SK13442000A3 - 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

The present invention relates to a novel process for the preparation of a tetrapeptide, more specifically the tetrapeptide H-Tyr-D-Ala-Phe (pF) -Phe-NH 2, or a pharmaceutically acceptable salt thereof. The present invention further relates to novel intermediates used in this process.

BACKGROUND OF THE INVENTION

The present invention relates to a novel process for the preparation of the peptide H-Tyr-D-AlaPhe (pF) -Phe-NH ₂ , or a pharmaceutically acceptable salt thereof.

WO 97/07129 discloses a process for the production of, inter alia, the peptide H-Tyr-D-AlaPhe (pF) -Phe-NH ₂ . The peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ is also disclosed in WO 97107130. Said peptide exhibits peripheral analgesic activity and selectivity for the µ opioid receptor subtype and is particularly suitable for the treatment of pain. In addition, it is prepared by solid phase synthesis according to established procedures in the art. The drawback of solid phase synthesis, which is a common and established method of peptide synthesis, is that it is difficult to use for large-scale production and is moreover expensive.

The process of the present invention provides the H-Tyr-D-Ala-Phe (pF) Phe-NH ₂ tetrapeptide with higher purity, a more cost-effective and better method compared to methods known in the art. Furthermore, the process of the present application gives the product in a higher yield.

It is therefore an object of the present invention to provide a novel process suitable for use in large-scale synthesis. Another object of the present invention is to provide a process comprising as few reaction steps as possible.

SUMMARY 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-NH ₂ , which is a peptide of formula I

Η ₂ Ν

or a pharmaceutically acceptable salt thereof.

The process of the present invention for preparing the above compound of formula (I) consists of the following reaction steps:

Step 1 (i) The coupling step, wherein the activated p-fluorophenylalanine derivative of formula III,

H

About where

A is an amino protecting group and

R is a moiety of an activating agent residue;

previously prepared in the 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 formula Phe-R ¹ where R ¹ is an ester or amide residue in the presence of a solvent a dipeptide derivative of formula IV (IV),

where

A is an amino protecting group and

R ¹ is an ester or amide residue;

(ii) deprotection, deprotecting the protected dipeptide derivative of formula IV prepared in the preceding step either by catalytic hydrogenation or by treatment with a base or acid depending on the amino protecting group used to give the dipeptide derivative of formula 5,

where

R ¹ is an ester or amide residue;

Step 2 (i) The coupling step, wherein the activated alanine derivative of formula VII,

A.

(VII), where

A is an amino protecting group and R is an activating agent residue group;

prepared beforehand in the pre-activation step or generated in situ is reacted with the product of step 1, i. a dipeptide derivative of formula 5 in the presence of a solvent to give a protected tripeptide derivative of formula VIII

(VIII), where

A is an amino protecting group and

R ¹ is an ester or amide residue;

(ii) Deprotection, deprotecting the protected tripeptide derivative of formula (VIII) prepared in the previous step either by catalytic hydrogenation or by treatment with a base or acid depending on the amino protecting group used to give the tripeptide derivative of formula (9)

where

R ¹ is an ester or amide residue;

Step 3 (i) The coupling step, wherein the activated tyrosine derivative of formula X,

(X) where

A is an amino protecting group,

R is a moiety of an activating agent residue and

R ² is H or a benzyl-like group;

prepared in advance in the pre-activation step or generated in situ is reacted with the tripeptide derivative of formula 9, i.e. the product of step 2, in the presence of a solvent to give the protected tetrapeptide derivative of formula XI

where

A is an amino protecting group and R ¹ is an ester or amide residue;

(ii) An optional transformation step carried out when the protected tetrapeptide derivative of formula XI prepared in the previous step (i) is an ester wherein the ester compound of formula XI reacts with ammonia in an organic alcohol, preferably ammonia in methanol, to afford the protected dipeptide derivative formula XII,

(XII), (iii) A deprotection step of deprotecting a protected tetrapeptide derivative of formula (XII) either by catalytic hydrogenation or by treatment with a base or acid depending on the amino protecting group used to give the final tetrapeptide of formula (I) which may be optionally converting to a salt of the tetrapeptide of Formula I.

The H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ peptide of Formula I can be reacted, if desired, with a pharmaceutically acceptable acid such as AcOH, H ₃ PO ₄ , citric acid, lactic acid, and HCl. HCl is the preferred acid for use in accordance with the present invention. Possible salts that can be used are described in SM Berge, LD Bighley and DC Monkhouse, J. Pharmaceut. Sci., 66 (1977) 1-19.

Thus, the process of the present invention described above may be schematically described as consisting of the following steps:

Step 1 (i) Coupling step, (ii) Step of deprotecting,

Step 2 (i) Coupling step, (ii) Step of deprotecting,

Step 3 (i) Coupling step, (ii) Optional transformation step, (iii) Protecting group removal step

The optional transformation step described above in step 3 (ii) could instead be performed after the coupling step (i) in step 1 or step 2. The transformation step is preferably performed after the coupling step (i) in step 1. Preferred method for carrying out the process according to the present invention could therefore be schematically described as consisting of the following steps:

Step 1 (i) Coupling step, (ii) Optional transformation step, (iii) Step of deprotecting,

Step 2 (i) Coupling step, (ii) Step of deprotecting

Step 3 (i) Coupling step, (ii) Step of deprotecting

The N'-amino protecting group may be selected from any of the protecting groups suitable for peptide synthesis, such as tert-butoxycarbonyl (Boe) or benzyloxycarbonyl, often Z-, these are just two of the many possible amino-protecting groups. However, benzyloxycarbonyl is particularly preferred for use in this synthesis since it is readily removed by catalytic hydrogenation and, unlike the Boe protecting group, does not require neutralization of the liberated amine. Those skilled in the art will recognize suitable amino and carboxyl protecting groups that can be used in accordance with the present invention. They can be found in the following sources: J. Meienhofer in The Peptides, Vol.

I, Eds .: E. Gross & J. Meienhofer, Academic Press, Inc., London 1979, p. 264-309; The peptides, Vol. 1-9, E. Gross & J. Meienhofer, Eds., Academic Press Inc., London, 1979-1987; Houben-Weyl, Methoden der Organischen Chemie, E. Muller, ed., Vol. 15, Part II, Thieme, Stuttgart 1974; and M. Bodanszky, Principles of Peptide Synthesis, Springer Verlag, Berlin 1984.

The pre-activation step preceding step 1-3, or the generation of an activated amino acid derivative in situ, is accomplished by an amino acid reaction wherein the amino group is protected with a suitable protecting group, e.g. tert-butoxycarbonyl (Boe) or benzyloxycarbonyl (Z), either commercially available or available known in the art, with an activating agent in the presence of a tertiary amine and an organic solvent to provide an activated amino acid derivative. A schematic representation of the pre-activation step is shown below.

amino acid derivative activated amino acid derivative wherein

A is an amino protecting group and

R is a moiety of an activating agent residue;

In the coupling step in steps 1-3 described above, a number of strong solvents can be used in which the amino acid component is substantially soluble and available for immediate reaction with the activated peptide derivative. Examples of suitable solvents for the coupling step are acetone, acetonitrile, DMF, N-methylpyrrolidone (NMP) and EtOAc or mixtures thereof.

As used herein, the term "benzyl-like group" refers to any substituted or unsubstituted benzyl group that is hydrogenolysed under similar reaction conditions to the benzyloxycarbonyl group.

The term "pF" refers to a para-fluoro substituent.

Possible as well as preferred reagents and reaction conditions at each step are as follows:

Pre-activation step

Suitable activating agents may be selected from those which form any of the commonly used amino acid derivatives, including but not limited to carbodiimides, activated esters, azides, or anhydrides. A preferred activating agent is isobutyl chloroformate (iBuOCOCI). When the activating agent is isobutyl chloroformate (iBuOCOCI), the activated peptide derivative will have the following structure, which is shown in the example of D-alanine:

The tertiary amine may be any tertiary amine. However, NMIVI (N-methylmorpholine), diisopropylethylamine and triethylamine are preferred. In addition, a secondary amine which is sterically hindered may also be used.

The organic solvent may be any organic solvent known to the person skilled in the art as suitable in peptide chemistry. However, ethyl acetate, acetonitrile, acetone and tetrahydrofuran are preferred in the pre-activation step.

Joining step; step 1 (i), step 2 (i) and step 3 (i)

The solvent used for the coupling step may be selected from a variety of solvents in which the amino acid component is substantially 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-methylpyrrolidone (NMP) and EtOAc or mixtures thereof, of which preferred solvents are acetone, EtOAc, NMP and DMF.

Any temperature at which the activated amino acid derivative does not decompose and at which the reaction rate is not too low can be used. A preferred interval is from 0 ° C to -20 ° C and particularly preferred is from -5 ° C to -15 ° C. The rate of addition is controlled to maintain the preferred temperature.

The step of removing the protecting group; step 1 (ii), step 2 (ii) and step 3 (iii)

The catalyst used for the hydrogenation can be selected from a variety of catalysts, as will be apparent to one skilled in the art. However, a preferred catalyst is 5% Pd on carbon. Any solvent that can dissolve at least a portion of the peptide, except ketones such as acetone, or those solvents that poison the catalyst or react with the components of the reaction mixture can be used. The choice of solvent will be readily apparent to those skilled in the art. The preferred solvent is DMF.

Optional step 3 (ii) is required only if the protected tetrapeptide derivative (XI) prepared in step 3 (i) is an ester. Thus, if an amide from phenylalanine is used, step 3 (ii) is excluded from the synthetic procedure.

If an acid is used to remove the α-protecting group, an equivalent molar amount of 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 with benzyloxycarbonyl as the N- ^α- amino protecting group, is activated as a mixed anhydride with isobutyloxycarbonyl chloride or a similar type of chloroformate. The method used is based on the general method reviewed by J. Meienhofer in The Peptides, Vol. 1, Eds .: E. Gross & I. Meienhofer, Academic Press, Inc., London 1979, p. 264-309.

We have surprisingly found that the activation time can be extended to at least 30 minutes at a temperature of about 0 to -15 ° C as opposed to the recommended 1 to 2 minutes at -15 ° C. We also found that strictly anhydrous conditions are not necessary, as otherwise recommended. This allows the use of this method for large-scale production, where longer reaction times allow a safe and reproducible process to be carried out. Stereochemical integrity was fully maintained and chemical purity as well as yields were typically over 90%. The formed mixed anhydride is combined by slowly adding the amino acid component (amino acid or peptide amide or ester) at about 0 to -15 ° C and the reaction mixture is then allowed to reach 20 to 30 ° C for about 30 to 60 minutes or longer until initiates crystallization of the product directly from the reaction mixture.

In addition, we have surprisingly found that a separate washing step prior to crystallization is not required when using this method and using suitably selected solvent combinations. Preferably DMF, acetonitrile, EtOAc and water are used. Controlled crystallization not only achieves an excellent purification but also shortens the filtration or centrifugation during processing as well as shortens the drying if dry intermediates are needed. One important factor is to create sufficiently large crystals with a relatively small size distribution so as not to clog the filter media or the centrifugal fabric. It is very common for peptides to form gels or amorphous crystals that are almost impossible to filter.

The tripeptide derivative of formula 9

where

R ¹ is an ester or amide residue;

is a useful intermediate for the preparation of the target compound of formula I.

DETAILED DESCRIPTION OF THE INVENTION

The preparation of the peptide H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ or a pharmaceutically acceptable salt thereof will now be described in more detail in the following examples, but this should not be construed as limiting the scope of the invention. In addition, Scheme 1 below provides a detailed overview of the synthetic process for preparing a peptide of Formula I of the present invention using a phenylalanine derivative wherein the carboxyl group is protected as an ester. Scheme 2 below provides a detailed overview of the synthetic process for preparing a peptide of formula I of the present invention using a phenylalanine derivative wherein the carboxyl group is protected as an amide. The compound numbers given in the detailed synthesis description in the examples below correspond to the numbering of the compounds in Schemes 1 and 2.

Scheme 1

H-Phe (pF) -Phe-NH, (1)

Scheme 1 (continued)

Ô V -OH (i) iBuOCOCI jjr (ii) NMM (6)

ACN

-10 ^e C 20 min / CH ₃ λΛΥΛ

Z-D-Ala

HD-Ala-Phe (pF) -Phe-NH ₂

Scheme 1 (continued)

(12)

Z-Tyr-D-Ala-Phe (pF) -Phe-NH

O CH,

H-Tyr-D-Ala-Phe {pF) -Phe-NH

HO / HJO

acetone

MIBK

H-Tyr-O-Ala-Phe (pF) -Phe-NH, x HCl

Step 1 (i) Preparation of Z-Phe (pF) -Phe-OMe (compound 3 in Scheme 1) Scale - 3.5 moles

Z-Phe (pF) (compound 1) (1 eq.) Was first dissolved in acetone (4.7 L / mol) and cooled before adding IBK (0.9-1.2 eq.) (Actually 1 eq.) .). The reaction is then controlled by the addition rate (about 20 minutes) of NMM (N-methylmorpholine) (0.9-1.2 eq) (actually 1 eq). A reaction temperature between 0 and -15 ° C (actually from 9 ° C to -14 ° C) is recommended, where the reaction occurs immediately after addition of NMM, but the mixed anhydride does not decompose rapidly.

H-Phe-OMe x HCl (0.9-1.3 eq.) (In fact 1.04 eq.) Was meanwhile mixed with acetone (2.6 L / mol), neutralized with NMM (0.9- 1.5 eq) (actually 1.04 eq) and cooled to 0 to -20 ° C (actually about -10 ° C). This suspension is added at the rate of activation that maintains a temperature of about -10 ° C (actually -8 ° C to -13 ° C) after the activation is complete (about 30 minutes). EtOAc (4 L / mol) was then added and the organic phase was washed with water (2x2 L / mol) followed by azeotropic distillation from ACN and dissolved in MeOH before the next step. 92% purity in methanol suspension.

shift multiplicity Integral H 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 with 3 2.99 m 3 2.67 m 1

(ii) Preparation of Z-Phe (pF) -Phe-NH ₂ (Compound 4 in Scheme 1)

Scale - 2.3 to 3.3 moles

To a solution of compound 3 prepared in the previous step (about 8 L MeOH / mol) is added ammonia at a pressure of between 1 and 4 bar at 15 to 40 ° C for more than 5 hours or until the reaction is almost complete (true conversion 99%). After completion, the ammonia is evaporated and the reaction mixture is cooled before filtration or centrifugation. The product was washed with MeOH and dried under vacuum at 20-50 ° C. Yield 74% calculated on the basis of compound 1 (Z-Phe (pF)) and 100% purity.

shift multiplicity Integral H 8.05 D 1 7.49 D 1 7.24 m 11 7.07 m 3 4.94 m 2 4.46 m 1 4,21 m 1 3.00 m 1 2.88 m 1 2.66 m 1

Preparation of H-Phe (pF) -Phe-NH ₂ (compound 5 in Scheme 1)

Scale - 4.3 moles

Compound 4 prepared in the previous step is mixed with DMF (4.2 L / mol) and Pd / C catalyst (actual Pd content 5%) (0.2 -10% by weight of LEF-581) (actually 7%) is added. and the resulting mixture is hydrogenated for more than 0.5 hours (actually 1.2 hours) at 25 ° C and 3 bar H ₂ . The reaction mixture is then filtered and cooled to about -15 ° C before the next step. 99.6% purity in solution.

Step 2 (i) Preparation of ZD-Ala-Phe (pF) -Phe-NH ₂ (Compound 8 in Scheme 1)

Scale - 4.4 moles

Z-Phe (pF) (compound 6) (1 eq.) Was first dissolved in acetone (4.7 L / mol) and cooled before addition of IBK (0.9-1.2 eq.) (1 eq. .). NMM (0.9-1.2 eq.) (1 eq. Used) was then added in the same manner as described above for the preparation of compound 3. A solution of compound 5 (24.5 L) was then added over about 30 minutes. minutes while maintaining the temperature at about -10 ° C (actually -8 ° C to -14 ° C). After the coupling was complete, the product crystallized from the reaction mixture by slow addition of water (3 x 3.6 L / mol + 1 x 1.3 L / mol) with a pause of about 25 min between each addition and at an initial temperature of about 30 ° C and final temperature. about 20 ° C. The crystals can then be centrifuged and washed with a mixture of water and acetonitrile (4: 1) and dried under vacuum at 2050 ° C. Yield 90% and 99.5% purity.

shift multiplicity Integral H 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-NH ₂ (Compound 9 in Scheme 1)

Compound 8 prepared in the previous step was mixed with DMF (4.2 L / mol) and Pd / C catalyst (actual Pd content 5%) (0.2 -10% by weight of compound 3) (actually 7%) was added and the resulting mixture was added. is hydrogenated for more than 0.5 hours (actually 1.2 hours) at 25 ° C and 3 bar H ₂ . The reaction mixture is then filtered and cooled to about -15 ° C before the next step. Purity 97%. Starting material conversion> 98%.

Step 3 (i) Preparation of Z-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ (Compound 12 in Scheme 1) A -4.1 mole range

The same method was used in this synthesis as in the previous two syntheses. Z-Tyr (compound 10) (1 eq) was first dissolved in ACN (2.3 L / mol) and cooled before addition of IBK (0.9-1.2 eq). NMM (0.9-1.2 eq.) Was then added in the same manner as described above for the preparation of compound 3. The solution of compound 3 from the previous step was then added over about 30 minutes while maintaining the temperature around -10 ° C (actually from -7 ° C to -14 ° C). Upon completion of the synthesis, the product crystallized from the reaction mixture by slowly adding acetonitrile and water (2 L / mol ACN + 0.3 L / mol 25% NH ₃ in H ₂ O, standing for 2 h, adding 1.3 L / mol ACN: H ₂ O (1: 1), standing for 1 h, raising the temperature to 35 ° C, adding seed crystals (about 1% by weight), standing for 1 h, adding 1.3 l / mol ACN: H ₂ O (1: 1), standing for 1 h, adding 1.2 l / mol of H ₂ O and standing for 0.5 h at 35 ° C, adding 1.2 l / mol of H ₂ O and standing for 2 h at 20 ° C, adding 1.2 l / mol of H ₂ O and standing for 1 h at 20 ° C and addition of 1.2 L / mol of H ₂ O and standing for 0.5 h at 20 ° C). Centrifuge and wash first with water and then ACN before drying under vacuum at 20-50 ° C.

Yield 81% calculated from compound 8. Purity 98.4%.

shift multiplicity Integral H 9.18 with 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 m 1 2.83 m 1 2.64 m 1 0.89 D 3

(ii) Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ (Compound I in Scheme 1)

Scale - 3.1 and 3.2 moles

Compound 4 is mixed with DMF (2-2.6 l / mol in actual experiment) and 5% Pd / C catalyst (actual content) (0.2-10% by weight of 3) is added (actually 6-7% ) and the resulting mixture is hydrogenated for more than 0.5 hours (in a real experiment 1-2 hours) at 20-40 ° C (in a real experiment 20 25 ° C) and 3 bars H ₂ . The reaction mixture was then filtered to remove Pd / C, and the product was crystallized by addition of EtOAc until the material crystallized (mostly 10 L / mol). The solid phase is separated by filtration or centrifugation, washed with EtOAc and dried under vacuum at 20-50 ° C.

Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ Hydrochloride

Scale-2.1 moles

Dissolve the free base of compound I in a mixture of water and acetone with one equivalent of HCl and filter to obtain a clear solution (146 g / mol, 25% HCl / H ₂ O, 2 L acetone / mol in a real experiment). The salt has limited solubility in acetone and therefore the filter is washed once with another portion of a 95: 5 mixture of acetone and water (0.5 L / mol). Crystallization is initiated by slow addition of acetone (3.4 L / mol) with vigorous stirring, and then at least 1% by weight of seed crystals are added. After 30 minutes, the first portion of MIBK (3 L / mol) was added slowly and the mixture was allowed to stir slowly until evidently thick. MIBK (3 L / mol) was added three more times with breaks of 30-60 minutes while maintaining the internal temperature of the reactor at about 20 ° C. The solid phase is then separated by centrifugation or filtration, washed with MIBK and dried under vacuum at 20-50 ° C for more than 16 hours or until the solvent level is lower than specified.

Scheme 2

Z-Phe (pF)

iBuOCOCI

NMM

EtOAc

Z-Phe (pF) -OCOOiBu Phe-NH ₂ x HCl • 10 ° C 10 min EtOAc +

Z-Phe (pF) -Phe-NH ₂

Π3)

Phe (pF) -Phe-NH ₂ (15)

Scheme 2 (continued)

Scheme 2 (continued)

OH

H-Tyr-D-Ala-Phe (F) -Phe-NH 3 m

HCl

acetone

3% H, O

MIBK

Z-Tyr-D-Ala-Phe (pF) -Phe-NH 3 X HCl

Step 1 (i) Preparation of Z-Phe (pF) -Phe-NH ₂ (Compound 13 in Scheme 2)

Scale - 6.7 moles

Z-Phe (pF) (1 eq) was first dissolved in acetonitrile (EtOAc) (1.7 L / mol) and cooled before addition of i-butyl chloroformate (0.9-1.2 eq) (actually 1 , 05 eq). The reaction is then controlled by the addition rate (about 20 minutes, actually 15 min) of NMM (N-methylmorpholine) (0.9-2.0 eq) (actually 1.4 eq). A reaction temperature between 0 and -15 ° C (in fact from -8 ° C to -11 ° C) is recommended, where the reaction occurs immediately after the addition of N -methylmorpholine, but the mixed anhydride does not decompose rapidly.

In the meantime, H-Phe-NH ₂ x HCl (0.9-1.3 eq.) (Actually 1.04 eq.) Was dissolved in DMF (4.0 L / mol), neutralized with N-methylmorpholine (O, 9-1.5 equiv) (actually 1.04 equiv) and cooled to 0 to -20 ° C (actually about -10 ° C). This suspension is added at the rate of activation that maintains a temperature of about -10 ° C (actually -6 ° C to -13 ° C) (about 15 minutes, in fact 8 minutes) after activation is complete.

Upon completion of the synthesis, the product was crystallized from the reaction mixture by slowly adding 50% ethanol / water (3.6 L / mol). After standing for 30 minutes, a total of 2.85 l / mol of water was added in three portions with a break of about 25 minutes at a temperature of about 20 ° C. The crystals may be filtered or centrifuged after about 17 hours and washed with a 50% ethanol / water mixture and then with several portions of acetonitrile and dried under vacuum at 20-60 ° C. Yield 90% and 99.9% purity.

Preparation of H-Phe (pF) -Phe-NH ₂ (Compound 14 in Scheme 2)

Scale - 6.7 moles

The Z-Phe (pF) -Phe-NH ₂ prepared in the previous step is mixed with DMF (3.5 L / mol) and the Pd / C catalyst (actual Pd content of 5%) (0.2-10% by weight of LEF-582) is added. ) (in fact 5%) and the resulting mixture is hydrogenated for more than 0.5 hours (actually 1.3 hours) at 25-30 ° C and 3 bars 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 the starting material.

Step 2 (i) Preparation of ZD-Ala-Phe (pF) -Phe-NH ₂ (Compound 15 in Scheme 2)

Scale - 5.9 moles

ZD-Ala-OH (compound x) (1.03 equiv) was first dissolved in acetonitrile (1.9 L / mol) and cooled before addition of i-butyl chloroformate (0.9-1.2 equiv) (used with 1.07 eq). N-methylmorpholine (0.9-2.0 eq.) (1.2 eq. Used) was then added in a similar manner as described above for the preparation of Z-Phe (pF) -Phe-NH ₂ . The H-Phe (pF) Phe-NH ₂ (25 L) solution was then added over about 15 minutes (actually 8 minutes) while maintaining the temperature at about -10 ° C (actually -8 ° C to -11 ° C). C). Upon completion of the synthesis, the product was crystallized from the reaction mixture by slowly adding water (4 x 1.9 L / mol) with a pause of about 15-30 min between each addition and at an initial temperature of about 20 ° C. The crystals can then be filtered off or centrifuged and washed with a mixture of water and acetonitrile (4: 1) and then acetonitrile and dried under vacuum at 20 60 ° C. Yield calculated on the basis of Z-Phe (pF) -Phe-NH ₂ 93.8% and 99.6% purity. Preparation of HD-Ala-Phe (pF) -Phe-NH ₂ (Compound 16 in Scheme 2)

Scale - 5.5 moles

The ZD-Ala-Phe (pF) -Phe-NH ₂ prepared in the previous step is mixed with DMF (2.9 L / mol) and the Pd / C catalyst (actual Pd content of 5%) (0.2-10% by weight of the compound) is added. 3) (actually 5%) and the resulting mixture is hydrogenated for more than 0.5 hours (actually 3 hours) at 25-35 ° C and about 3 bars H ₂ . The reaction mixture is then filtered and cooled to about -15 ° C before the next step. Purity 99.4%. Starting material conversion> 99%.

Step 3 (i) Preparation of Z-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ (Compound 17 in Scheme 2)

Scale - 5.5 moles

A similar method to the two previous syntheses was used in this synthesis. Ty-Tyr (compound x) (1.05 eq) was dissolved in MeCN (1.9 L / mol) and cooled before addition of i-butyl chloroformate (0.9-1.2 eq) (in fact 1, 05). N-methylmorpholine (0.9-2.0 eq.) (Actually 1.3 eq.) Was then added in a similar manner as described above for the preparation of Z-Phe (pF) -Phe-NH ₂ . The HD-AlaPhe (pF) -Phe-NH _{2 solution} from the previous step was then added over about 20 minutes (actually 6 minutes) while maintaining the temperature at about -10 ° C (actually -8 ° C to -9 ° C). C). Upon completion of the synthesis, the product crystallized from the reaction mixture at about 20-45 ° C by the slow addition of acetonitrile and water (3.4 L / mol MeCN + 0.9 L / mol 15% NH ₃ in H ₂ O, standing for 5 min and seeding. , standing 4-24 h, then adding a total of 13.9 l / mol H ₂ O in four portions with breaks of about 30 min or more). Filtering or centrifuging and washing first with water and then MeCN before optionally drying under vacuum at 20-60 ° C. Yield 87.7% calculated based on ZD-Ala-Phe (pF) -Phe-NH ₂ and 95.1% purity. It has been found that yield and purity can be increased by heating the reaction mixture to about 60 ° C with the addition of ammonia to a pH of about 9 for two hours. This converts the most important impurity, Z-Tyr (O - (t-butyloxycarbonyl)) - D-Ala-Phe (pF) -PheNH ₂ , to the product.

(ii) Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ (Compound 1 in Scheme 2)

Scale - 5.4 moles

Z-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ was mixed with DMF (2.6 L / mol in actual experiment) and 5% Pd / C catalyst (actual content) (0.2-10%) was added. by weight of compound 3) (actually 6.4%) and the resulting mixture is hydrogenated for more than 0.5 hours (in a real experiment of 1.8 hours) at 20-40 ° C (in a real experiment of 20-25 ° C) and about 3 hours. bar H ₂ . The reaction mixture is then filtered to remove Pd / C, and the product is crystallized by addition of EtOAc until the substance is completely crystallized (mostly about 14 L / mol). The solid phase is separated by filtration or centrifugation, washed with EtOAc and dried under vacuum at 20-50 ° C. Purity 96.7%. Starting material conversion> 99%.

Preparation of H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ hydrochloride

Scale - 4.6 moles

H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ free base was dissolved in a mixture of water and acetone with one equivalent of HCl and filtered to give a clear solution (146 g / mol, 25% HCl). H ₂ O, 2 L acetone / mol in actual experiment). The salt is almost insoluble in acetone and therefore the filter is washed once with a further portion of a 95: 5 mixture of acetone and water (0.5 L / mol). Crystallization is initiated by the slow addition of acetone (3.4 L / mol) with vigorous stirring, and then about 1% by weight of seed crystals are added. After 30 minutes, the first portion of MIBK (3 L / mol) was added slowly and the mixture was allowed to stir slowly until evidently thick. MIBK (3 L / mol) was added three more times with breaks of 30-60 minutes while maintaining the internal temperature of the reactor at about 20 ° C. The solid phase is then separated by centrifugation or filtration, washed with MIBK and dried under vacuum at 2050 ° C for more than 16 hours or until the solvent level is lower than specified. Yield 95.8% and purity 99.89%.

Reprocessing

A product that does not meet the drug specifications can be recrystallized by the same procedure as described above for crystallization of compound I, but without the addition of HCl.

Assignment of NMR spectra for H-Tyr-D-Ala-Phe (F) -Phe-NH ₂ x HCl, ie for compound I in the form of the hydrochloride

NMR spectra were obtained from a solution of 36 mg of the compound in approximately 0.7 mL of DMSO-d ₆ (99.95 atomic% D) at 27.0 ° C on a Varian UMTY plus 400 MHz instrument. The chemical shift reference for proton spectra was the mean peak of the DMSO-d ₆ multiplet taken as 2.49 ppm. Reference for carbon spectra was the middle peak of the multiplet _DMS0-d6 taken as 39.5 ppm.

The numbering of the atoms used in the assignment is arbitrary and refers to the figure above.

Proton spectra

One-dimensional proton spectrum allows group assignment of alpha protons (3.9-4.4 ppm), CH ₂ protons of benzyl (2.6-3.1 ppm), amide NH and phenol OH protons (8.2-8.5 ppm) as well as the specific assignment for the alanine (14-CH ₃ ) CH ₃ protons (0.74 ppm).

The two-dimensional DQFCOSY spectrum allows for group assignment of spin systems (alpha, beta, and NH protons) at each amino acid residue and group assignment of aryl protons in each aromatic ring. It is also possible to specifically assign all protons in the Ala residue.

Carbon spectra

The one-dimensional carbon spectrum allows for the group assignment of alpha carbons, CH ₂ protons of benzyl, carbonyl and aryl carbons, and of course the specific assignment of C-14. The APT spectrum allows the assignment of CH multiplicity for each carbon. Line splitting due to CF interaction allows specific assignment of carbons in the fluorinated aromatic ring.

Two-dimensional heterocorrelated spectra

The two-dimensional carbon-proton correlated (HMQC) spectrum gives correlation between protonated carbons and direct coupled protons. It is also possible to specifically assign all protonated carbons in the Ala residue.

The two-dimensional carbon-proton multiply correlated (HMBC) spectrum gives a correlation between the carbon and protons located at a distance of two to three bonds. This allows the amino acid sequence to be assigned through the alpha hydrogens and the carbonyl group of the adjacent amino acid residue (trivalent correlation), as well as through the NH and the carbonyl group of the adjacent amino acid residue (bivalent correlation). Similarly, the two- and three-bond correlations between CH ₂ benzyl and aryl carbons as well as between aryl protons and CH ₂ benzyl allow specific assignment of aryl protons and carbons of individual aromatic amino acids. In this way, all (non-replacing) protons and carbons in all four amino acid residues can be specifically and unambiguously assigned.

Table 1

Assignment of protons

Chemical shift (ppm) integral multiplicity assignment 9.4 1 H with 300 H 8.48 1 H D 9NH 8.37 1 H D 1 NH 8.35 1 H D 8 NH 8.26 2H with 7.53 1 H with 7.28 2H m 18 H, 19 H 7.26 2H m 21 H, 22 H 7.22 2H m 6H, 7H 7.18 1 H m 24 H 7.12 1 H with 7.04 2H m 10H, 11H 6.98 2H m 26H, 27H 6.68 2H m 28H, 29H 4.43 1 H m 1 H 4.39 1 H m 8 H

Chemical shift (ppm) integral multiplicity assignment 4.26 1 H m 9H 3.96 1 H m 20 H 3.39 HDO 3.02 1 H m 12 Hb 2.99 1 H m 2 Hb 2.87 2H m 23 Ha, 23 Hb 2.85 1 H m 12 Ha 2.67 1 H m 2 Ha 0.74 3H D 14 H

Table 2

Carbon assignment

Chemical shift (ppm) multiplicity assignment Jc-F 172.99 with 13 171.08 with 5 170.81 with 3 167.39 with 17 162.11 with 15 241 Hz 159.70 with 15 241 Hz 156.50 with 30 137.98 with 16 133.96 with 4 3.1 Hz 133.93 with 4 3.1 Hz 131.16 D 6.7 8.4 Hz 131.08 D 6.7 8.4 Hz 130.46 D 26,27 129.26 D 18.19 128.09 D 21.22 126.30 D 24 124.73 with 25 115.16 D 28.29 114.63 D 10.11 213 Hz 114.41 Γ d 10.11 213 Hz 54.33 D 8 54.01 D 1 53.37 D 20 47,96 D 9 37.57 T 12 36.83 T 2 36.17 T 23

Claims (12)

PATENT CLAIMS A process for the preparation of H-Tyr-D-Ala-Phe (F) -Phe-NH ₂ tetrapeptide of formula I or a pharmaceutically acceptable salt thereof, characterized in that it comprises the following steps: (i) a coupling step, wherein the activated tyrosine derivative of formula X, (X). where A is an amino protecting group, R 2 is a moiety of an activating agent moiety and R ² is H or a benzyl-like group; reacts with a tripeptide derivative of the formula ιχ Η ₂ Ν where R ¹ is an ester or amide residue; in the presence of a solvent under standard conditions to provide the protected tetrapeptide derivative of formula XI (XI). where A is an amino protecting group and R ¹ is an ester or amide residue; (ii) a deprotection step of removing a protecting group from a protected tetrapeptide derivative of formula XII either by catalytic hydrogenation or by treatment with a base or acid under standard conditions in the presence of a solvent to give formula I. The method of claim 1, further comprising the following additional steps: (i) a coupling step, wherein the activated alanine derivative of formula VII, QH ₃ where A is an amino protecting group and R is an activating agent residue group; reacted with a dipeptide derivative of formula 5 (5), wherein R ¹ is an ester or amide residue in the presence of a solvent under standard conditions to provide the protected tripeptide derivative of formula VIII (VIII) wherein: A is an amino protecting group and R ¹ is an ester or amide residue; (ii) deprotection, deprotecting the protected tripeptide derivative of formula (VIII) prepared in the previous step either by catalytic hydrogenation or by treatment with a base or acid in the presence of a solvent under standard conditions to give a tripeptide derivative of formula (9) wherein R ¹ is an ester or amide residue. The method of claim 2, further comprising the following additional steps: (i) a coupling step, wherein the activated p-fluorophenylalanine derivative of formula III, wherein A is an amino protecting group and R is a moiety of an activating agent residue; reacts with the amino group of phenylalanine, wherein the carboxyl group is protected as an ester or amide, in the presence of a solvent under standard conditions, to provide a protected dipeptide derivative of formula IV wherein: A is an amino protecting group and R ¹ is an ester or amide residue; (ii) deprotection, deprotecting the protected dipeptide derivative of formula IV prepared in the previous step either by catalytic hydrogenation or by treatment with a base or acid in the presence of a solvent under standard conditions to give a dipeptide derivative of formula 5, wherein R ¹ is an ester or amide residue. Process according to claims 1 to 3, characterized in that one of the coupling steps (i) is followed by an optional transformation step which is carried out when the carboxyl group of the amino acid derivative is protected as an ester, the ester reacting with ammonia in an organic alcohol. Process according to claims 1 to 3, characterized in that the activated amino acid derivative used in at least one of the coupling steps is selected from the group consisting of carbodiimide, activated ester, azide or anhydride. The process according to claim 5, wherein the activating agent is isobutyl chloroformate. Process according to any one of claims 1 to 6, characterized in that the solvent used in at least one of the coupling steps is acetone, acetonitrile, NMP, DMF or EtOAc. Process according to claims 1 to 3, characterized in that the solvent in the coupling step is DMF. 9th 10th 11th 12th 9th 10th 11th 12th Process according to any one of claims 1 to 3, characterized in that the deprotection step is carried out by means of Pd on activated carbon. Process according to any one of claims 1 to 9, characterized in that at least one of the reactions is carried out at a temperature from 0 ° C to -20 ° C. Process according to claim 10, characterized in that the temperature is from -5 ° C to -15 ° C. A peptide derivative of formula 9 wherein R ¹ is an ester or amide residue.