MXPA00008828A - Process for the preparation of a tetrapeptide - Google Patents

Abstract

The present invention discloses a new and improved process for the preparation of the tetrapeptide H-Tyr-D-Ala-Phe(F)-Phe-NH2 which 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 suitable for large scale production.

Description

PROCESS FOR THE PREPARATION OF A TETRAPEPTIDE 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 other aspects, the present invention also relates to new intermediary products that are used in the process.

BACKGROUND AND PRIOR ART The present invention relates to a novel process for the preparation of the peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2, or a pharmaceutically acceptable salt thereof. WO 97/07129 describes a process for producing inter alia, the peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2. The peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2 is also described in WO 97/07130. This peptide exhibits an analgesic peripheral activity and a selectivity for the μ subtype of the opioid receptors, and REF .: 122763 is particularly suitable in pain therapy. In addition, it is prepared using a solid phase synthesis, according to the procedures established in the art. The disadvantage with solid phase synthesis, which is a common and well-established method for peptide synthesis, is that it is difficult to use for large scale production, besides being expensive. The process for the present invention provides the tetrapeptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2, in higher purity, in an economical and more environmentally friendly manner, compared to the methods known in the technique. In addition, the process of the present methodology provides the product to greater production. Therefore, the aim of the present invention is to provide a novel process, suitable for use in large-scale synthesis. Another objective of the present invention is to provide a process that contains as few reaction steps as possible.

Description of the Invention The present invention provides a novel process for the large-scale preparation of the peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2, which is a peptide of the formula (I) or a pharmaceutically acceptable salt thereof. The process according to the present invention for preparing a compound of the formula (I) above, comprises the following reaction steps; Step 1 (i) a step of copulation where an activated derivative of p-fluorophenylalanine (III), wherein A is an amino protecting group, and R is an activating agent of a residual group; which is prepared previously by the preactivation step or is generated in situ, is reacted with the amino group of phenylalanine, wherein the carboxyl group is protected as an ester or amide group, ie, a compound of the formula Phe- R1, wherein R1 is the residual ester or amide group, in the presence of a solvent, which provides a protected dipeptide derivative (IV) wherein A is an amino protecting group, and R1 is an ester or a residual amide group; (ii) a step of deprotection wherein a protected dipeptide derivative (IV) which is prepared in the previous step is deprotected, either by catalytic hydrogenation, a treatment with an acid or a base depending on the amino protecting group used, and which provides the dipeptide derivative (5), wherein R1 is a residual amide or ester group; Step 2 (i) a step of copulation where an activated derivative of alanine (VII), wherein A is an amino protecting group, and R is an activating agent of a residual group; which is prepared in advance by the preactivation step or is generated in itself, is reacted with the product of step 1, ie, the dipeptide derivative (5) in the presence of a solvent, which provides the protected tripeptide derivative (VIII ) wherein A is an amino protecting group, and R1 is an ester or a residual amide group; (ii) a deprotection step wherein a protected tripeptide derivative (VIII) which is prepared in the previous step, is deprotected either by catalytic hydrogenation, or an acid treatment, depending on the amino protecting group that is used, which provides the tripeptide derivative (9), wherein R 1 is an ester or a residual amide group; Step 3 (i) a step of copulation where- an activated derivative of tyrosine (X), wherein A is an amino protecting group, and R is an activating agent of a residual group, and R2 is H or a benzyl-like group; which is previously prepared by the preactivation step, or generated in itself, and reacted with the tripeptide derivative (9) and is the product of step 2, in the presence of a solvent, which provides the protected derivative of the tetrapeptide (XI) wherein, A is an amino protecting group, and R 1 is an ester or a residual amide group; (ii) an optional transformation step that is carried out if the protected tetrapeptide derivative (XI), which is prepared in the previous step (i), is an ester, wherein the ester compound (XI) is reacted with the ammonia in an organic alcohol, preferably ammonia in methanol, which provides the protected derivative of the dipeptide (XII), (iii) a deprotection step wherein a protected derivative of a tetrapeptide (XII) is deprotected, either by catalytic hydrogenation, or a treatment with an acid or a base, depending on the amino protecting group that is used, which provides the final tetrapeptide (I), which optionally can be converted into a salt of the tetrapeptide (I). The peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2 (I), if desired, can be reacted with a pharmaceutically acceptable salt, such as AcOH, H3PO4, citric acid, lactic acid and HCl. HCl is the preferred acid to be used in accordance with the present invention. Possible salts that can be used are described in S. M. Berge, L. D. Boghley and D. C. Monkhouse, J. Pharmaceut. Sci. , 66 ^ (1977) 1 -19. The process according to the present invention, described above, can therefore be described schematically by understanding the following steps; Step 1 (i) one step of copulation, (ii) one step of deprotection. Step 2 (i) a coupling step, (ii) a deprotection step Step 3 (i) a coupling step (ii) an optional transformation step (iii) a deprotection step The optional transformation step described above, in step 3 (ii) may instead be carried out after the coupling step (i), in Step 1, or in step 2. Preferably, the transformation step is carried out after the coupling step (i) in step 1. Therefore, the preferred way of carrying out the process of the present invention can be described systematically to understand the Next steps; Step 1 (i) a coupling step, (ii) an optional transformation step, (iii) a deprotection step, Step 2 (i) a coupling step, (ii) a deprotection step Step 3 (i) a step of coupling, (ii) a step of detachment The amino-Na protecting group can be selected from any suitable protecting group in the synthesis of peptides, such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl, which is commonly abbreviated as Z-, only to mention two possible amino protecting groups. However, benzyloxycarbonyl is particularly preferred for use in the present synthesis since it is easily removed by a catalytic hydrogenation, and contrary to the Boc protecting group, it does not require neutralization of the amine that is released. Suitable amino and carboxyl protecting groups that can be used in accordance with the present invention are appreciated by one skilled in the art. Reference is made to J. Mei enhofer 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. Mei enhofer, Eds. , Academic Press Inc., London, 1979-1987; Houben-Weyl, Methoden der organischen Chemie, E. Muller, ed. , Vol. 15m Part I-II, Thieme, Stuttgart 1974; and M. Bodanszky, Principies of peptide Synthesis, Springer Verlag, Berlin 1984. The pre-activation step that precedes Steps 1-3, or the in-generation of the activated derivative of the activated amino acid, is achieved by reacting an amino acid, where the amino function has been protected by a suitable protecting group, such as terbutoxycarbonyl (Boc) or benzyloxycarbonyl (Z), which, whether commercially available or available by methods known in the art, with an activating agent in the presence of a tertiary amine and a organic solvent, which provides the activated derivative of the amino acid. Below is a schematic representation of the pre-activation step (II) (ni) Derivative of Activated Amino Acid Derivative Amino Acid wherein A is an amino protecting group, and R is an activating agent of a residual group; For the step of copulation, in Steps 1-3 described above, a variety of potent solvents can be used, so long as the amino component is essentially soluble and available for immediate reaction with the activated derivative of the peptide. Examples of suitable solvents for the coupling step are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP) and EtOAc, or mixtures thereof. As used herein, the term "benzyl-like group" denotes any substituted or unsubstituted benzyl group that is hydrogenated under reaction conditions similar to those of the benzyloxycarbonyl group.The term * pF "denotes a para-fluoro substituent.

The possible reactants and reaction conditions, as well as those preferred in each step, are as follows.

The Pre-Activation Step Suitable activating agents may be selected from those which generate any of the activated derivatives of commonly used amino acids including, but not limited to carbodiimides, activated esters, azides, or anhydrides. Isobutylchloroformate (iBuOCOCl) is the preferred activating agent. When the Isobutylchloroformate (iBuOCOCl) is the activating agent, the activated derivative of the peptide has the following structure, which is exemplified in D-alanine, The tertiary amine can be selected from any tertiary amine. However, NMM (N-ethylmorpholine), di-isopropylethylamine and triethylamine are preferred. In addition, a secondary amine that is sterically inhibited can also be used. The organic solvent can be any organic solvent that is known to one skilled in the art, because it is suitable in peptide chemistry. However, ethyl acetate, acetonitrile, acetone and tetrahydrofuran are the preferred solvents in the pre-activation step.

The copulation step; Step 1 (i), step 2 (i) and step 3 (i) The solvent that is used for the coupling step can be selected from a variety of solvents, as long as the amino component is essentially soluble and available for an immediate reaction with the activated amino acid residue. Examples of suitable solvents for the copula steps are acetone, acetonitrile, DMF, N-methylpyrrolidone (NMP) and EtOAc, or mixtures of these, of which, acetone, EtOAc, NMP and DMF They are the preferred ones. Any temperature can be used where the activated derivative of the amino acid is not degraded or where the reaction rate is too slow. The preferred range is from 0 ° C to -20 ° C, and particularly preferred ranges from -5 ° C to -15 ° C. The rate of addition is adjusted such that the preferred temperature is maintained.

The deprotection step; Step I (ii), step 2 (ii) and step 3 (iii) The catalyst used for hydrogenation can be selected from a wide variety of catalysts as appreciated by one skilled in the art. However, 5% Pd in carbon is preferred. It is possible to use any solvent that can dissolve at least some of the peptide except the ketones, such as acetone, or those solvents that deplete the catalyst or react with the reaction components. The choice of solvent is appreciated by someone skilled in the art. The preferred solvent is DMF. Optional step 3 (ii) is only required if the protected tetrapeptide derivative (XI) which is prepared in step 3 (i) is an ester. Therefore, if an amide or a phenylalanine is used, step 3 (ii) of the synthetic process is excluded. If an acid is used for the removal of the protective group-a, an equivalent molal amount of a base is required to deprotonate the amino group of the peptide derivative. In a preferred embodiment of the present invention, the protected amino acid, preferably when using the benzyloxycarbonyl, in the manner of a Na-amino protecting group, is activated in the form of a mixed anhydride with isobutyloxycarbonylchloride, or a chloroformate of similar type. The method used is based on the general method reviewed by J. Meienhofer in The Peptides, Vol. 1, Eds .: E. Gross &; J. Meienhofer, Academic Press, Inc., London 1979, pp. 264-309. Surprisingly, we have found that the activation time can be extended for at least 30 minutes at a temperature from about 0 ° C to -15 ° C, contrary to the recommended duration of 1-2 minutes at -15 ° C. We have also found that strictly anhydrous conditions are not necessary, as otherwise recommended. This allows the current method to be used in a large scale production, where the longer reaction times allow a safe and reproducible process to be carried out. The stereochemical integrity is completely maintained and the chemical purity, as well as the production, are typically over 90%. The mixed anhydride that is generated is bound together with the amino component to a slow addition (amino acid / peptide amide or ester) at about 0 ° C to -15 ° C and then the reaction mixture is allowed to reach 20-30. ° C for about 30-60 minutes or more, before starting the crystallization of the product directly from the reaction mixture.

Surprisingly, we have also found that when the current method is used, if appropriately selected combinations of the solvent are used, there is no need for a separate wash step prior to crystallization. Preferably, DMF, acetonitrile, and EtOAc and water are used. Controlled crystallization not only achieves excellent purification, but also decreases the filtration or centrifugation time during the preparation, as well as decreases the drying time, if intermediate drying steps are required. An important factor is to generate sufficiently large crystals, with a relatively narrow distribution size, so as not to block the filtering medium or the centrifuge cloth. It is very common, in particular for peptides, to generate amorphous gels or crystals that are almost impossible to filter. The tripeptide derivative (9) wherein R 1 is an ester or a residual amide group; It is a useful intermediate for the preparation of the objective compound (I).

Detailed Description of the Invention The preparation of the peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH2 or a pharmaceutically acceptable salt thereof is now described in greater detail by the following Examples, which, however, are not should consider as limiting the invention. In addition, the following Reaction Scheme 1 provides a detailed review of the synthetic route followed for the preparation of the peptide of formula (I), according to the present invention, by using a derivative of phenylalanine, wherein a carboxyl group is protected way of an ester. The following Reaction Scheme 2 provides a detailed review of the synthetic routes followed for the preparation of the peptide of the formula (I), according to the present invention, by using a derivative of the phenylalanine wherein the carboxyl group is protected in the manner of a amide. The numbers of the compounds referred to in the detailed synthesis of the description of the following Examples correspond to the numbering of the compounds in Reaction Schemes 1 and 2.

Reaction Scheme I H-PhßfoF ^ Pttß-NH, Reaction Scheme I (Cont.) H-I Ia-PhßÍpFJ-Phß-NHj Reaction Scheme I (Cont.) HCWjO Aceten »MIBK xHCI Step 1 (i) Preparation of Z-Phe (pF) -Phe-OMe (Compound 3 in Reaction Scheme 1) 3.5 mol scale First, Z-Phe (pF) (compound 1) (1 eq.) is dissolved in acetone (4.7 L / mol) and cooled before the addition of IBK (0.9-1.2 eq.) (1 eq. Current). Then the reaction is controlled by the rate of addition (for about 20 minutes) of NMM (N-methylmorpholine) (0.9-1.2 eq.) (1 eq. Current). A reaction temperature between 0 and -15 ° C (from -9 ° C to -14 ° C current) where the reaction occurs immediately upon adding the NMM, however, prevents the mixed anhydride from decomposing too quickly. Meanwhile, H-Phe-OMe x HCl (0.9-1.3 eq.) (1.04 eq. Current) is mixed with acetone (2.6L / mol), neutralized with NMM (0.9-1.5 eq.) (1.04 eq. current) and cooled between 0 and -20 ° C (approximately -10 ° C current). This aqueous suspension is over the term of the activation that is added at a rate that keeps the temperature around -10 ° C (from -8 ° C to -13 ° C current) (for about 30 minutes). Then EtOAc (4L / mol) is charged and the organic phase is washed with water (2 x 2L / mol) followed by azeotropic distillation of ACN and a solution in MeOH before the next step. A 92% purity is obtained in an aqueous methanol slurry.

Multiple Conversion. H Integral 8.5. d 1 7.49 d 1 7.31 m 7 7.24 m 5 7.08 m 2 4.93 m 2 4.5 m 1 4.26 m 1 3.58 s 3 2.99 m 3 2.67 m 1 (ii) Preparation of Z-Phe (pF) -Phe-NH2 (compound 4 in Reaction Scheme 1) 2.3-3.3 mol scale Ammonia is charged into the solution of compound 3 prepared in the previous step (about 8L MeOH / mol) at a pressure between 1-5 bar at 15 to 40 ° C for more than 5 hours or until the reaction is near completion (current conversion 99%). At the end, the ammonia is evaporated and the reaction is cooled before filtration or centrifugation. The product is washed with MeOH and dried under vacuum at 20-50 ° C. The production is 74% which is calculated from compound 1 (Z-Phe (pF)) and has a purity of 100%. Conversion Multiplicity H Integral 8.05 d 1 7.49 d 1 7.24 m 11 7.07 m 3 4. 94 m 2 4. 4 6 m 1 4. 21 m 1 3. 00 m 1 2. 88 m 1 2. 66 m 1 Preparation of H-Phe (pF) -Phe-NH2 (compound 5 in Reaction Scheme 1) 4.3 mol scale Compound 4 that is prepared in the previous step is mixed with DMF (4.2L / mol) and a Pd / C catalyst (5% Pd current content) is added (0.2-10% w / w / LEF-581) (7% current) and the resulting mixture is hydrogenated for more than 0.5 hours (1.2 current hours) at 25 ° C and 3 bar H2. The reaction mixture is then filtered and cooled to about -15 ° C before the next step. 99.6% purity is produced in the solution.

Step 2 (i) Preparation of ZD-Ala-Phe (pF) -Phe-NH2 (compound 8 in Reaction scheme 1) 4.4 scale mol Dissolve ZD-Ala (compound 6) (1 eq.) In acetonitrile ( ACN) (2.3L / mol) and cooled before the addition of IBK (0.9-1.2 eq.) (1 eq. Used). NMM (0.9-1.2 eq.) 1 eq. used) and then added in the same manner as described above for the preparation of compound 3. Then the solution of compound 5 (24.5L) is charged for approximately 30 minutes, the temperature is maintained around -10 ° C (from -8 ° C to -14 ° C current). After finishing the copulation, the product is crystallized from the reaction mixture by the slow addition of water (3 x 3.6L / mol + 1 x 1.3 L / mol) with a wait of approximately 25 minutes between each addition and an initial temperature of about 30 ° C and a final temperature of about 20 ° C. The crystals can be centrifuged and washed with water / acetonitrile (4: 1) before being dried under vacuum at 20-50 ° C. The production is 90% and has 99.5% purity.

Conversion Multiplicity H Integral 8.14 d 1 8.05 d 1 7.43 d 1 7.29 m 12 7.05 m 2 5.01 m 2 4.44 m 2 4. 01 m 1 2. 92 m 3 2. 73 m 1 0. 96 d 3 Preparation of HD-Ala-Phe (pF) -Phe-NH2 (compound 9 in Reaction Scheme 1) Compound 8 which is prepared in the previous step is mixed with DMF (4.2L / mol) and a Pd / C catalyst (5% Pd current content) is added (0.2-10% p / p / compound 3) (7% current) and the resulting mixture is hydrogenated for more than 0.5 hours (1.2 current hours) at 25 ° C and 3bar H2 . Then, the reaction mixture is filtered and cooled to about -15 ° C before the next step. Purity of 97%. Conversion of raw material > 98%.

Step 3 (i) Preparation of Z-Tyr-D-Ala-Phe (pF) -Phe-NH2 (compound 12 in Reaction Scheme 1) 4.1 mol scale Copulation uses the same method as the two previous copulas. Dissolve Z-Tyr (compound 10) (1 eq.) In ACN (2.3 L / mol) and cool before addition of IBK (0.9-1.2 eq.): Then add NMM (0.9-1.2 eq.) in the same manner as described above under compound 3. Then the solution of compound 3, from the previous step, is charged for about 30 minutes, keeping the temperature at about -10 ° C (from -7 ° C to -14 ° C). Current C). After completion, the product is crystallized from the reaction mixture by slow addition of acetonitrile and water (2L / mol ACN + 0.3L / mol 25% NH3 in H20), maintained for 2 hours, added 1.5L / mol of ACN: H20 (1: 1), it is maintained for one hour, - the temperature is increased to 35 ° C, the seeded crystals are added (approximately 1% w / w), it is maintained for one hour, 1.3L / mol of ACN: H20 (1: 1) is added, it is maintained for one hour, 1.2L / mol of H20 is added, and it is maintained during 0.5 hours at 35 ° C, 1.2L / mol of H20 is added , and it is kept for 2 hours at 20 ° C, 1.2L / mol of H20 is added, and it is kept for 1 hour at 20 ° C and 1.2L / mol of H20 are added and it is maintained during 0.5 hours at 20 ° C . It is centrifuged and washed first with water and then with ACN before being dried under vacuum at 20-50 ° C.

Production of 81% calculated from compound 8 Purity of 98.4%.

Conversion Multiplicity H Integral 9.18 s 1 8.18 d 1 8.11 d 1 8.05 d 1 7.43 d 1 7.24 m 12 .7.04 m 4 6.63 d 2 4.92 m 2 4.45 m 2 4.22 m 2 3.00 1 2.83 m 1 2.64 m 1 0.89 d 3 (ii) Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH2 (compound I in Reaction Scheme 1) 3.1 and 3.2 mol scale Compound 4 is mixed with DMF (2-2.6 L / mol current series) and a catalyst of 5% Pd / C (current content) is added (0.2-10% w / w / compound 3) (6-7% current) and the resulting mixture is hydrogenated for more than 0.5 hours (1 -2 hours current series) at 20-40 ° C (20-25 ° C current series) and 3bar H2. The reaction mixture is then filtered to remove the Pd / C before crystallizing the product by the addition of EtOAc until all the substance has crystallized (usually lOL / mol). The solid is separated by filtration or centrifugation and washed with EtOAc before being dried under vacuum at 20-50 ° C. (iii) Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH2 hydrochloride 2.1 mol scale Free base compound I is dissolved in a mixture of water and acetone with one equivalent of HCl which is added and it is filtered by filtration (146 g / mol 25% HC1 / H20, 2 L Acetone / mol in the current series). The salt has a limited solubility in acetone and therefore, the filter is washed once with an additional amount of a mixture of acetone / water (95: 5) (0.5L / mol). Crystallization is initiated by slow addition of acetone (3.4L / mol) at a high rate of agitation, and at least 1% w / w of seed crystals is added. After 30 minutes, the first amount of MIBK (3L / mol) is slowly loaded and maintained with a slow stirring until the batch is clearly thickened. The MIBK (3L / mol) is charged three more times, which is separated during intervals of 30-60 minutes while maintaining the interior temperature of the bioreactor at approximately 20 ° C. The solid is then separated by filtration or centrifugation and washed with MIBK before, vacuum drying at 20-50 ° C for more than 16 hours or until the solvent levels are lower than those specified in the commercialization specifications.

Scheme of Reaction 2 Z-Phß. { pF) iBuocoa NMM (14) Reaction Scheme 2 (Cont.) -D-Ala-OCQOiBu Reaction Scheme 2 (Cont.) H-Tyr-D-AÍ »-Phß. { F) -Phß-NH, O) HCl Step 1 (i) Preparation of Z-Phe (pF) -Phe-NH2 (compound 13 in Reaction Scheme 2) 6.7 mol scale. The Z-Phe (pF) (1 eq.) Is first dissolved in acetonitrile (EtOAc) (1.7L / mol) and cooled before the addition of? -Butylchloroformate (0.9-1.2 eq.) (1.05 eq. Current) . Then the reaction is controlled by the rate of addition, (for about 20 minutes) 15 minutes present, of N-Methylmorpholine (0.9-2.0 eq.) (1.4 eq. Current). A reaction temperature between 0 and -15 ° C (from -8 ° C to -11 ° C) is recommended where the reaction occurs immediately after the addition of N-Methylmorpholine, but prevents the mixed anhydride from decomposing quickly. Meanwhile, H-Phe-? H2 X HCl (0.9-1.3 eq.) (1.04 eq. Current) is dissolved in DMF ((4.0 L / mol), neutralized with N-Methylmorpholine (0.9-1.5 eq.) ( 1.04 eq. Current) and cooled from 0 to -20 ° C (about -10 ° C current) This thick aqueous suspension is at the end of activation, and is added at a rate that keeps the temperature around -10 ° C (from -6 ° C to -13 ° C current) (for approximately 15 minutes) for 8 current minutes After the end of copulation, the product is crystallized from the reaction mixture by a slow addition of 50 % Ethanol / water (3.6L / mol) After a wait of 30 minutes, a total of 2.85L / mole of water is loaded in three portions during a wait of approximately 25 minutes between each addition and at a temperature of approximately 20 minutes. C. Crystals can be filtered after approximately 17 hours or centrifuged and washed with 50% Ethanol / water followed by several portions of acetonitrile before being dried under vacuum at 20-60 ° C. Production of 90% and 99.9% purity. (ii) Preparation of H-Phe (pF) -Phe-NH2 (compound 13 in Reaction Scheme 2) 6.7 mol scale The Z-Phe (pF) -Phe-NH2 which is prepared in the previous step, is mixed with DMF (3.5L / mol) and a Pd / C catalyst (5% Pd current content) are added (0.2-10% p / p) LEF-582) (5% current) and the resulting mixture is hydrogenated for more than 0.5 hours (1.3 hours current) at 25-30 ° C and approximately 3bar H2. The reaction mixture is then filtered and cooled to approximately -15 ° C before the next step. 99.6% purity in a solution and > 99% conversion of the raw material.

Step 2 (i) Preparation of ZD-Ala-Phe (pF) -Phe-NH2 (compound 15 in Reaction Scheme 2) 5.9 mol scale Dissolve ZD-Ala-OH (compound x) (1.03 eq. Used) in acetonitrile (1.9 L / mol) and cooled before the addition of i-Butylchloroformate (0.9-1.2 eq.) (1.07 eq. used). Then - N-Methylmorpholine (0.9-2.0 eq.) (1.2 eq. Used) is added in a similar manner as described above for the preparation of Z-Phe (pF) -Phe-? H2. The solution of H-Phe (pF) -Phe-? H2 (25L) is charged for approximately 15 minutes (8 current minutes), and the temperature is maintained around -10 ° C (from -8 ° C to -11 ° C current). After the end of copulation, the product is crystallized from the reaction mixture by a slow addition of water (4 x 1.9 L / mol) for approximately 15-30 minutes of waiting between each addition and at a temperature of about 20 minutes. ° C. The crystals can then be filtered or centrifuged and washed with water / acetonitrile (4: 1) followed by acetonitrile before optional drying under vacuum at 20-60 ° C. The calculated production for Z-Phe (pF) -Phe-NH2 is 93.8% and 99.6% pure.

Preparation of HD-Ala-Ph (pF) -Phe-NH2 (compound 16 in Reaction Scheme 2) 5.5 scale mol The ZD-Ala-Phe (pF) -Phe-NH2 which is prepared in the previous step is mixed with DMF (2.9L / mol) and one is added, a Pd / C catalyst (5% Pd current content) (0.2-10% w / w / compound 3) (5% current) and the resulting mixture is hydrogenated for more from 0.5 hours (3 current hours) to 25-35 ° C and approximately 3bar H2. The reaction mixture is then filtered and cooled to about -15 ° C before the next step. Purity of 99.4%. Conversion of raw material > 99% Step 3 (i) Preparation of Z-Tyr-D-Ala-Phe (pF) -Phe-NH2 (compound 17 in Reaction Scheme 2) 5.5 scale mol This copula uses a method similar to the two previous copulas. Z-Tyr (compound x) (1.05 eq.) Is dissolved in MeCN (1.9 L / mol) and cooled before the addition of i-Butylchloroformate (0.9-1.2 eq.) (1.05 current). Then N-Methylmorpholine (0.9-2.0 eq.) (1.3 current) is added in a similar manner as described above for the preparation of Z-Phe (pF) -Phe-? H2. After the HD-Ala-Phe (pF) -Phe-? H2 solution from the previous step is loaded for approximately 20 minutes (6 minutes), the temperature is maintained around -10 ° C (from -8 ° C to - 9 ° C current). After the end of copulation, the product is crystallized from the reaction mixture, at approximately 20-45 ° C by a slow addition of acetonitrile and water (3.4L / mol MeC? + 0.9 L / mol%? H3 in H20) and it is maintained during 5 minutes and it is sown, it is maintained during 4 hours, and then a total of 13.9L / mol of H20 is added in four portions during a wait of 30 minutes or more, between each one. It is filtered and centrifuged and washed first with water and then with MeCN before optional drying under vacuum at 20-60 ° C. Production of 87.7% calculated for Z-D-Ala-Phe (pF) -Phe-NH2 and 95.1% pure. It is observed that production purity is increased and by heating the reaction to about 60 ° C with the addition of ammonia at a pH of about 9 for 2 hours. This converts the highest impurity, Z-Tyr (0- (i-Butyloxycarbonyl) -D-Ala-Phe (pF) -Phe-NH2, into a product. (ii) Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH2 (compound I in Reaction scheme 2) 5.4 mol scale Z-Tyr-D-Ala-Phe (pF) - Phe-NH2 with DMF (2.6L / mol current series) and a 5% catalyst is added Pd / C (current content) (0.2-10% w / w / compound 3) (6.4% current) and the resulting mixture is hydrogenated for more than 0.5 hours (1.8 hours current series) at 20-40 ° C (20-40 ° C). 25 ° C current series) and approximately at 3bar H2. The reaction mixture is then filtered to remove the Pd / C before crystallizing the product by the addition of EtOAc until all the substance has crystallized (typically, at about 14 L / moles): the solid is separated by filtration or centrifugation and washed with EtOAc before being dried under vacuum at 20-50 ° C. Purity of 96.7%. Conversion of raw material > 99% Preparation of Hydrochloride of H-Tyr-D-Ala-Phe (pF) -Phe- NH2 4.6 scale mol The free base H-Tyr-D-Ala-Phe (pF) -Phe-NH2 is dissolved in a mixture of water and acetone with one HCl equivalent that is added and purified by filtration (146 g / mol 25% HC1 / H20, 2 L Acetone / mol in current series). The salts are almost insoluble in acetone, and therefore, the filter is washed once with an amount of a mixture of acetone / water (95: 5) (0.5L / mol). The crystallization is initiated by a slow addition of acetone (3.4L / mol) at a high stirring speed and then about 1% w / w of seed crystals is added. After 30 minutes, the first amount of MIBK (3L / mol) is slowly loaded and left in slow agitation until the batch thickened markedly. The MIBK (3L / mol) is charged three additional times which are separated during 30-60 minute intervals, while maintaining the internal temperature of the bioreactor at approximately 20 ° C. The solid is then separated by filtration or centrifugation and washed with MIBK before being dried in a vacuum at 20-50 ° C for more than 15 hours or until the solvent levels are lower than those specified in the commercialization specifications. Production of 95.8% and purity of 99.8%.

Reprocessing The product that does not achieve the specifications of the substance of the drug can be recrystallized again by the same procedure described above for the crystallization of compound I, but without the addition of HCl.

Assignment of the spectrum for NMR (nuclear magnetic resonance) of H-Tyr-D-Ala-Phe (pF) -Phe-NH2 x HCl, ie for compound I in its hydrochloride form The NMR spectra are obtained from of a solution of 36mg of the compound in about 0.7 ml of DMSO-de (.99.95 atomo-% D) at 27.0 ° C in a Varian UNITY plus instrument of 400 MHz. The reference of the chemical conversion for the protonic spectrum is the peak mean of the multiple DMSO-dβ which is taken as 2.49 ppm. The reference for the carbon spectrum is the average peak of the multiple DMSO-of which is taken as 39.5 ppm.

The atomic numbering used in the subject is arbitrary and refers to the previous figure.

Proton Spectrum The one-dimensional protonic spectrum allows the subject, in group form, of the alpha protons (3.9-4.4 ppm), benzyl-CH? (2.6-3.1 ppm), amide-NH and phenol-OH (8.2-8.5 ppm) and also the specific subject for Ala-CH3 (14-CH3) (0.74 ppm). The DQFCOSY two-dimensional spectrum allows the group subject for the spinaural systems (alpha, beta and NH protons) in each amino acid residue, and the group subject of the aryl protons in each aromatic ring. All protons in the Ala residue can also be assigned in a specific way. Carbon Spectrum The one-dimensional carbon spectrum allows the group subject of the alpha carbons, the benzyl-CH2, the carbonyls and the aryl carbons and of course the specific subject of C-14. The spectrum for APT allows the subject of multiplicity-CH for each carbon. The linear unfolding, due to the C-F couplings, allows the specific subject of the carbons in the fluoroaromatic ring.

Two-dimensional Hetero-correlated Spectrum The carbon-proton correlated two-dimensional spectrum (HMQC) gives us a correlation between the protonated carbons and all directly bonded protons. All the protonated carbons in the Ala residue can be specifically assigned. * "The correlated two-dimensional spectrum of multiple carbon-proton copulas (HMBC) gives us a correlation between the carbons and protons located in two or three separate copulas.This allows the subject of the amino acid sequence by means of the alpha hydrogens and the carbonyl group of the surrounding acid residue (correlation of three copulas), as well as by means of the NH and the carbonyl group of the surrounding amino acid residue (correlation of two copulas) Similarly, the correlations of two and three copulas between benzyl CH2 and the aryl carbons as well as between the protons of aryl and benzyl-CH2 allow the specific subject of the protons of the aryl and the carbons of individual aromatic amino acids, in this way, all the protons (not interchangeable) and carbons in all the Four amino acid residues can be assigned in a specific manner in an unambiguous manner.

Table 1 Proton Subject Table 2 Carbon Subjects It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (12)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

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, characterized in that it comprises the following steps: (i) a coupling step wherein the activated derivative of tyrosine (X), wherein: A is an amino protecting group, R is an activating agent of a residual group, and R2 is H or a group similar to benzyl; it is reacted with the tripeptide derivative (9) wherein: R1 is an ester or a residual amide group; in the presence of a solvent under normal conditions, which provides the protected derivative of the tetrapeptide (XI) wherein: A is an amino protecting group, R1 is an ester or a residual amide group; (ii) a deprotection step wherein the protected derivative of a tetrapeptide (XII) is deprotected either by catalytic hydrogenation, a treatment with an acid or a base under normal conditions in the presence of a solvent to give (I).

2. A process according to claim 1, characterized in that it comprises the additional steps of: (i) a coupling step wherein an activated derivative of alanine (VII), wherein: A is an amino protecting group, and R is an activating agent of a residual group; it is reacted with the dipeptide derivative (5) wherein: R1 is an ester or a residual amide group; in the presence of a solvent under conventional conditions, which provides the protected derivative of the tripeptide (VIII) wherein: A is an amino protecting group, and R1 is an ester or a residual amide group; (ii) a deprotection step wherein a protected derivative of a tripeptide. (VIII) which is prepared in the previous step, is deprotected either by catalytic hydrogenation, an acid or alkaline treatment, in the presence of a solvent under conventional conditions, which provides the tripeptide derivative O) wherein: R1 is an ester or a residual amide group;

3. A process according to claim 2, characterized in that it comprises the additional steps of: (i) a coupling step wherein an activated derivative of p-fluorophenylalanine (III), (III) wherein: A is an amino protecting group, and R is an activating agent of a residual group; it is reacted with the amino group of phenylalanine, wherein the carboxyl group is protected as an ester or amide, in the presence of a solvent under normal conditions, which provides a protected dipeptide derivative (IV) wherein: A is an amino protecting group, R1 is an ester or a residual amide group; (ii) a step of deprotection wherein a protected derivative of a dipeptide (IV) which is prepared in the previous step, is deprotected either by catalytic hydrogenation, a treatment with an acid or a base, in the presence of a low solvent normal conditions, which provides the dipeptide derivative (5), wherein: R1 is an ester or a residual amide group.

4. A process according to claims 1-3, characterized in that one of the copula steps (i) is followed by an optional step of transformation, which is carried out on the carboxyl group of the amino acid derivative which is protected in the manner of a derivative of an ester, wherein the ester compound is reacted with the ammonia in an organic alcohol.

5. A process according to claims 1-3, characterized in that the activated derivative of the amino acid used in at least one of the coupling steps is selected from the group consisting of a carbodiimide, an activated ester, an azide, or an anhydride

6. A process according to claim 5, characterized in that the activating agent is isobutylchloroformate.

7. A process according to any of claims 1-6 / characterized in that the solvent used in at least one of the coupling steps is acetone, acetonitrile, NMP, DMF or EtOAc.

8. A process according to claims 1-3, characterized in that the solvent in the coupling step is DMF.

9. A process according to any of claims 1-3, characterized in that the deprotection step is carried out using Pd in carbon.

10. A process according to any of claims 1-9, characterized in that at least one of the reactions is carried out at the temperature ranging from 0 ° C to -20 ° C.

11. A process according to claim 10, characterized in that the temperature ranges from -5 ° C to -15 ° C.

12. A peptide derivative of the formula (9) wherein: R1 is an ester or a residual amide group,