AU2968899A - Process for the preparation of a tetrapeptide - Google Patents

Description

WO 99/47548 PCT/SE99/00414 1 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, 5 more specifically the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe- NH2, or a pharmaceutically acceptable salt thereof. In further aspects, the present invention also relates to new intermediates used in process. 10 BACKGROUND AND PRIOR ART The present invention relates to a new process for the preparation of the peptide H-Tyr-D Ala-Phe(pF)-Phe-NH 2 , or a pharmaceutically acceptable salt thereof. WO 97/07129 discloses a process for producing inter alia the peptide 15 H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 . The peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 is also disclosed in WO 97/07130. Said peptide exhibits peripheral analgesic activity and selectivity for the g subtype of opioid receptors, and is suitable in particularly in pain therapy. Furthermore, it is prepared using solid phase synthesis according to procedures well established in the art. The drawback with solid phase synthesis, which is a common 20 and well established method for peptide synthesis, is that it is difficult to use for large scale production, in addition to being expensive. The process of the present invention provides the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe

NH

2 with a higher purity, in a more cost effective and environmentally better way 25 compared to methods known in the art. Furthermore, the process of the present application provides the product in a higher yield. Thus, the object of the present invention is to provide a novel process suitable for use in large scale synthesis. A further object of the present invention is to provide a process 30 containing as few reaction steps as possible.

WO 99/47548 PCT/SE99/00414 2 OUTLINE OF THE INVENTION The present invention provides a new process for large scale preparation of the peptide H Tyr-D-Ala-Phe(pF)-Phe-NH 2 , which is a peptide of the formula (I) 5 OH F o o NH : N NH H 2 0 CH H 0 or a pharmaceutically acceptable salt thereof. The process according to the present invention for preparing a compound of the formula (I) 10 above, comprises the following reaction steps; Step 1 (i) A coupling step wherein an activated p-fluorophenylalanine derivative (III), F A R I) 5 15 A-N wherein A is an amino protecting group, and R is an activating agent residue group; WO 99/47548 PCT/SE99/00414 3 previously prepared by a pre-activation step or generated in situ, is reacted with the amino group of phenylalanine, wherein the carboxyl group is protected as an ester or amide, i.e. a compound of the formula Phe-R 1 , wherein R 1 is the ester or amide residue group, in the presence of a solvent, providing a protected dipeptide derivative (IV) 5 F 0 A , R 1 0 (IV) wherein A is an amino protecting group, and R1 10 R is an ester or an amide residue group; (ii) A deprotection step wherein a protected dipeptide derivative (IV) prepared in the previous step, is deprotected by either catalytic hydrogenation, base or acid treatment, depending on the amino protecting group used, providing the dipeptide derivative (5), 15 F O (5) 0 WO 99/47548 PCT/SE99/00414 4 wherein

R

1 is an ester or an amide residue group; Step 2 5 (i) A coupling step wherein an activated alanine derivative (VII),

H

3 Q A.N OR (VII) H O 10 wherein A is an amino protecting group, and R is an activating agent residue group; previously prepared by a pre-activation step or generated in situ, is reacted with the product 15is of step 1, i.e. the dipeptide derivative (5) in the presence of a solvent, providing the protected tripeptide derivative(VIII) F 0 11o AR (VIII) CH 0 3 wherein 20 A is an amino protecting group, and R is an ester or an amide residue group; WO 99/47548 PCT/SE99/00414 5 (ii) A deprotection step wherein a protected tripeptide derivative (VIII) prepared in the previous step, is deprotected either by catalytic hydrogenation or acid treatment, depending on the amino protecting group used, providing the tripeptide derivative (9) F 0 0

H

2 N O R (9) 0 5 ~ C H 3 5 wherein R is an ester or an amide residue group; Step 3 10 (i) A coupling step wherein an activated tyrosine derivative (X),

OR

2 Os* (X) A-N is wherein A is an amino protecting group, WO 99/47548 PCT/SE99/00414 6 R is an activating agent residue group, and

R

2 is H or a benzyl-like group; previously prepared by a pre-activation step or generated in situ, is reacted with the tripeptide derivative (9) and being the product of step 2, in the presence of a solvent, 5 providing the protected tetrapeptide derivative (XI) OH F 0 0 : O O R (XI) 0 0

CH

3 wherein A is an amino protecting group, and 10 R 1 is an ester or an amide residue group; (ii) A optional transformation step performed if the protected tetrapeptide derivative (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, providing the 15 protected dipeptide derivative (XII), OH F 0 0 A-N I O NH ( ( XII) 0 CH

OH

3 WO 99/47548 PCT/SE99/00414 7 (iii) A deprotection step wherein a protected tetrapeptide derivative (XII) is deprotected either by catalytic hydrogenation, base or acid treatment, depending on the amino protecting group used, providing the final tetrapeptide (I), which optionally may be converted to a salt of the tetrapeptide (I). 5 The peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 (I), may if desired be reacted with a pharmaceutically acceptable acid, such as AcOH, H 3

PO

4 , citric acid, lactic acid and HC1. HCI is the preferred acid to use in accordance with the present invention. Possible salts which may be used are described in S. M. Berge, L. D. Bighley and D. C. Monkhouse, J. 1o Pharmaceut. Sci., 66(1977) 1-19. The process according to the present invention described above can therefore schematically be described as comprising the following steps; is Step 1 (i) A coupling step, (ii) A deprotection step, Step 2 20 (i) A coupling step, (ii) A deprotection step Step 3 (i) A coupling step, 25 (ii) An optional transformation step, (iii) A deprotection 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. Preferably the transformation step is 30 performed after the coupling step (i) in step 1. The preferred way of performing the process WO 99/47548 PCT/SE99/00414 8 of the present invention could therefore schematically be described as comprising the following steps; Step 1 5 (i) A coupling step, (ii) An optional transformation step, (iii) A deprotection step, Step 2 to (i) A coupling step, (ii) A deprotection step Step 3 (i) A coupling step, 15 (ii) A deprotection step The Na-amino protecting group may be selected from any protecting group suitable in peptide synthesis, such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl, often abbreviated Z-, just to mention two possible amino protecting groups. However, 20 benzyloxycarbonyl is particularly preferred to use for the present synthesis since it is easily removed by catalytic hydrogenation, and contrary to the protecting group Boc, it does not require neutralization of the liberated amine. Suitable amino and carboxyl protecting groups which may be used in accordance with the present invention will be appreciated by a person skilled in the art. Reference is made to J. Meienhofer in The Peptides, Vol.1, Eds.: 25 E. Gross & J. Meienhofer, Academic Press, Inc, London 1979, pp. 264-309; The peptides, Vol. 1-9, E. Gross & J. Meienhofer, Eds., Academic Press Inc., London, 1979-1987; Houben-Weyl, Methoden der organischen Chemie, E. Muller, ed., Vol. 15, Part I-II, Thieme, Stuttgart 1974; and M. Bodanszky, Principles of peptide Synthesis, Springer Verlag, Berlin 1984. 30 WO 99/47548 PCT/SE99/00414 9 The pre-activation step preceding Step 1-3, or the in situ generation of the activated activated amino acid derivative, is achieved by reacting an amino acid, wherein the amino function has been protected by a suitable protecting group, such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z), which are either commercially available or available by 5 techniques known in the art, with an activating agent in the presence of a tertiary amine and an organic solvent, providing the activated amino acid derivative. A schematic representation of a pre-activation step is shown below F A F S/ F activating agent N__ H OHO O A-N (II) (II) aminoacid derivative activated aminoacid derivative 10 wherein A is an amino protecting group, and R is an activating agent residue group; For the coupling step, in Step 1-3 described above, a variety of powerful solvents may be is used, as long as the amino component is essentially soluble and available for immediate reaction with the activated peptide derivative. Examples of suitable solvents for the coupling step are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP) and EtOAc, or mixtures thereof. 20 As used herein, the term " benzyl-like group " denotes any substituted or un-substituted benzyl group that is hydrogenolyzed under similar reaction conditions as the benzyloxycarbonyl group.

WO 99/47548 PCT/SE99/00414 10 The term "pF" denotes a para-fluoro substituent. Possible as well as preferred reagents and reaction conditions in each step are the following. 5 The pre-activation step Suitable activating agents may be selected from those that generates any of the commonly used activated amino acid derivatives including, but not limited to, carbodiimides, lo activated esters, azide, or anhydrides. Isobutylchloroformiate (iBuOCOCI) is the preferred activating agent. When isobutylchloroformiate (iBuOCOC1) is the activating agent, the activated peptide derivative will have the following structure, exemplified on D-alanine,

H

3 q -iBu A' 0 0 15 The tertiary amine may be selected from any tertiary amine. However, NMM (N-methylmorpholine), di-isopropylethylamine and triethylamine are preferred. Furthermore, a secondary amine which is sterically hindered may also be used. 20 The organic solvent may be any organic solvent known to a person skilled in the art to be suitable in peptide chemistry. However, ethyl acetate, acetonitrile, acetone and tetrahydrofurane are preferred solvents in the pre-activation step. 25 The coupling step; Step 1(i), step 2i) and step 3 (i) The solvent used for the coupling step may be selected from a variety of solvents, as long as the amino component is essentially soluble and available for immediate reaction with the WO 99/47548 PCT/SE99/00414 11 activated amino acid residue. Examples of suitable solvents for the coupling steps are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP) and EtOAc, or mixtures thereof, of which acetone, EtOAc, NMP and DMF are preferred. 5 Any temperature where the activated amino acid derivative is not degraded or the reaction rate is too slow may be used. The preferred range is from OC to -20'C, and particularly preferred is from -5 0 C to -15 0 C. The rate of addition is adjusted so that the preferred temperature is maintained. 10 The deprotection step: Step 1(ii), step 2(ii), and step 3 (iii) The catalyst used for hydrogenation may be selected from a great variety of catalysts as will be appreciated by a person skilled in the art. However 5% Pd on carbon is preferred. Any solvent that can dissolve at least some of the peptide is possible to use except ketones, such Is as acetone, or those solvents that poison the catalyst or react with the components of the reaction. The choice of solvent will be appreciated by a person skilled in the art. DMF is the preferred solvent. The optional Step 3(ii) is only required if the protected tetrapeptide derivative (XI) 20 prepared in step 3(i) is an ester. Thus, if an amide of phenylalanine is used, step 3(ii) will be excluded from the synthetic procedure. If an acid is used for removal of the oc-protecting group, an equivalent molar amount of a base is required to deprotonate the amino group of the peptide derivative. 25 In a preferred embodiment of the present invention the protected amino acid, preferably using Benzyloxycarbonyl- as Na-amino protecting group, is activated as a mixed anhydride with isobutyloxycarbonylchloride, or a similar type of chloroformate. The method employed is based on the general method reviewed by J. Meienhofer in The Peptides, 30 Vol.1, Eds.: E. Gross & J. Meienhofer, Academic Press, Inc, London 1979, pp. 264-309.

WO 99/47548 PCT/SE99/00414 12 We have surprisingly found that the activation time can be extended to at least 30 min at a temperature about 0 - -15oC, contrary to the recommended 1-2 min at -15 0 C. We also found that strictly anhydrous conditions are not necessary as otherwise is recommended. This allows the present method to be used for large scale production where the longer 5 reaction times allow a safe and reproducible process to be carried out. The stereochemical integrity has been completely maintained and the chemical purity as well as yields have been typically over 90%. The generated mixed anhydride is coupled with the slow addition of the amino component (amino acid/ peptide amide or ester) at about 0 - -15 0 C and the reaction mixture is then allowed to reach 20-30'C in about 30-60 min. or longer before o10 crystallization of the product is initiated directly from the reaction mixture. We have also surprisingly found that when using the present method, if appropriately selected solvent combinations is used, there is no need for a separate washing step prior to crystallization. DMF, acetonitrile, EtOAc and water are preferably used. A controlled 15 crystallization not only achieves an excellent purification but also shortens the filtering or centrifugation time during work up as well as shortens the drying time, if dry intermediates are required. One important factor is to generate sufficiently large crystals with a relatively narrow size distribution not to block the filter medium or centrifugation cloth. It is very common for peptides in particular to generate gels or amorphous crystals that are almost 20 impossible to filter. The tripeptide derivative (9) F 0 0

H

2 o R (9) 0H3

OH

3 WO 99/47548 PCT/SE99/00414 13 wherein

R

1 is an ester or an amide residue group; 5 is a useful intermediate for the preparation of target compound (I). DETAILED DESCRIPTION OF THE INVENTION The preperation of the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 or a pharmaceutically to acceptable salt thereof, will now be described in more detail by the following Examples, which however should not be construed as limiting the invention. Furthermore, Scheme 1 below provides a detailed overview of the synthetic route followed for the preparation of the peptide of the formula (I) according to the present invention using a phenylalanine derivative, wherein the carboxyl group is protected as an ester. Scheme 2 below provides a 15is detailed overview of the synthetic route followed for the preparation of the peptide of the formula (I) according to the present invention using a phenylalanine derivative wherein the carboxyl group is protected as an amide. The compound numbers referred to in the detailed synthesis description in the Examples below, correspond to the compound numbering in Schemes 1 and 2. 20 WO 99/47548 PCT/SE99/00414 14 Scheme 1 / \ (i) CH, - - F N F (H 3 iO 0 F (1 iBuOCOCI CI / OH Z-Phe(pF) (ii) Acetone 0 AcetonCH 3 N -10 0 nC 0 (2) 20 min "-0 \

H

3 NMM Z-Phe(pF)-OCOOiBu (i) Phe-OMe x HCI F (ii) NMM / (a)-10 'C 30 min (b) 25 'C 1h 0

CH

3 O (3) S Z-Phe(pF)-Phe-OMe NHz MeOH 2-3 atm F F

H

2 Pd/C O 0 3bar NH 2 N NH DMF H 2 N O O 2 25 °C 1 h 25 0 C~0 (4) Nz Z-Phe(pF)-Phe-NH 2 (5) H-Phe(pF)-Phe-NH 2 5 WO 99/47548 PCT/SE99/00414 15 Scheme 1 (cont'd) H 3 (i) iBuOCOCI / H OHC (ii) NMM

H

3 CH (6) OO -1OH 1C 20 min OCH Z-D-Ala O O l3 (a) -10 oC 30 min (7) Z-D-Ala-OCOOiBu (b) 25 C 1 h ACN + DMF H-Phe(pF)-Phe-NH 2 (5) F 0 0 H2 O5 % Pd/C S NH 3 bar / F C - DMF (8) H 3 25 0 C 1 h Z-D-Ala-Phe(pF)-Phe-NH 2

H

2 N NH 0 2 OH 3 - H CH -D (9) H-D-Ala-Phe(pF)-Phe-NH 2 WO 99/47548 PCT/SE99/00414 16 Scheme 1 (Cont'd) OH OH W iiBuOCOCIL \/(ii) NMM CH 3 ACN 0 0 0OH -10COC

CH

3 Ip0 i (a) -10 'C- 30 min (1)ZTr(b) 25 00 1 h (11) Z-Tyr-OCOOiBu H-D-Ala-Phe(pF)-PheNH 2 (9) OQH F 0 0 0

CH

3 -(12) Z-Tyr-D-Ala-Phe(pF)-Phe-NH 2 5 %Pd/C

H

2 DM 3 ba r 2 0 C1 h OH F 00

NH

2 M' N (I)H 0 OH 3 H 0 H-Tyr-D-Ala-Phe(Pp)-Phe.NH 2 iHCV/H 2 0 Acetone MIBK H-Tyr-D-Aa-Phe(pF)Phe.NH 2 x HCI WO 99/47548 PCT/SE99/00414 17 Step 1 (i) Preparation of Z-Phe(pF)-Phe-OMe (Compound 3 in Scheme 1) 3.5 mole scale Z-Phe(pF) (compound 1)(1 eq.) is first dissolved in acetone (4.7L/mole) and cooled before 5 addition of IBK (0.9-1.2 eq.)(leq actual). The reaction is then controlled by the rate of addition (about 20 minutes) of NMM (N-methylmorpholine) (0.9-1.2 eq.) (leq actual). A reaction temperature between 0 and -15 0 C is recommended (from -9 0 C to -14 0 C actual) where the reaction occurs immediately upon addition of NMM, yet prevents the mixed anhydride from decomposing to rapidly. 10 H-Phe-OMe x HCI (0.9-1.3 eq.) (1.04 eq actual) is meanwhile mixed with acetone (2.6L/mole), neutralized with NMM (0.9-1.5 eq.) (1.04eq actual) and cooled to 0 - -20 0 C (about -10 0 C actual). This slurry is upon completion of the activation added at a rate that maintains the temperature around -10 0 C (from -8 0 C to -13 0 C actual) (about 30 minutes). 15is EtOAc (4L/mole) is then charged and the organic phase washed with water (2x2L/mole) followed by azeotrop distillation from ACN and dissolution in MeOH prior to the next step. 92% purity in a methanol slurry.

WO 99/47548 PCT/SE99/00414 18 Shift Multiplicity Integral H 8.5 d 1 7.49 d 1 7.31 m 7 5 7.24 m 5 7.08 m 2 4.93 m 2 4.5 m 1 4.26 m 1 o10 3.58 s 3 2.99 m 3 2.67 m 1 15 (ii) Preparation of Z-Phe(pF)-Phe-NH 2 (compound 4 in Scheme 1) 2.3-3.3 mole scale Ammonia is charged to the solution of compound 3 prepared in the previous step (about 8L MeOH/mole) at a pressure between 1-5 bar at 15 to 40 0 C and for more than 5 hours or 20 until the reaction is close to completion (actual conversion 99%). Upon completion the ammonia is evaporated and the reaction cooled before filtration or centrifugation. The product is washed with MeOH and dried under vacuum at 20-50 OC. Yield 74% calculated from compound 1 (Z-Phe(pF) ) and 100% purity.

WO 99/47548 PCT/SE99/00414 19 Shift Multiplicity Integral H 8.05 d 1 7.49 d 1 5 7.24 m 11 7.07 m 3 4.94 m 2 4.46 m 1 4.21 m 1 o10 3.00 m 1 2.88 m 1 2.66 m 1 15 Preparation of H-Phe(pF)-Phe-NH (compound 5 in Scheme 1) 4.3 mole scale Compound 4 prepared in the previous step is mixed with DMF (4.2L/mole) and and a Pd/C 20 catalyst(5% Pd actual content) is added (0.2-10% w/w / LEF-581) (7% actual) and the resulting mixture hydrogenated for more than 0.5 hours (1.2h actual) at 25 0 C and 3bar H2. The reaction mixture is then filtered and cooled to about -15 0 C before the next step. 99.6% purity in solution. 25 Step 2 (i) Preparation of Z-D-AIa-Phe(pF)-Phe-NH (compound 8 in Scheme 1) 4.4 mole scale Z-D-Ala (compound 6) (1 eq.) was dissolved in acetonitrile (ACN) (2.3L/mole) and cooled 30 before addition of IBK (0.9-1.2 eq.)(leq used). NMM (0.9-1.2 eq.) (leq used) was then WO 99/47548 PCT/SE99/00414 20 added in the same manner as described above for the preparation of compound 3. The solution of compound 5 (24.5L) was then charged during about 30 minutes, maintaining the temperature around -10 0 C (from -8 0 C to -14C actual). After completion of the coupling the product was crystallized from the reaction mixture by slow addition of water 5 (3x3.6L/mole + lx 1.3L/mole) with about 25 min wait between each addition and at a starting temperature of about 30oC and an ending temp of about 20oC. The crystals can then be centrifuged and washed with water/acetonitrile (4:1) before drying under vacuum at 20-50 0 C. Yield 90% and 99.5% purity. 10 Shift Multiplicity Integral H 8.14 d 1 8.05 d 1 7.43 d 1 7.29 m 12 15is 7.05 m 2 5.01 m 2 4.44 m 2 4.01 m 1 2.92 m 3 20 2.73 m 1 0.96 d 3 WO 99/47548 PCT/SE99/00414 21 Preparation of H-D-AIa-Phe(pF-Phe-NH (compound 9 in Scheme 1) Compound 8 prepared in the previous step is mixed with DMF (4.2L/mole) and a Pd/C catalyst (5% Pd actual content) is added (0.2-10% w/w / compound 3)(7% actual) and the 5 resulting mixture hydrogenated for more than 0.5 hours (1.2h actual) at 25oC and 3bar H2. The reaction mixture is then filtered and cooled to about -15'C before the next step. Purity 97%. Conversion of starting material >98% 10 Step 3 (i) Preparation of Z-Tyr-D-Ala-Phe(pF)-Phe-NH2 (compound 12 in Scheme 1) 4.1 mole scale This coupling utilized the same method as the previous two couplings. Z-Tyr 15 (compound 10) (1 eq.) was dissolved in ACN ( 2 .3L/mole)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 under compound 3. The solution of compound 3 from the previous step was then charged during about 30 minutes, maintaining the temperature around -10 0 C (from -7C to -14°C actual). After completion of the coupling the product was crystallized from the 20 reaction mixture by slow addition of acetonitrile and water (2L/mole ACN + 0.3L/mole 25% NI-I 3 in H 2 0 ,hold 2h, add 1.5L/mole

ACN:H

2 0 (1:1), hold 1 h, increase temp to 35 0 C, add seeding crystals (about 1% w/w), hold 1 h, add 1.3L/mole

ACN:H

2 0 (1:1), hold 1 h, add 1.2L/mole

H

2 0 and hold 0.5h at 35 0 C, add 1.2L/mole H20 and hold 2h at 20 0 C, add 1.

2 L/mole

H

2 0 and hold lh at 20 0 C and add 1.2L/mole

H

2 0 and hold 0.5h at 20 0 C. 25 Centrifuge and wash first with water and then ACN before drying under vacuum at 20-50 0 C. Yield 81% calculated from compound 8. 98.4% pure.

WO 99/47548 PCT/SE99/00414 22 Shift Multiplicity Integral H 9.18 s 1 8.18 d 1 5 8.11 d 1 8.05 d 1 7.43 d 1 7.24 m 12 7.04 m 4 io 6.63 d 2 4.92 m 2 4.45 m 2 4.22 m 2 3.00 m 1 15is 2.83 m 1 2.64 m 1 0.89 d 3 20 (ii) Preparation of H-Tyr-D-AIa-Phe(pF)-Phe-NH (compound I in Scheme 1) 3.1 and 3.2 mole scale Compound 4 is mixed with DMF (2-2.6L/mole actual runs) and a 5 % Pd/C (actual content) catalyst is added (0.2-10% w/w / compound 3) (6-7% actual) and the resulting mixture hydrogenated for more than 0.5 hours(1-2h actual run) at 20-40 0 C (20-25 0 C actual 25 runs) and 3bar H 2 . The reaction mixture is then filtered to remove the Pd/C before crystallizing the product by addition of EtOAc until all substance has crystallized (typically 10L/mole). The solid is separated by filtration or centrifugation and washed with EtOAc prior to drying under vacuum at 20-50 0

C.

WO 99/47548 PCT/SE99/00414 23 Preparation of H-Tvr-D-AIa-Phe(pF)-Phe-NH 2 hydrochloride 2.1 mole scale The free base compound I is dissolved in a mixture of water and acetone with one 5 equivalent HCI added and clear filtered (146g/mole 25% HCl/H 2 0, 2L Acetone/mole in actual run). The salt has a limited solubility in acetone and therefore the filter is washed once with an additional amount of the acetone/water(95:5) mixture (0.5L/mole). The crystallization is initiated by a slow addition of acetone (3.4L/mole) at high agitation rate and then at least 1% w/w of seeding crystals is added. After 30 minutes the first amount of io MIBK (3L/mole) is slowly charged and left with slow stirring until the batch clearly thickens. MIBK (3L/mole) is charged three additional times separated by 30-60 minutes while maintaining the reactor inner temperature at about 20 0 C. The solid is then separated by centrifugation or filtration and washed with MIBK before drying under vacuum at 20-50oC for more than 16 hours or until the solvent levels are lower than specified in the 15 release specifications.

WO 99/47548 PCT/SE99/00414 24 Scheme 2 F

CH

3 H1C N O 0N OH + H 3 C O0 + H CI 0 Z-Phe(pF) IBuOCOCl NMM EtOAc -10°C 20 min. F cl CH 3 0 CH 3 N O O O~ OH3 OO O

NH

2 NMM Z-Phe(pF)-OCOOiBu Phe-NH 2 x HCI -10*C 25 0 C 10min +1h EtOAc + DMF F F 0 H 2 -NH 2 O NH 2

H

2 N N-- DMF H 250C O 0 -lh Z-Phe(pF)-Phe-NH 2 Phe(pF)-PheNH 2 (13) (14) WO 99/47548 PCT/SE99/00414 25 Scheme 2 (cont'd) OH

H

3 C) iBuOCOCI OH / ~ii) NMM-OH NH 0 0 NH 0 MeCN00 0 .1000 C/.~Or H 3 Z-D-Ala Z-D-Ala-OCOOiBu H-Phe(pF)-Phe-NH 2 (I14) i) -1000C, 10 min ii) 2 500 , 1lh MeCN 00 0

N

2 5%H2/ H-D-Ala-Phe(pF)-Phe-NH 2 00 -DMF (16) CH3 Z-D-Ala-Phe(pF)-Phe-NH 2 (15) WO 99/47548 PCT/SE99/00414 26 Scheme 2 (cont'd) OH OH j) iBuOCOCI O ii) NMM 0 0 CH 3 NH MeCN CH 100~ 0 0 CH 3 Z-Tyr Z-Tyr-OCOOiBu H-D-Aia-Phe(pF)-Phe-NH2 (16) 0) -1 O'C, 10 min ii) 2500, lh 7MeCN ~OH F 0 0 0 N N , N O,. Hj N 2 0 0 0

CH

3 Z-Tyr-D-Aa-Phe(pF)-Phe.NH2

H

2 (17) 5%P/C DMF H-Tyr-D-Ala-Phe(p)-PheNH2 LiCJ 3%eH20 MIBK Z-Tyr-D-Ala-Phe(p).PheNH2 x HCI WO 99/47548 PCT/SE99/00414 27 Step 1 (i) Preparation of Z-Phe(pF)-Phe-NH (Compound 13 in Scheme 2) 6.7 mole scale 5 Z-Phe(pF) (1 eq.) is first dissolved in acetonitrile (EtOAc)(1.7L/mole) and cooled before addition of i-Butylchloroformiate (0.9-1.2 eq.)(1.05eq actual). The reaction is then controlled by the rate of addition, (about 20 minutes) 15 min actual, of N Methylmorpholine (0.9-2.0 eq.) (1.4eq actual). A reaction temperature between 0 and 10 15 0 C is recommended (from -8 0 C to -11 0 C actual) where the reaction occurs immediately upon addition of N-Methylmorpholine, yet prevents the mixed anhydride from decomposing to rapidly. H-Phe-NH 2 x HCI (0.9-1.3 eq.) (1.04 eq actual) is meanwhile dissolved in DMF 15 (4.0L/mole), neutralized with N-Methylmorpholine (0.9-1.5 eq.) (1.04eq actual) and cooled to 0 - -20 0 C (about -10 0 C actual). This slurry is upon completion of the activation added at a rate that maintains the temperature around -10°C (from -6 0 C to -13oC actual) (about 15 minutes) 8 min actual. After completion of the coupling the product was crystallized from the reaction mixture by 20 slow addition of 50% Ethanol/water (3.6L/mole). After 30 min wait a total of 2.85L/mole water in three portions were charged with about 25 min wait between each addition and at temperature of about 20 0 C. The crystals can after about 17 hours be filtered or centrifuged and washed with 50% Ethanol/water followed by several portions of acetonitrile before drying under vacuum at 20-60 0 C. Yield 90% and 99.9% purity. 25 Preparation of H-Phe(pF)-Phe-NH 2 (compound 14 in Scheme 2) 6.7 mole scale Z-Phe(pF)-Phe-NH 2 prepared in the previous step is mixed with DMF (3.5L/mole) and a 30 Pd/C catalyst (5% Pd actual content) is added (0.2-10% w/w / LEF-582) (5% actual) and WO 99/47548 PCT/SE99/00414 28 the resulting mixture hydrogenated for more than 0.5 hours (1.3h actual) at 25-30 0 C and about 3bar H 2 . The reaction mixture is then filtered and cooled to about -15 0 C before the next step. 99.6% purity in solution and >99% conversion of starting material. 5 Ste2 (i) Preparation of Z-D-AIa-Phe(pF)-Phe-NH (compound 15 in Scheme 2) 5.9 mole scale Z-D-Ala-OH (compound x) (1.03 eq. used) was dissolved in acetonitrile (1.9L/mole) and o10 cooled before addition of i-Butylchloroformiate (0.9-1.2 eq.)(1.07eq used). N Methylmorpholine (0.9-2.0 eq.) (1.2eq used) was then added in a similar manner as described above for the preparation of Z-Phe(pF)-Phe-NH 2 . The solution of H-Phe(pF) Phe-NH 2 (25L) was then charged during about 15 minutes (8min actual), maintaining the temperature around -10 0 C (from -8 0 C to -11 0 C actual). After completion of the coupling 5is the product was crystallized from the reaction mixture by slow addition of water (4x1.9L/mole) with about 15-30 min wait between each addition and at a temperature of about 20 0 C. The crystals can then be filtered or centrifuged and washed with water/acetonitrile (4:1) followed by acetonitrile before an optional drying under vacuum at 20-60'C. Yield calculated from Z-Phe(pF)-Phe-NH 2 93.8% and 99.6% purity. 20 Preparation of H-D-AIa-Phe(pF)-Phe-NH (compound 16 in Scheme 2) 5.5 mole scale Z-D-Ala-Phe(pF)-Phe-NH 2 prepared in the previous step is mixed with DMF (2.9L/mole) 25 and a Pd/C catalyst (5% Pd actual content) is added (0.2-10% w/w / compound 3)(5% actual) and the resulting mixture hydrogenated for more than 0.5 hours (3h actual) at 25 35 0 C and about 3bar H 2 . The reaction mixture is then filtered and cooled to about -15oC before the next step. Purity 99.4%. Conversion of starting material >99% 30 WO 99/47548 PCT/SE99/00414 29 Step 3 (i) Preparation of Z-Tyr-D-Ala-Phe(pF)-Phe-NH (compound 17 in Scheme 2) 5.5 mole scale 5 This coupling utilized a similar method as the previous two couplings. Z-Tyr (compound x) (1.05 eq.) was dissolved in MeCN (1.9L/mole)and cooled before addition of i-Butylchloroformiate (0.9-1.2 eq.)(1.05 actual). N-Methylmorpholine (0.9-2.0 eq.)(1.3 actual) was then added in a similar manner as described above for the preparation of Z Phe(pF)-Phe-NH 2 . The solution of H-D-Ala-Phe(pF)-Phe-NH 2 from the previous step was 10 then charged during about 20 minutes (6min actual), maintaining the temperature around 10 0 C (from -8'C to -9 0 C actual). After completion of the coupling the product was crystallized from the reaction mixture at about 20-45oC by slow addition of acetonitrile and water (3.4L/mole MeCN + 0.9L/mole 15% NH 3 in H 2 0 hold 5min and seed, hold 4-24h, then add a total of 13.9L/mole H 2 0 in four portions with about 30min or longer hold in 15is between each. Filter or centrifuge and wash first with water and then MeCN before optional drying under vacuum at 20-60 0 C. Yield 87.7% calculated from Z-D-Ala-Phe(pF)-Phe-NH 2 and 95.1% pure. The yield and purity were found to be increased by heating the reaction to about 60'C with addition of ammonia to a pH of about 9 for two hours. This will convert the major 20 impurity, Z-Tyr(O-(i-Butyloxycarbonyl))-D-Ala-Phe(pF)-Phe-NH 2 , to product. (ii) Preparation of H-Tyr-D-AIa-Phe(pF)-Phe-NH (compound I in Scheme 2) 5.4 mole scale Z-Tyr-D-Ala-Phe(pF)-Phe-NH 2 is mixed with DMF (2.6L/mole actual run) and a 5 % Pd/C 25 (actual content) catalyst is added (0.2-10% w/w / compound 3) (6.4% actual) and the resulting mixture hydrogenated for more than 0.5 hours(1.8h actual run) at 20-40 0 C (20 25 0 C actual runs) and about 3bar H 2 . The reaction mixture is then filtered to remove the Pd/C before crystallizing the product by addition of EtOAc until all substance has crystallized (typically about 14L/mole). The solid is separated by filtration or centrifugation WO 99/47548 PCT/SE99/00414 30 and washed with EtOAc prior to drying under vacuum at 20-50 0 C. Purity 96.7%. Conversion of starting material >99% 5 Preparation of H-Tvr-D-Ala-Phe(pF)-Phe-NH hydrochloride 4.6 mole scale The free base H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 is dissolved in a mixture of water and acetone with one equivalent HCI added and clear filtered (146g/mole 25% HCI/H 2 0, 2L o10 Acetone/mole in actual run). The salt is almost insoluble in acetone and therefore the filter is washed once with an additional amount of the acetone/water(95:5) mixture (0.5L/mole). The crystallization is initiated by a slow addition of acetone (3.4L/mole) at high agitation rate and then about 1% w/w of seeding crystals is added. After 30 minutes the first amount of MIBK (3L/mole) is slowly charged and left with slow stirring until the batch clearly 15is thickens. MIBK (3L/mole) is charged three additional times separated by 30-60 minutes while maintaining the reactor inner temperature at about 20 0 C. The solid is then separated by centrifugation or filtration and washed with MIBK before drying under vacuum at 20 50 0 C for more than 16 hours or until the solvent levels are lower than specified in the release specifications. Yield 95.8% and a purity of 99.8%. 20 Reprocessing Product that fail the specifications for the drug substance may be recrystallized by the same procedure as described above for the crystallization of the compound I, but without the HCI addition. 25 WO 99/47548 PCT/SE99/00414 31 Assignment of NMR spectra for H-Tyr-D-Ala-Phe(F)-Phe-NH, x HCI, i.e for compound I in its hydrochloride form. 5 NMR spectra were obtained on a solution of 36mg of the compound in approx. 0.7 ml DMSO-d 6 (99.95 atom-% D) at 27.00 C on a Varian UNITY plus 400 MHz instrument. Chemical shift reference for proton spectra was the middle peak of the DMSO-d 6 multiplet taken as 2.49 ppm. Reference for carbon spectra was the middle peak of the DMSO-d 6 multiplet taken as 39.5 ppm. 10 29 2 OH F 2 5 4 110,0 N 2 HN 1 1H0( 23 H 02 1 20 119 0 OH 3 H 0 110182 14 321 1 24 22 Atom numbering used in assignment is arbitrary and refers to the figure above. PROTON SPECTRA 15is The one dimensional proton spectrum allows groupwise assignment of alpha protons (3.9 4.4 ppm), benzyl-CH 2 (2.6-3.1 ppm), amide-NH and phenol-OH (8.2-8.5 ppm) and also specific assignment for Ala-CH 3 (14-CH 3 ) (0.74 ppm). The two dimensional DQFCOSY spectrum allows for groupwise assignment of the spin 20 systems (alpha, beta and NH protons) in each amino acid residue, and groupwise assignment of aryl protons in each aromatic ring. All protons in the Ala residue can also be specifically assigned.

WO 99/47548 PCT/SE99/00414 32 CARBON SPECTRA The one dimensional carbon spectrum allows for groupwise assignment of alpha carbons, benzyl-CH 2 , carbonyls and aryl carbons and of course specific assignment of C-14. The APT spectrum allows assignment of CH-multiplicity for each carbon. Line splittings due to 5 C-F couplings allows specific assignment of the carbons in the fluoroaromatic ring. TWO DIMENSIONAL HETEROCORRELATED SPECTRA The two dimensional carbon-proton correlated (HMQC) spectrum gives a correlation 10 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 correlated (HMBC) spectrum gives a correlation between carbons and protons situated two to three bonds apart. This allows 5is assignment of amino acid sequence via alpha hydrogens and the carbonyl group of the neighboring amino acid residue (three-bond correlation), as well as via NH and the carbonyl group of the neighboring amino acid residue (two-bond correlation). Similarly two- and three-bond correlations between benzyl-CH 2 and aryl carbons as well as between aryl protons and benzyl-CH 2 allows specific assignment of aryl protons and carbons of the 20 individual aromatic amino acids. In this way all (non-exchanging) protons and carbons in all four amino acid residues can be specifically assigned in an unambiguous way.

WO 99/47548 PCT/SE99/00414 33 Table 1 Proton assignments Chemical shift (ppm) Integral Multiplicity Assignment 9.4 1H s 300H 8.48 1H d 9NH 8.37 1H d 1NH 8.35 1H d 8NH 8.26 2H s 7.53 1H s 7.28 2H m 18H, 19H 7.26 2H m 21H, 22H 7.22 2H m 6H, 7H 7.18 1H m 24H 7.12 1H s 7.04 2H m 10H, 11H 6.98 2H m 26H, 27H 6.68 2H m 28H, 29H 4.43 1H m 1H 4.39 1H m 8H 4.26 1H m 9H 3.96 1H m 20H 3.39 HDO 3.02 1H m 12Hb 2.99 1H m 2Hb 2.87 2H m 23Ha, 23Hb 2.85 1H m 12Ha 2.67 1H m 2Ha 0.74 3H d 14H WO 99/47548 PCT/SE99/00414 34 Table 2 Carbon assignments Chemical shift (ppm) Multiplicity Assignment JC-F 172.99 s 13 171.08 s 5 170.81 s 3 167.39 s 17 162.11 s 15 241 Hz 159.70 s 15 241 Hz 156.50 s 30 137.98 s 16 133.96 s 4 3.1 Hz 133.93 s 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 s 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 WO 99/47548 PCT/SE99/00414 35 Table 2 (contd.) Carbon assignments Chemical shift (ppm) Multiplicity Assignment JC-F 47.96 d 9 37.57 t 12 36.83 t 2 36.17 t 23 18.45 q 14 5

Claims (14)

1. A process for the preparation of the tetrapeptide H-Tyr-D-Ala-Phe(F)-Phe-NH2 of the formula (I) 5 OH F O o NH 2 N NH 2 (I) O CH 3 O or a pharmaceutically acceptable salt thereof, comprising the following steps: (i) A coupling step wherein an activated tyrosine derivative (X), 10 OR 2 (X) A-. 0 R wherein A is an amino protecting group, 15is R is an activating agent residue group, and R is H or a benzyl-like group; is reacted with the tripeptide derivative (9) WO 99/47548 PCT/SE99/00414 37 F 0 0 HHN R (9) 0 OH 3 wherein R1 is an ester or an amide residue group; in the presence of a solvent under standard conditions, providing the protected tetrapeptide 5 derivative (XI) OH F O 0 A----- O O(XI) o 0 CH 3 wherein A is an amino protecting group, and 10 R is an ester or an amide residue group; (ii) A deprotection step wherein the protected tetrapeptide derivative (XII) is deprotected either by catalytic hydrogenation, base or acid treatment under standard conditions in the presence of a solvent to give (I). 15

2. A process according to claim 1, characterized in comprising additional steps of: WO 99/47548 PCT/SE99/00414 38 (i) A coupling step wherein an activated alanine derivative (VII), H

3 C A R (VII) 5 wherein A is an amino protecting group, and R is an activating agent residue group; is reacted with the dipeptide derivative (5) F 0 H 2 N N 0 - (5) 10 wherein R 1 is an ester or an amide residue group in the presence of a solvent under standard conditions, providing the protected tripeptide 15 derivative (VIII) WO 99/47548 PCT/SE99/00414 39 F 0 0 H R (VIII) CH 0 3 wherein A is an amino protecting group, and R 1 is an ester or an amide residue group; 5 (ii) A deprotection step wherein a protected tripeptide derivtive (VIII) prepared in the previous step, is deprotected either by catalytic hydrogenation, base or acid treatment, in the presence of a solvent under standard conditions, providing the tripeptide derivative (9) F O 0 H 2 N I (9) 0 CH 3 10 wherein R 1 is an ester or an amide residue group. 15 3. A process according to claim 2, characterized in comprising additional steps of: (i) A coupling step wherein an activated p-fluorophenylalanine derivative (III), WO 99/47548 PCT/SE99/00414 40 F ON A- 0 R(I wherein A is an amino protecting group, and S R is an activating agent residue group; 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 standard conditions, providing a protected dipeptide derivative (IV) F 0 0 A (IV) 10 wherein A is an amino protecting group, and R 1 is an ester or an amide residue group; is (ii) A deprotection step wherein a protected dipeptide derivative (IV) prepared in the previous step, is deprotected by either catalytic hydrogenation, base or acid treatment, in the presence of a solvent under standard conditions, providing the dipeptide derivative (5), WO 99/47548 PCT/SE99/00414 41 F O R H 2 N N H - (5) 0 wherein R is an ester or an amide residue group. 5

4. A process according to claim 1-3, characterized in that one of the coupling steps (i) is followed by an optional transformation step, performed if the carboxyl group of the amino acid derivative is protected as an ester derivative, wherein the ester compound is reacted with ammonia in an organic alcohol. 10

5. A process according to claims 1-3, characterized in that the activated amino acid derivative used in at least one of the coupling steps is selected from a group consisting of a carbodiimide, an activated ester, an azide, or an anhydride. 15

6. A process according to claim 5, wherein the activating agent is isobutylchloroformiate.

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. 20

8. A process according to claims 1-3, wherein 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 performed using Pd on charcoal. WO 99/47548 PCT/SE99/00414 42

10. A process according to any of claims 1-9, characterized in that at least one of the reactions are performed at a temperature of from 0 0 C to -20 *C.

11. A process according to claim 10, wherein the temperature is from -5 oC to -15 oC. 5

12. A peptide of the formula I OH F I O NH 2 N NH () O CH 3 H prepared according to the process of claim 1. 10

13. A peptide according to claim 12, in form of the hydrochloride salt.

14. A peptide derivative of the formula (9) F O 0 2 O R (9) 0 OH 3 15 wherein R 1 is an ester or an amide residue group.