CZ20003342A3 - Process for preparing a tetrapeptide - Google Patents

Abstract

An improved process for the preparation of the tetrapeptide H-Tyr-D-Ala-phe(F)-Phe-NH2, which is a peptide of formula (I), or its pharmaceutically acceptable salts, as well as novel intermediates for its preparation. The novel process is suitable for large-scale production.

Description

Field of the invention

The present invention is directed to a novel process for the preparation of a tetrapeptide, more specifically to the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe-NH2 or a pharmaceutically acceptable salt thereof. In further aspects, the present invention relates to novel intermediates used in the process.

State of the art

The present invention relates to a novel process for preparing the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NHh or a pharmaceutically acceptable salt thereof.

WO 97/07129 describes a method for preparing, 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. Said peptide exhibits peripheral analgesic activity and selectivity for the μ subtype of opioid receptors, and is particularly useful in the treatment of pain. Furthermore, it is prepared using solid phase synthesis according to methods well established in the art. A disadvantage of solid phase synthesis, which is a common and well established method for peptide synthesis, is that it is difficult to use for large-scale preparation, and is also expensive.

The process of the present invention provides the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe-NH2 of higher purity, in a more cost-effective and environmentally preferable manner, compared to methods known in the art. In addition, the process of the present application provides the product in higher yield.

• 4

It is therefore an object of the present invention to provide a new 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.

The essence of the invention

The present invention provides a novel method for the large-scale production of the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH ₂ . This peptide is

or a pharmaceutically acceptable salt thereof.

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

Step 1 (i) A condensation step in which the activated p-fluorophenylalanine derivative of general formula (III),

AND

O (III) • · • · · · · » · · · » • 9 9 · · · • · · · · • ·« · » It » · · · fc « · · · · » , · ♦ ·· w where

A is an amino protecting group and

R is the residue group of the activating agent, previously prepared by a pre-activation step or formed in situ, is allowed to react with the amino group of phenylalanine, while the carboxyl group is protected in the form of an ester or amide, i.e. as a compound of the formula Phe-R ¹ , where R ¹ is the residue of an ester or amide group, in the presence of a solvent, providing a protected dipeptide derivative of the general formula (IV)

(IV) where

A is an amino protecting group and _r

R ¹ is the residue of an ester or amide group;

(ii) A deprotection step, wherein the protected dipeptide derivative of general formula (IV) prepared in the previous step is deprotected either by catalytic hydrogenation, basic or acidic treatment, depending on the amino protecting group used, to obtain a dipeptide derivative of general formula (5),

« *

9

999

AND

I · *

9· · · • · 9

9 9 9 * * where

R ¹ is the residue of an ester or amide group;

Step 2 (i) A condensation step in which an alanine derivative of general formula (VII),

H ₃ Q ^A ~'N ^Z '~'V

HO(VII) where

A is an amino protecting group and

R ¹ is a residual group of the activating agent;

previously prepared by the pre-activation step or formed in situ, is reacted with the product of step 1, i.e. the dipeptide derivative of general formula (5) in the presence of a solvent, to obtain the protected tripeptide derivative of general formula (VIII)

(VIII) where

A is an amino protecting group and

R ¹ is the residue of an ester or amide group;

• » • · · • · · · · * ·» • · i* • · · ·

9 9 9 (ii) A deprotection step in which the protected tripeptide derivative of general formula (VIII) prepared in the previous step is deprotected either by catalytic hydrogenation or by acidic treatment, depending on the amino protecting group used, to obtain a tripeptide derivative of general formula (9)

where

R ¹ is the residue of an ester or amide group;

Step 3 (i) A condensation step in which the activated tyrosine derivative of general formula (X),

(X)

9 » ·

• 9 9 · · » · • · · · · · · · • · 9 9 9 9

9 * · · · · « · *99* •999999 99 ·· where

A is an amino protecting group,

R is the residual group of the activating agent and

R ² is H or a benzyl-like group;

previously prepared by a pre-activation step or formed in situ, is reacted with the tripeptide derivative of general formula (9) , which is the product of step 2, in the presence of a solvent, to form a protected tetrapeptide derivative of general formula (XI)

where

A is an amino protecting group and

R ¹ is the residue of an ester or amide group;

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

I

9 9

9 999 4 • ·

9 9999 • 9

OH

F

AND

NH ₂ (XII) (iii) A deprotection step in which the protected tetrapeptide derivative of general formula (XII) is deprotected either by catalytic hydrogenation, basic or acidic treatment, depending on the amino protecting group used, to give the final tetrapeptide of general formula (I), which may optionally be converted to a salt of the tetrapeptide of general formula (I).

The peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH2 (I) can be reacted, if desired, with a pharmaceutically acceptable acid such as AcOH, H3PO4, citric acid, lactic acid and HCl. According to the present invention, the use of HCl is preferred. Possible salts that can be used are described in S.M. Berge, L.D. Bighley and D.C. Monkhouse, J. Pharmaceut. Sci., 66, 1-19, (1977).

The method according to the present invention described above can therefore be schematically described as a method consisting of the following steps:

Step 1 (i) Condensation step, (ii) Deprotection step,

Step 2 (i) Condensation step,

(ii) Step to cancel protection, 8 4 > · 4 4 4 4 4 4 4« • 4 · 4 4 4 · ♦ · · • 4 4 4 4 4 4 4 4 4 • 4 4 4 4 4 4 <444 « 4 4 4 4 4 4 9 4 4 444*4 44 4 4 Step 3 (i) Condensation step, (ii) A possible transformational step, (iii)Step of cancellation of protection. Possible transformation step described above in step 3(ii) can to be carried out instead in step 1 or 2 after condensation step (i). Preferably transformational step performs after

condensation step (i) in step 1. A preferred route of carrying out the process of the present invention could therefore be schematically described as consisting of the following steps:

Step 1 (i) Condensation step, (ii) Optional transformation step, (iii) Deprotection step,

Step 2 (i) Condensation step, (ii) Deprotection step,

Step 3 (i) Condensation step, (ii) Deprotection step.

The protecting group of the N-amino group can be selected from any protecting group suitable for peptide synthesis, such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl, often abbreviated to Z-, to mention two possible amino protecting groups. However, the use of benzyloxycarbonyl is particularly preferred for the present synthesis, as it is «» «

B » · • · · *

Β Β ΒΒΒΒΒΒ * • · · • · * * < » · » · · ·»· · » 9 9 9 • · Β β · · • ΒΒΒΒΒΒΒ • · ·. Β · « »···♦· · Β ·« easily removed by catalytic hydrogenation, and in contrast to the Boc protecting group, does not require neutralization of the liberated amine. Suitable protecting groups for amino groups and carboxyl groups which can be used according to the present invention will be appreciated by those skilled in the art. References are made to J. Meienhofer in The Peptides, vol.l, ed.: E. Gross & J. Meienhofer, Academie Press lne., London, pp.264 to 309, 1979; The peptides, vol. 1 to 9, E. Gross & J. Meienhofer, ed., Academie Press lne., London, 1979 to 1987; Houben-Weyl, Methoden der organischen Chemie, E. Muller, ed., vol. 15, parts I to II, Thieme, Stuttgart 1974; M. Bodanszky, Principles of peptide Synthesis, Springer Verlag, Berlin, 1984.

The pre-activation step preceding steps 1 to 3, or the formation of an activated derivative of an activated amino acid in situ, can be achieved by reacting an amino acid, wherein the amino function is protected by a suitable protecting group, such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z), which are either commercially available or obtainable by techniques known in the art, with an activating agent in the presence of a tertiary amine and an organic solvent, to obtain an activated amino acid derivative. A schematic example of a pre-activation step is shown below

(II) amino acid derivative

activated amino acid derivative

4 4 · 4» 44 44

4* 4 4 4 4-4 4 44 4 • 444 · 4 4444

444444 4 4 V· 44 4

444 4* 4444

4 4444444 · · ·· where

A is an amino protecting group, and

R is the residual group of the activating agent.

For the condensation step described above in steps 1 to 3, a variety of strong solvents can be used, as long as the amino moiety is substantially soluble and available for immediate reaction with the activated peptide derivative. Examples of suitable solvents for the condensation step are acetone, acetonitrile, N,N-dimethylformamide (DMF), N-methylpyrrolidine (NMP) and EtOAc (ethyl acetate), or mixtures thereof.

When used herein, the term benzyl-like group means any substituted or unsubstituted benzyl group that is hydrogenated under similar reaction conditions as a benzylcarbonyl group.

The term pF means a fluorine substituent in the para position.

Possible, as well as preferred reagents and reaction conditions in each step are as follows.

Pre-activation step

Suitable activating agents may be selected from those that form any of the commonly used activated derivatives of amino acids including, but not limited to, carbodiimides, activated esters, azides or anhydrides. Isobutyl chloroformate (iBuOCOCl) is a preferred activating agent. When the activating agent is isobutyl chloroformate (iBuOCOCl), the activated peptide derivative will have the following structure, shown for example on D-alanine φ · · φ φφφ φ φφφφφ

A

The tertiary amine may be selected from any tertiary amine. However, NMM (N-methylmorpholine), diisopropylethylamine is preferred. In addition, a secondary amine which is sterically hindered may also be used.

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

Condensation step; Step l(i), step 2(i) and step 3(i)

The solvent used in the condensation step can be selected from a variety of solvents as long as the amine component is substantially soluble and available for immediate reaction with the activated amino acid residue. Examples of suitable solvents for the condensation steps are acetone, acetonitrile, DMF, N-methylpyrrolidone (NMP) and EtOAc, or mixtures thereof, of which acetone, EtOAc, NMP and DMF are preferred.

Any temperature at which the activated amino acid derivative is not degraded or the reaction rate is not too slow can be used. The preferred range is from 0 °C to -20 °C, and the most preferred range is from -5 °C to -15 °C. The rate of addition of the reactants is adjusted to maintain the preferred temperature.

• ·

4 4 4 4 · 4 4 4 4 • «44444 · · 4 4 4 4 · • · · 44 4444

4 4 4444444 · 4 44

Deprotection step; Step 1(ii), Step 2(ii) and Step 3(ii)

The catalyst used for the hydrogenation can be selected from a wide variety of catalysts, as will be appreciated by those skilled in the art. However, 5% Pd on carbon is preferred. Any solvent capable of dissolving at least some of the peptides can be used, except ketones, such as acetone, or those solvents that poison the catalyst or react with the reaction components. The choice of solvent will be appreciated by those skilled in the art. The preferred solvent is DMF.

The optional step 3(ii) is only required when the hindered tetrapeptide derivative of general formula (XI) prepared in step 3(i) is an ester. Thus, when phenylalanine amide is used, step 3(ii) will be eliminated from the synthetic process.

When 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 hindered amino acid, preferably benzyloxycarbonyl is used as the Na-amino-protecting group, is activated as a mixed anhydride with isobutoxycarbonyl chloride, or a similar type of chloroformate. The method used is based on the general method described by J. Meierhofer in The Peptides, Vol. I, eds.: E. Gross & J. Meienhofer, Academie Press, lne, London, pp. 264-309, 1979.

The inventors 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 and 2 minutes at -15’C. The inventors have also found that strictly anhydrous conditions are not necessary as

• · · · 1 • · otherwise recommended. This allows the present process to be used for large-scale production, where the longer reaction time allows for a safe and reproducible process. Stereochemical integrity and chemical purity are maintained, as is the yield, which is usually greater than 90%. The mixed anhydride formed is condensed with slow addition of the amine component (amino acid/amide or peptide ester) at about 0 to -15 ’C and then the reaction mixture is allowed to reach 20 to 30 ’C over about 30 to 60 minutes or longer, before crystallization of the product is initiated directly from the reaction mixture.

The inventors have also surprisingly found that when the present method is used, if a suitably selected combination of solvents is used, a separation washing step prior to crystallization is not necessary. DMF, acetonitrile, EtOAc and water are preferably used. Controlled crystallization not only achieves excellent purification, but also reduces filtration or centrifugation times as well as drying times when drying of intermediates is required. One important factor is the formation of sufficiently large crystals with a relatively narrow size distribution to avoid blocking the filter medium or centrifuge membrane. It is very common, especially for peptides, to form gels or amorphous crystals that are almost impossible to filter.

Tripeptide derivative of general formula (9)

O (9) ··· · · · · · · · · • · · · · · · · · · • ······ · · · · ·« # ··· · · φ · · · tt * ··««··φ «· ·Φ where

R ¹ is the residue of an ester or amide group, is a useful intermediate for the preparation of the target compound of general formula (I).

Detailed description of the invention

The preparation of the peptides H-Tyr-D-Ala-Phe(pF)-Phe-NH2 or its pharmaceutically acceptable salts will now be described in more detail in the following examples, which are not intended to be limiting of the invention. In addition, Scheme 1 below provides a detailed overview of the synthetic routes followed for the preparation of peptides of general formula (I) using a phenylalanine derivative according to the present invention, wherein the carboxyl group is protected as an ester. Scheme 2 below provides a detailed overview of the synthetic route followed for the preparation of peptides of formula (I) using a phenylalanine derivative according to the present invention, wherein the carboxyl group is protected as an amide. The numbers of the compounds referred to in the detailed description of the synthesis in the examples below correspond to the numbering in Schemes 1 and 2.

· • ·

Scheme 1 · ·

9 9

9 9 9

999999

9 9

9 9

99 9

H-Phe(pF)-Phe-NHj • ······ • * · « · · ······· ·· 9 9 ··

Scheme 1 (continued) iBuOCOCI (ii)NMM

ACN

-10°C 20 minutes

(7) Z-D-Ala-OCOOiBu u

Z-D-Ala (a) -10 °C 30 min/ (b) 25 ’C 1 h /

ACN + DMF/ H-Phe(pF)-Phe-NH ₂ Z (5)

«Λ

CH, (8)

Z-D-Ala-Phe(pF)-Phe-NH2

(9)

HD-Ala-Phe(pF)-Phe-NH _z

• · ·

Scheme 1 (continued)

OH

HCl/H2O

Acetone

MIBK

H-Tyr-D-Ala-Phe(pF)-Phe-NH ₂ x HCl f>V2m- JMZ,

C-S.

JUDr. Pavel ZELENÝ attorney *

- Halkova 2, Prague 2

Examples of embodiments of the invention

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

Molar scale 3.5

Z-Phe(pF) (compound 1) (1 equivalent) is first dissolved in acetone (4.7 liters/mol) and cooled before the addition of IBK (0.9 to 1.2 equiv.) (1 equivalent actually). The reaction is then controlled by the rate of addition (about 20 minutes) of NMM (N-methylmorpholine) (0.9 to 1.2 equiv.) (1 equivalent actually). A reaction temperature between 0 and -15°C (-9°C to -14°C actually) is recommended, at which the reaction occurs immediately after the addition of NMM, which protects the mixed anhydride from rapid decomposition.

Η-Phe-OMe x HCl (0.9 to 1.3 eq.) (1.04 eq. actual) is meanwhile mixed with acetone (2.6 liter/mol), neutralized with NMM (0.9 to 1.5 eq.) (1.04 eq. actual) and cooled to a temperature of 0 to -20 ’C (around -10 ’C actual). This thin suspension is added after activation is complete at a rate that maintains the reaction temperature around -10 ’C (from -8 'C to -13 ’C actual) (about 30 minutes). EtOAc (4 liter/mol) is then added and the organic phase is washed with water (2x2 liter/mol), followed by azeotropic distillation from ACN and dissolution in methanol, before the next step, which results in a 92% purity of the methanol suspension.

Shift_Multiplicity_Total H

8.5

7.49

7.31 py fy)oo- 33 (J. MM)

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)

Molar scale 2.3 to 3.3

Ammonia is added to the solution of compound 3 prepared in the previous step (about 8 liters of MeOH/mol) at a pressure between 1-5.10 ² kPa at 15 to 40 °C for more than 5 hours or until the reaction is close to completion (actual conversion 99%) and the ammonia evaporates.

After completion of the reaction, it is cooled by centrifugation, vacuumed at 20 to 50°C and the reaction mixture is evaporated before filtration or The product is washed with MeOH and dried under 100°C.

The yield calculated from compound 1 (Z-Phe(pF)) was 74% at 100% purity.

Shift Multiplicity Total 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

• · · • ·· ·» and· • · · · · · · · • · · · · · • · · · · · · • · · · · · ······· · · ··

3.00m 1

2.88m 1

2.66m 1

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

Molar scale 4.3

Compound 4 prepared in the previous step is mixed with DMF (4.2 liter/mol) and Pd/C catalyst (actual Pd content is 5%) and (0.2 to 10% w/w/LEF-581)(7% actual) are added and the resulting mixture is hydrogenated for more than 0.5 hours (actually 1.2 hours) at 25 °C and 3.10 ² kPa H2. The reaction mixture is then filtered and cooled to about -15 °C before the next step. 99.6% purity of the substance in solution is achieved.

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

Molar scale 4.4

Z-D-Ala (compound 6) (1 eq.) was dissolved in acetonitrile (ACN) (2.3 liter/mol) and cooled before addition of IBK (0.9 to 1.2 eq.) (1 eq. used). NMM (0.9 to 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 liters) was then added over about 30 minutes, maintaining the temperature at around -10 °C (-8 C to -14 °C in reality). After completion of the condensation step, the product was crystallized from the reaction mixture by slow addition of water (3x3.6 liter/mol + 1x1.3 liter/mol) with a waiting time of about 25 minutes between each addition, while

·· initial temperature around 30 °C and final temperature around 20 °C.

They crystallized then they can centrifuge and wash with water/acetonitrile (4:1) before before they were vacuum dried at a temperature of 20 to 50 ’C. The yield was 90% and 99.5% purity. Shift Multiplicity Total 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 liter/mol) and Pd/C catalyst (actual content 5% Pd) was added and (0.2 to 10% w/w of compound 3) (7% actually) was added and the resulting mixture was hydrogenated for more than 0.5 h (1.2 h actually) at 25 °C and 3x10 ² kPa H2 pressure. The reaction mixture was then filtered and cooled to about -15 °C before the next step. The purity was 97%. The conversion of the starting material was >98%.

Step 3 • 4 • · • · · • · · · • ····· · ♦ 4 4

4 ··· ·«*·

99 • 9 9 4 • · 4 4

9 9 9 • · 9 9

99 (i) Preparation of Z-Tyr-D-Ala-Phe(pF)-Phe-NH ₂ (compound 12 in Scheme 1)

Molar scale 4.1

This condensation uses the same method as the previous two. Z-Tyr (compound 10) (1 eq.) is dissolved in ACN (2.3 liter/mol) and cooled before adding IBK (0.9 to 1.2 eq.). NMM (0.9 to 1.2 eq.) is then added, in the same manner as described above under compound 3. The solution of compound 3 from the previous step is then added over about 30 minutes, maintaining the temperature around -10 °C (actually from -7 'C to -14 'C). After completion, the condensation product is crystallized from the reaction mixture by slow addition of acetonitrile and water (2 liter/mol ACN + 0.3 liter/mol 25% NH3 in H2O, time 2 h., add 1.5 liter/mol ACN: _H2O (1:1) , time 1 h., increase the temperature to 35 'C, add seed crystals (about 1% weight/weight) , time 1 h., add 1.3 liter/mol ACN:H2O(1:1) , time 1 h., add 1.2 liter/mol H2O with a hold of 0.5 h. at 35 ”C, add 1.2 liter/mol _H2O with a hold of 2 h. at 20 °C, add 1.2 liter/mol _H2O with a hold of 1 h. at 20 'C and add 1.2 liter/mol _H2O with a hold of 0.5 h at 20°C. The product is centrifuged and washed first with water and then with acetonitrile before vacuum drying at 20-50°C.

A yield of 81% was achieved, based on compound 8.

Product purity 98.4%.

Shift

9.18

8.18 8.11 8.05

Multiplicity with

d d

d

Total H

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

(ix) Preparation of H-Tyr-D-Ala-Phe(pP)-Phe-NH ₂ (compound X in Scheme 1)

Molar scale 3.1 and 3.2

Compound 4 is mixed with DMF (2 to 2.6 liter/mol actual) and 5% Pd/C (actual content) catalyst (0.2 to 10% w/w/compound 3) (6 to 7% actual) is added and the resulting mixture is hydrogenated for over 0.5 h. (1 to 2 h. actual) at 20 to 40 °C (20 to 25 °C actual) and a H ₂ pressure of 3x10 ² kPa. The reaction mixture is then filtered to remove Pd/C before crystallizing the product by adding EtOAc until all the material has crystallized (usually 10 liter/mol). The solid is collected by filtration or centrifugation and washed with EtOAc before vacuum drying at 20 to 50 °C.

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

Molar scale 2.1

« · · · ·

The free base compound I is dissolved in a mixture of water and acetone with the addition of 1 equivalent of HCl and clarified by filtration (146 g/mol 25% HCl/H2O, 2 liters acetone/mol in the actual operation). The salt has limited solubility in acetone, so the filter is washed once with an additional amount of acetone/water (95:5) (0.5 liter/mol). Crystallization is initiated by slowly adding acetone (3.4 liter/mol) at high stirring speed and then adding at least 1% w/w of seed crystals. After 30 minutes, the first amount of MIBK (3 liter/mol) is slowly added and allowed to stir slowly until the batch thickens noticeably. MIBK (3 liter/mol) is added 3 times at intervals of 30 to 60 minutes while maintaining the internal temperature of the reactor at about 20 °C. The solid phase is then separated by centrifugation or filtration and washed with MIBK before vacuum drying, at 20 to 50°C, for more than 16 hours until the solvent contents are lower than stated in the related descriptions.

• »

&

• · 9

Scheme 2

Z-Phe(pF) IBuOCOCI

CH,

NMM

EtOAc

Z-Phe(pF)-OCOOiBu

Phe-NH ₂ x HCl

NMM

-10’C 10 minutes

EtOAc + DMF

Z-Phe(pF)-Phe-NH ₂ (13)

Phe(pF)-Phe-NH ₂ (14) «·»

Scheme 2 (continued)

(15) • ·

* • f it · * • Φ · 9 » • » Φ φ · • < « · φ Φ

Φ Φ φ Φ Φ • · · Α Φ» Φ <

Scheme 2 (continued)

i) ÍBuOCOCI ii) NMM

MeCN

-10’C

Z-Tyre

Z-Tyr-OCOOiBu

HD-Ala-Phe(pF)-Phe-NH ₂ (16)

i) -10'C, 10 min ii) 25°C, 1h MeCN

5% Pd/C DMF

H-Tyr-D-Ala-Phe(F)-Phe-NHj

0)

HCI

IN

Acetone 3% _H2O MIBK

Z-Tyr-D-Ala-Phe(pF)-Phe-NH ₂ x HCl ·' • φ

• · · • « · • φ · · φ · φ φ φ φ φ φ φ · ·>

«

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

Molar scale 6.7

Z-Phe(pF) (1 eq.) is first dissolved in acetonitrile (EtOAc) (1.7 liter/mol) and cooled before the addition of isobutyl chloroformate (0.9 to 1.2 eq.) (1.05 eq. actual). The reaction is then monitored by the rate of addition (around 20 minutes, 15 minutes actual), of N-methylmorpholine (0.9 to 2.0 eq.) (1.4 eq. actual). A reaction temperature between 0 and -15 °C (-8 °C to -11 °C actual) is recommended, with the reaction occurring immediately after the addition of N-methylmorpholine to prevent the mixed anhydride from decomposing too quickly.

H-Phe-NH ₂ xHCl (0.9 to 1.3 eq., 1.04 eq. actual) is meanwhile dissolved in DMF (4.0 liter/mol), neutralized with N-methylmorpholine (0.9 to 1.5 eq., 1.04 eq. actual) and cooled to 0 to -20 °C (around -10 ^° C actual). This thin suspension is added at a rate that maintains the temperature around -10 °C (from -6 °C to -13 °C actual) for about 15 minutes (8 minutes actual) until activation is complete.

After the condensation is complete, the product is crystallized from the reaction mixture by slow addition of 50% ethanol in water (3.6 liters/mol). After waiting 30 minutes, a total of 2.85 liters/mol of water is added in three portions, with about 25-minute intervals between each addition, at a temperature of about 20 °C. The crystals can be filtered off or centrifuged after about 17 hours and washed with a 50% mixture of ethanol and water and then with several portions of acetonitrile before vacuum drying at a temperature of 20 to 60 °C.

The yield was 90% and the purity was 99.9%.

• · 4 » ·* » · 4 » • · · · 4 Λ · · 4 · 4 ···· * · · · » · • »···»* · 9. 9 9 9 9 * • 9 9 9 9 9 9 9

999 9999 99 99

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

Molar scale 6.7

The Z-Phe(pF)-Phe-NH2 prepared in the previous step was mixed with DMF (3.5 liters/mol) and a Pd/C catalyst (actual content 5% Pd) (0.2 to 10% w/w/LEF-582) (5% actual) was added and the resulting mixture was hydrogenated for more than 0.5 hours (1.3 hours actual) at 25 to 30 °C and a H2 pressure of 3x10 ² kPa. The reaction mixture was then filtered and cooled to about -15 °C before the next step. The purity of the solution was 99.6% and >99% conversion of the starting material.

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

Molar scale 5.9

Z-D-Ala-OH (compound X) (1.03 eq. used) was dissolved in acetonitrile (1.9 liter/mol) and cooled before addition of isobutyl chloroformate (0.9 to 1.2 eq., 1.07 eq. used) followed by addition of N-methylmorpholine (0.9 to 2.0 eq., 1.2 eq. used) in the same manner as above for the preparation of ZPhe(pF)-Phe-NH2. The H-Phe(pF)-Phe-Nh2 solution (25 liters) was then added over about 15 minutes (8 minutes actual) while maintaining the temperature at around -10°C (-8°C to -11°C actual). After the condensation is complete, the product is crystallized from the reaction mixture by slow addition of water (4x1.9 liter/mol) with a waiting time of about 15 to 30 minutes between each addition at about 20 °C. The crystals can then be filtered off or centrifuged and washed with water/acetonitrile (4:1) followed by acetonitrile before optional vacuum drying at 20 to 60 °C. The yield calculated from Z-Phe(pF)-Phe-NH2 was 93.8% and the purity was 99.6%.

• 99 9

9 9 9 9

9 9 9 9

9 9 9 9 9 « 9 • 99 9

9,999

9 9 * 9 9

9 9 9 9 9

9 9 9 9

9 9 9 9 9 • 9 · * 9

9999 99 99

Preparation of HD-Ala-Phe(pF)-Phe-NH ₂ (compound 16 in Scheme 2)

Molar scale 5.5

ZD-Ala-Phe(pF)-Phe-NH2 prepared in the previous step was mixed with DMF (2.9 liter/mol) and Pd/C catalyst (actual content 5% Pd) (0.2 to 10% w/w/compound 3) (5% actual) was added and the resulting mixture was hydrogenated for more than 0.5 hours (3 h actual) at 25 to 35 °C and a H2 pressure of about 3x10 ² kPa. The reaction mixture was then filtered and cooled to about -15 °C before the next step. The purity was 99.4%, the conversion of the starting material was >99%.

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

Molar scale 5.5

This condensation reaction uses a similar method to the previous two. Z-Tyr (compound X) (1.05 eq.) is dissolved in MeCN (1/9 liter/mol) and cooled before the addition of isobutyl chloroformate (0.9 to 1.2 eq., 1.05 actual). N-methylmorpholine (0.2 to 2.0 eq., 1.3 actual) is then added in a similar manner to that described above for the preparation of Z-Phe(pF)Phe-NH ₂ . The HD-Ala-Phe(pF)-Phe-NH ₂ solution from the previous step is then added over about 20 minutes (6 minutes actual), while maintaining the temperature at around -10 °C (-8 °C to -9 °C actual). Upon completion, the condensation product is crystallized from the reaction mixture at about 20 to 45 °C by slow addition of acetonitrile and water (3.4 liter/mol MeCN +0.9 liter/mol 15% NH3 in

H2O hold for 5 minutes and seeds, hold for 4 to 24 h., then add a total of 13.9 liter/mol _H2O in 4 portions with a hold of about 30 minutes • · · · «9 » · > · · · · · · • * · · · 9 • · · 9 9 • « · · » · f « or longer between each addition. Filter or centrifuge and wash first with water and then with MeCN before optional vacuum drying at 20 to 60 'C.

Yield 87.7%, calculated from ZD-Ala-Phe(pF)-Phe-NH ₂ and purity 95.1%.

Applicants have found that the yield and purity are increased by heating the reaction to about 60°C with the addition of ammonia to a pH of about 9 for 2 hours. This converts most of the impurity, ZTyr(O-iso-butylcarbonyl))-D-Ala-Phe(pF)-Phe-NH ₂ to product.

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

Molar scale 5.4

Z-Tyr-D-Ala-Phe(pF)-Phe-NH ₂ was mixed with DMF (2.6 liter/mol actual) and 5% Pd/C (actual content) catalyst (0.2 to 10% w/w/compound 3) (6.4% actual) was added and the resulting mixture was hydrogenated for over 0.5 h (1.8 h actual) at 20 to 40 °C (20 to 25 °C actual) and a H ₂ pressure of about 3x10 ² kPa. The reaction mixture was then filtered to remove Pd/C, before crystallizing the product, adding ETOAc until all the material had crystallized (usually about 14 liter/mol). The solid phase was separated by filtration or centrifugation and washed with EtOAc before vacuum drying at 20 to 50 °C. The purity was 96.7%, starting material conversion >99%.

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

Molar scale 4.6 φ· φ 4 4 · · 4 «φ φ 4

ΦΦ · φ φ φ φ φ φ

Φ · ·? 4 Φ Φ * • · * Φ φ Φ

4 4 4 4 4 φ * 44

The free base compound H-Tyr-D-Ala-Phe(pF)-Phe-NKh is dissolved in a mixture of water and acetone with the addition of 1 equivalent of HCl and filtered to clarify (146 g/mol 25% HCl/H ₂ O, 2 liters Acetone/mol in the actual embodiment). The salt is almost insoluble in acetone and therefore the filter is washed once with an additional amount of acetone/water (95:5) (0.5 liter/mol). Crystallization is initiated by slowly adding acetone (3.4 liter/mol) at high stirring speed and then about 1% w/w seed crystals are added. After 30 minutes, the first amount of MIBK (3 liter/mol) is slowly added and left under slow stirring until the batch thickens noticeably. MIBK (3 liters/mol) was added 3 times at 30-60 minute intervals while maintaining the internal temperature of the reactor at about 20°C. The solid particles were then separated by centrifugation or filtration and washed with MIBK before vacuum drying at 20-50°C for more than 16 hours or until the solvent content was lower than that stated in the related descriptions. The yield was 95.8% and the purity was 99.8%.

Reprocessing

The product that does not meet the specifications for drug substances can be recrystallized in the same manner as described above for the crystallization of compound I, but without the addition of HCl.

Assignment of the NMR spectrum for H-Tyr-D-Ala-Phe (P) -Phe-NH ₂ xHCl, i.e. for compound I in its hydrochloride form

The NMR spectrum was obtained on a solution of 36 mg of compound in approximately 0.7 mL of DMSO-d6 (99.95 atom % D) at 27.0 °C on a Varian UNITY plus 400 MHz instrument. The reference chemical shift for the proton spectrum was the mean peak of the DMSO-d6 multiplet obtained as 2.49 ppm. The reference for the carbon spectrum was the mean peak of the DMSO-d6 multiplet obtained as 39.5 ppm.

* · ·

9 9 9

9 9 9

9 9 9 » · »

9 9 9

(AND)

The atom numbering used in the assignment is arbitrary and corresponds to the above figure.

Proton spectrum

The one-dimensional proton spectrum allows group assignments of alpha protons (3.9 to 4.4 ppm), benzyl-CH2 (2.6 to 3.1 ppm), amide-NH and phenol-OH (8.2 to 8.5 ppm) as well as specific assignments for Ala-CH3 (I4-CH3) (0.74 ppm).

The two-dimensional DQFCOSY spectrum allows group assignment of the spin system (alpha, beta and NH protons) in each amino acid residue, and group assignment of the aryl protons in each aromatic ring. All protons in the Ala residue can also be specifically assigned.

Carbon spectrum

The one-dimensional carbon spectrum allows group assignment of alpha carbons, benzyl-CH2, carbonyl and aryl carbons and of course specific assignment of C-14. The APT spectrum allows ♦ · » · · · « » · · · • * · · B « • · · « · * 9 · · · ·t 9· for each carbon. The splitting of the lines due to specific assignment of carbons in « · » B • » ···· » · · · · CH-multiplicity assignment allows C-F condensation to the fluoroaromatic ring.

Two-dimensional heterocorrelated spectrum

A two-dimensional carbon-proton correlation spectrum (HMQC) gives a correlation between protonated carbons and all directly bound protons. All protonated carbons in the Ala residue can be specifically assigned.

The two-dimensional carbon-proton multiple bond correlation (HMBC) spectrum gives the correlation between carbons and protons located 2 to 3 bonds apart. This allows the assignment of amino acid sequences via the alpha hydrogens and carbonyl groups of the adjacent amino acid residue (3-bond correlation), as well as via the NH and carbonyl groups of the adjacent amino acid residue (2-bond correlation). Similarly, the 2- and 3-bond correlations between benzyl-CH2 and aryl carbons as well as between aryl protons and benzyl-CH2 allow the specific assignment of aryl protons and carbons of individual aromatic amino acids.

In this way, all (non-exchangeable) protons and carbons in all 4 residues of an amino acid can be specifically assigned in a reliable manner.

Table 1

Proton assignment

Chemical Shift(ppm)_Total H_Multiplicity Assignment

9.4

8.48

OH

NH

4 4 « 4 4 * · 4 · • · · · · 4 4 4 · 4 ·

4 4 4 4 « 4444

4 44 44 * · 444444

444 * 4 4444 > 4444444 44 44

8.37 8.35 1 1 d d 1 NH 8 NH 8.26 2 with 7.53 1 with 7.28 2 m 18 H, 19 H 7.26 2 m 21 H, 22 H 7.22 2 m 6 AM, 7 PM 7.18 1 m 24 hours 7.12 1 with 7.04 2 m 10 AM, 11 AM 6, 98 2 m 26th, 27th 6.68 2 m 28th, 29th 4.43 1 m 1 H 4.39 1 m 8 H 4.26 1 m 9 H 3.96 1 m 20 H 3.39 HDO 3.02 1 m 12 Hb 2.99 1 m 2 Hb 2, 87 2 m 23 Ha,23 Hb 2.85 1 m 12 Ha 2.67 1 d 2 Ha 0.74 3 d 14 H

♦ · ·» · • · • · a · · a « · · ♦ · ··

B 9 9 9 • 9 9 9

9 9 9 ·· B·

Table 2

Carbon assignment

Chemical shift(ppm) Multiplicity Assignment Jc-p 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 21.12 126.30 d 24 124.73 with 25 115.16 d 28.29 114.63 d 10.11 21.3 Hz 114.41 d 10.11 21.3 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 18.45 2 14

Claims (14)

PATENTOVÉ Claims A process of formula (I) for preparing a tetrapeptide H-Tyr-D-Ala-Phe (pF) -Phe-NH ₂ or a pharmaceutically acceptable salt thereof, comprising the following steps: (i) a condensation step wherein the activated tyrosine derivative of general formula (X) (X) in which A is an amino protecting group, R 2 is a residual activating agent group and R ² is H or a benzyl-like group; φ · φ φ φ · · · φ φ φ φ φ · · · · φ φ φ φ φ φ φ φ φ φ · · φ φ · · · · · · Φ φ is reacted with a tripeptide derivative of general formula (9) in which: R ¹ is an ester or amide residue; in the presence of a solvent under standard conditions to provide a protected tetrapeptide derivative of the general formula (XI) in which: A is an amino protecting group and R ¹ is an ester or amide residue; (ii) a deprotection step wherein the protected tetrapeptide derivative of formula (XII) is deprotected by either catalytic hydrogenation, basic or acid treatment under standard conditions in the presence of a solvent to obtain a compound of formula (I). 9 9 . 9 99999 9 9 9 9 9 9,999 · 9 9 9 9 9 9 9 • · · · 9 The method of claim 1, comprising the additional steps of: (i) a condensation step, wherein the activated alanine derivative of formula (VII) H, C O * AND R O. (VII) wherein A is an amino protecting group and R is a residual group of activating agent; is reacted with a dipeptide derivative of general formula (5) in which: R ¹ is a residue of an ester or amide group, in the presence of a solvent under standard conditions, to provide a protected tripeptide derivative of general formula (VIII) in which: A is an amino protecting group and R ¹ is an ester or amide residue; (ii) a deprotection step wherein the protected tripeptide derivative of formula (VIII), prepared in the previous step, is deprotected by either catalytic hydrogenation, basic or acid treatment, in the presence of a solvent under standard conditions, to yield the tripeptide derivative of formula (9) ) in which R ¹ is a residue of an ester or amide group. The method of claim 2, comprising the additional steps of: 4 4 · 4 4 4 4 4 4 5 4 44 * 444 4 4 4 4 4 44 4 4444444 ♦ · (i) a condensation step, a fluorophenylalanine derivative in which the general formula is activated by p (III), (III) in which A is an amino protecting group and R ¹ is an ester or amide residue; is reacted with the amino group of phenylalanine, the carboxyl group being protected as an ester or amide, in the presence of a solvent under standard conditions, wherein to obtain a protected dipeptide derivative of the general formula (IV) in which: • · · · · · · <<<· · A is an amino protecting group and R ¹ is an ester or amide residue; (ii) a deprotection step wherein the protected dipeptide derivative of formula (IV), prepared in the previous step, is deprotected by either catalytic hydrogenation, basic or acid treatment, in the presence of a solvent under standard conditions, to obtain the dipeptide derivative of formula (5) ), in which R ¹ is a residue of an ester or amide group. The process according to claims 1 to 3, characterized in that one of the condensation steps (i) is followed by an optional transformation step which is performed when the carboxyl group of the amino acid derivative is protected as an ester derivative, wherein the ester compound is reacted with ammonia in the organic alcohol. Method according to claims 1 to 3, characterized in that the method according to claims 1 to 3 is 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. 6. The process of claim 5 wherein the activating agent is isobutyl chloroformate. The process according to any one of claims 1 to 6, wherein the solvent used in at least one of the condensation steps is acetone, acetonitrile, NMP, DMF or EtOAc. Process according to claims 1 to 3, characterized in that the solvent in the condensation step is DMF. Method according to any one of claims 1 to 3, characterized in that the deprotection step is carried out using 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 of from 0 ° C to -20 ° C. The method of claim 10, wherein the temperature is from -5 ° C to -15 ° C. A peptide of formula I prepared according to the method of claim 1. The peptide of claim 12, which is in the form of the hydrochloride salt. A peptide derivative of the general formula (9) in which R ¹ is a residue of an ester or amide group.