to room temperature and 2M HCl (40 ml, 80 mmol) added followed by water (365 ml). The mixture was heated to reflux, ca 70°C, and acetone removed. The precipitate was filtered off and washed with acetonitril/water 3:2 (250 ml x 2) and dried to yield 18.9 g (77 %) of the title compound. Step 4 Preparation of H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 (compound I in Scheme 1) Example 9 Compound 4 is mixed with DMF (2-2.6L/mole) and a 5 % Pd/C catalyst is added (6-7%, by weight) and the resulting mixture hydrogenated for more than 0.5 hours at 20-50°C and 3bar H 2 . The reaction mixture is then filtered to remove the Pd/C before crystallizing the product by addition of EtOAc until all substance has crystallized (typically lOL/mole). The solid is separated by filtration or centrifugation and washed with EtOAc prior to drying under vacuum at 20-60°C. Preparation of H-Tyr-D-Ala-Phe(pF)-Phe-NH? hvdrochloride Example 10 The free base compound I is dissolved in a mixture of water and acetone with one equivalent HCl added and clear filtered (146g/mole 25% HCl/H 2 O, 2L Acetone/mole in actual run). The salt has a limited solubility in acetone and therefore the filter is washed once with an additional amount of the acetone/water (95:5) mixture (0.5L/mole). The crystallization is initiated by a slow addition of acetone (3.4L/mole) at high agitation rate and then 1% w/w of seeding crystals is optionally added. After 30 minutes the first amount of MIBK (3L/mole) is slowly charged and left with slow stirring until the batch clearly thickens. MLBK (3L/mole) is charged three additional times separated by 30-60 minutes while maintaining the reactor inner temperature at about 20°C. The solid is then separated by centrifugation or filtration and washed with MLBK before drying under vacuum at 20- 50°C for more than 16 hours or until the solvent levels are lower than specified in the release specifications.

Claims: CLAIMS 1. A process for the preparation of the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe-NH2 of the formula (I) or a pharmaceutically acceptable salt thereof, comprising a coupling step wherein an activated dipeptide derivative (V), wherein A is an amino protecting group, R is an activating agent residue group, and 2 R is H or a benzyl-like group is reacted with the amino group of a compound (IX) in the presence of a solvent under standard conditions, to give a protected tetrapeptide derivative (X) wherein A is an amino protecting group, R is -NH2 or an ester residue group, and 2 R is H or a benzyl-like group which is thereafter deprotected either by catalytic hydrogenolyzis, aqueous base or aqueous acid treatment, under standard conditions, to give the tetrapeptide (I). . 2. A process according to claim 1 , characterized in comprising an additional coupling step wherein an activated tyrosine derivative (II), wherein A is an amino protecting group, and R is an activating agent residue group, and R is H or a benzyl-like group; is reacted with the amino group of D-alanine, D-Ala-R 1 (III), wherein R is an ester group, in the presence of a solvent under standard conditions, to give a protected dipeptide derivative (IV) wherein A is an amino protecting group, R is an ester residue group, and 2 R is H or a benzyl-like group; which is thereafter a) deprotected by aqueous base treatment, under standard conditions, and b) treated with an activating agent to give an activated dipeptide derivative (V). 3. A process according to claim 1, characterized in comprising an additional coupling step wherein an activated tyrosine derivative (II), wherein A is an amino protecting group, and o R is an activating agent residue group, and 2 R is H or a benzyl-like group; is reacted with the amino group of D-alanine, D-Ala-R 1 (III), wherein R .1 i .s OH, 5 in the presence of a solvent under standard conditions, to give dipeptide derivative (V), o wherein A is an amino protecting group, and 2 R is H or a benzyl-like group; which is thereafter treated with an activating agent to give an activated dipeptide derivative (V). 4. A process according to claim 1 , characterized in comprising an additional coupling step wherein an activated -fluorophenylalanine derivative (VI), wherein A is an amino protecting group, and R is an activating agent residue group; is reacted with the amino group of phenylalanine Phe-R 1 (VII) wherein R is -NH2 or an ester residue group, in the presence of a solvent under standard conditions, providing a protected dipeptide derivative (VIII) wherein A is an amino protecting group, and R is -NH2 or an ester residue group; which is thereafter deprotected, under standard conditions, to give (IX). 5. A process according to claim 1, characterized in that the activated amino acid derivative used in at least one of the coupling steps is selected from a group consisting of a carbodiimide, an activated ester, an azide, or an anhydride. 6. A process according to claim 1, characterized in that benzyloxycarbonyl is the amino protecting group, methyl ester is the carboxyl protecting group, -NH2 is the phenylalanine carboxyl protecting group, the activated amino acid derivative is prepared from isobutylchloroformiate or 2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and no tyrosine side-chain protecting group is used. 7. A process according to claim 1, characterized in that the solvent used in at least one of the coupling steps is acetone, acetonitrile, NMP, DMF, EtOAc or a mixture thereof. 8. A process according to claims 1, wherein the solvent used in at least one of the coupling steps is DMF. 9. A process according to claim 1 characterized in that the deprotection step of the amino protecting group is performed using palladium on charcoal. 10. A process according to claim 1, characterized in that at least one of the coupling reactions are performed at a temperature from 0°C to -20 °C. 1 1. A process according to claim 10, wherein the temperature is from -5 °C to -15 °C. 12. A peptide of the formula (I) or a pharmaceutically acceptable salt thereof, prepared according to the process of claim 1. 13. A peptide according to claim 12, in the form of a hydrochloride salt. 14. A protected peptide derivative of the formula (X') wherein A is tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Z) or a substituted benzyloxycarbonyl derivative; R is -NH2, Ci-Cό alkyl ester, benzyl ester, OH, 9-fluorenylmetyl ester or a substituted benzyl ester derivative: 2 R is H, a benzyl-like group, tert. butyl group, or 9-flourenylmethyl group,

Inventors: ELLBURG MAARTEN, FRANZEN HENRY, NILSSON MATHS, ELLBURG, MAARTEN, FRANZEN, HENRY, NILSSON, MATHS
Applicants: ASTRAZENECA AB, ELLBURG MAARTEN, FRANZEN HENRY, NILSSON MATHS, ELLBURG, MAARTEN, FRANZEN, HENRY, NILSSON, MATHS
Application year: 2000
Main classification: C07K5
IPC codes: A61K38, A61P29, A61P43, C07K1, C07K5