Process for preparing D-arginyl-2,6-dimethyl-L-tyrosyl-L-lysyl-L-phenylalaninamide

Abstract

The invention relates to a process for solution-phase synthesis of D-Arginyl-2,6-dimethyl-L-tyrosyl-L-lysyl-L-phenylalaninamide, an active ingredient used for both common and rare diseases including a mitochondrial targeted therapy for ischemia reperfusion injury.

Claims

1. A liquid-phase process for the production of H-D-Arg-(2,6-Dimethyl)Tyr-Lys-Phe-NH2 of formula (I), in the form of the acetic acid salt, ##STR00014## which comprises the following steps: coupling compound (II) H-Phe-NH2: ##STR00015## with compound (III) Boc-Lys(Z)-OH: ##STR00016## to obtain a compound of formula (IV), Boc-Lys(Z)-Phe-NH2: ##STR00017## reacting compound (IV) with methanesulfonic acid (V)
MeSO3H(V) to obtain the free amine salt (VI) MeSO3H.H-Lys(Z)-Phe-NH2: ##STR00018## reacting the salt (VI) with the protected amino acid Boc-Dmt(Bzl)-OH (VII) ##STR00019## to obtain the protected tripeptide (VIII): ##STR00020## treating the compound (VIII) with methanesulfonic acid (V) to obtain the corresponding salt (IX), MeSO3.H-Dmt(Bzl)-Lys(Z)-Phe-NH2: ##STR00021## coupling the acid salt (IX) with Z-D-Arg-ONa (X) ##STR00022## to form the protected tetrapeptide (XI), Z-D-Arg-Dmt(Bzl)-Lys(Z)-Phe-NH2: ##STR00023## and deprotecting compound (XI) by hydrogenolysis to obtain the tetrapetide H-D-Arg-(2,6-Dimethyl)Tyr-Lys-Phe-NH2 (I) and further salifying it with acetic acid to form the corresponding salt.

2. A process according to claim 1 wherein the coupling between (II) and (III) and/or the coupling between (VI) and (VII) and/or the coupling between (IX) and (X) is performed in the presence of N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate.

3. A process according to claim 2 wherein the coupling between (II) and (III) and/or the coupling between (VI) and (VII) and/or the coupling between (IX) and (X) is performed in the presence of a tertiary amine, preferably selected from N-methyl morpholine, triethylamine or diisopropylethylamine.

4. A process according to claim 1 wherein the coupling between (II) and (III) and/or the coupling between (VI) and (VII) and/or the coupling between (IX) and (X) is performed in organic polar solvents.

5. A process according to claim 1 wherein the coupling between (II) and (III) is performed in a temperature range between 0 C. and 60 C.

6. A process according to claim 1 wherein the formation of methanesulfonic salt (VI) is obtained in methylene chloride as solvent and crystallized from the same solvent.

7. A process according to claim 1 wherein the coupling between (VI) and (VII) is performed in a temperature range between 0 C. and 60 C.

8. A process according to claim 1 wherein the coupling between (IX) and (X) is performed in a temperature range between 0 C. and 60 C.

9. A process according to claim 1 wherein the step of deprotecting compound (XI) is performed by hydrogenation.

10. A compound of formula (VIII): Boc-(2,6-dimethyl)Tyr(Bzl)-Lys(Z)-Phe-NH2 ##STR00024##

11. A compound of formula (XIV) H-(2,6-dimethyl)Tyr(Bzl)-Lys-Phe-NH2 or a salt thereof: ##STR00025##

12. A compound according to claim 11 in form of the mesylate salt.

13. A compound of formula (XI) Z-D-Arg-(2,6-dimethyl)Tyr(Bzl)-Lys(Z)-Phe-NH2 ##STR00026##

14. A process according to claim 4, wherein said organic polar solvents is selected from dimethylformamide, acetonitrile, tetrahydrofuran, 2-methyl tetrahydrofuran, or a mixture thereof.

Description

DETAILED DESCRIPTION

(1) The present invention provides, in a first aspect, a novel and efficient process that leads to a SS-31 salt, especially the acetic acid salt, which is convenient for the industrial scale and provides the desired product in good yields. In particular, the inventors found that SS-31 acetate salt can be advantageously obtained with a process, in which the overall deprotection step is the n1 step of the process.

(2) Accordingly, it is an object of the present invention to provide a process for preparing H-D-Arg-Dmt-Lys-Phe-NH.sub.2 of formula (I) as the acetic acid salt.

(3) ##STR00003##

(4) which comprises the steps of: coupling compound (II) H-Phe-NH.sub.2:

(5) ##STR00004##

(6) with compound (III) Boc-Lys(Z)-OH:

(7) ##STR00005##

(8) to obtain a compound of formula (IV), Boc-Lys(Z)-Phe-NH.sub.2:

(9) ##STR00006##

(10) and reacting compound (IV) with methanesulfonic acid (V)
MeSO.sub.3H(V)

(11) to obtain the free amine salt (VI) MeSO.sub.3H.H-Lys(Z)-Phe-NH.sub.2:

(12) ##STR00007##

(13) The salt (VI) is reacted with the protected amino acid Boc-Dmt(Bzl)-OH (VII)

(14) ##STR00008##

(15) to obtain the protected tripeptide (VIII):

(16) ##STR00009##

(17) which is treated with methanesulfonic acid (V) to obtain the corresponding salt (IX), MeSO.sub.3.H-Dmt(Bzl)-Lys(Z)-Phe-NH.sub.2:

(18) ##STR00010##

(19) The acid salt (IX) is coupled with Z-D-Arg-ONa (X)

(20) ##STR00011##

(21) to form the protected tetrapeptide (XI), Z-D-Arg-Dmt(Bzl)-Lys(Z)-Phe-NH.sub.2:

(22) ##STR00012##

(23) The tetrapeptide (I) is obtained by hydrogenolysis of (XI) and further reacted with acetic acid to form the corresponding salt.

(24) In one embodiment of the process, the coupling between (II) and (III) is performed in the presence of N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (known as TBTU) and an organic base belonging to the class of tertiary amines such as NMM, triethylamine and diisopropylethylamine as well as a polar solvent such as DMF, acetonitrile, tetrahydrofuran (THF), 2-methyl-tetrahydrofuran (ME-THF) etc.

(25) In one embodiment, the coupling between (II) and (III) is performed in a temperature range between 0 C. and 60 C., preferably between 20 C. and 30 C.

(26) In another embodiment, the formation of methanesulfonic salt (VI) is obtained in methylene chloride as solvent and crystallized from the same solvent. Other solvents suitable for crystallization are THF, ethyl acetate and acetonitrile.

(27) In one embodiment of process the coupling reaction between compound (VI) and compound (VII) is performed in the presence of N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (known as TBTU) and a base of tertiary amine class such as N-methyl morpholine (NMM), triethylamine and diisopropylethylamine as well as a polar solvent as DMF, acetonitrile, THF, 2-ME-THF etc.

(28) In another embodiment, the formation of methanesulfonic salt (IX) is obtained in methylene chloride as solvent and crystallized from the same solvent. Other suitable solvents for crystallization are THF, ethyl acetate and acetonitrile.

(29) In one embodiment of process the coupling reaction between compound (IX) and compound (X) is performed in the presence of N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (known as TBTU) and a base of tertiary amine class such as NMM, triethylamine and diisopropylethylamine as well as a polar solvent as DMF, acetonitrile, THF, 2-ME-THF etc.

(30) In one embodiment the hydrogenolysis of (XI) is performed with heterogeneous catalyst Pd on C and acetic acid as solvent. Other suitable catalysts are Pd(OH).sub.2 on carbon, Pd (and or PdCl.sub.2) on SiO.sub.2, Al.sub.2O.sub.3 or polymer, and solvents such as methanol, ethanol, isopropanol, DMF, THF and acetonitrile.

(31) The intermediates can be isolated or not from the reaction mixture.

(32) In one aspect of the process, the intermediates (IV), (VI), (VIII), (IX) and (XI) are isolated and crystallized. When the intermediates are isolated, their purity exceeds 98%.

(33) In one preferred aspect, the crystallization of intermediate (VIII), Boc-Dmt(Bzl)-Lys(Z)-Phe-NH.sub.2 is able to avoid the transfer of a critical impurity to the following process steps.

(34) In one preferred aspect, the critical impurity is compound (XII), H-D-Dmt(Bzl)-Lys(Z)-Phe-NH.sub.2.

(35) ##STR00013##

(36) In another preferred aspect, the crystallization process for the protected tetrapeptide (XI), Z-D-Arg-Dmt(Bzl)-Lys(Z)-Phe-NH.sub.2 allows to obtain the product as a solid with a purity close to 99%.

(37) In another preferred aspect, the final deprotection is performed by simple hydrogenation and allows to obtain the final product in solid form of acetate salt after simple crystallization without any need of HPLC purification or any freeze-drying, a purification and isolation process extremely expensive but commonly used in the manufacture of peptide as drug.

(38) In a preferred aspect, the process allows to scale-up an efficient process to obtain the peptide as a solid which can be used in formulation as such or can be easily converted in any other salt if required. The purity of the obtained final compound is higher than 98.5%, usually 99% and each impurity is close to 0.2% or below.

EXAMPLES

Example 1: Preparation of Boc-Lys(Z)-Phe-NH2

(39) Charge 200 mL of DMF, 44 g of Boc-Lys(Z)-OH and 15.6 g of H-Phe-NH.sub.2 in a flask. Stir the mixture at room temperature for 10 min. Add 19.2 g of N-methylmorpholine and 32.1 g of TBTU successively at room temperature. Stir the mixture at room temperature for 1 h. Add 500 mL of water into the reaction mixture to precipitate the product at room temperature. Filter the mixture to isolate the solid product and wash the filter cake with water. Transfer the filter cake into a flask containing 360 mL of ethyl acetate and heat the mixture at 50 C. till all the solid is dissolved. Separate the organic phase of product and discard the small aqueous phase. Concentrate the organic phase at 4045 C. and under vacuum to remove the solvent till lots of solid is formed. Filter the residue to isolate the solid product. Transfer the filter cake into a flask containing 2000 mL of MTBE and heat the mixture at refluxing for 20 min. Then, cool down the mixture to room temperature. Filter the mixture to isolate the solid product. Dry the filter cake at 30 C. and under vacuum to give 35 g of solid product.

Example 2: Preparation of H-Lys(Z)-Phe-NH2.MeSO3H

(40) Charge 26.3 g of Boc-Lys(Z)-Phe-NH.sub.2, 200 mL of methylene chloride and 9.6 g of methanesulfonic acid. Stir the mixture at 1520 C. for 18 h. Add 100 mL of MTBE into the mixture and stir at 1520 C. for 1 h. Filter the mixture to isolate the solid product. Dry the wet cake in air at room temperature to give 26.4 g of white solid product.

Example 3: Preparation of Boc-DMeTyr(Bzl)-Lys(Z)-Phe-NH2

(41) Charge 8.4 g of Boc-DMeTyr(Bzl)-OH, 11 g of H-Lys(Z)-Phe-NH.sub.2.MeSO.sub.3H, 7.4 g of TBTU and 80 mL of THF in a flask. Stir the mixture at room temperature for 15 min, and then cool down to 10 C. Add 6.36 g of N-methylmorpholine and stir the mixture at 20-25 C. for 3 h. Add the reaction mixture into a flask containing 240 mL of water. Add 32 mL of methylene chloride into the mixture obtained in the previous operation of. Stir the resultant mixture at room temperature for 20 min. Filter the mixture to isolate the solid product and wash the filter cake with acetone (300 mL2). Dry the filter cake in air at room temperature to give 14.3 g of white solid product.

Example 4: Preparation of H-DMeTyr(Bzl)-Lys(Z)-Phe-NH2.MeSO3H

(42) Charge 14 g of Boc-BMeTyr(Bzl)-Lys(Z)-Phe-NH.sub.2, 280 mL of methylene chloride and 3.3 g of methanesulfonic acid in a flask. Stir the mixture at 1822 C. for 10 h. Add 560 mL of heptanes into the mixture and stir the mixture at room temperature for 30 min. Filter the mixture to isolate the solid product. Dry the wet cake in air at room temperature to give 14 g of white solid product.

Example 5: Preparation of Z-D-Arg-DMeTyr(Bzl)-Lys(Z)-Phe-NH2

(43) Charge 6.34 g of Z-D-Arg-ONa, 100 mL of DMF and 2.0 g of methanesulfonic acid in a flask. Stir the mixture at room temperature till a clear solution was formed. Add 14 g of H-DMeTyr(Bzl)-Lys(Z)-Phe-NH.sub.2.MeSO.sub.3H and cool down the mixture to 10 C. Add 6.15 g of TBTU and 9.67 g of N-methylmorpholine successively. Stir the mixture at room temperature for 4 h. Add aqueous solution of LiOH prepared by dissolving 2.9 g of LiOH.H.sub.2O in 8 mL of water. Stir the mixture for 30 min. Add the resultant mixture slowly into a flask containing 420 mL of water under stirring. Add 56 mL of methylene chloride into the mixture. Filter the mixture to isolate the solid product. Transfer the filter cake into a flask containing 150 mL of acetic acid, and heat the mixture at 3540 C. till most of the solid was dissolved. Add 450 mL of MTBE into the mixture and cool down the mixture under stirring to room temperature. Filter the mixture to isolate the solid product. Dry the filter cake in air at room temperature to give 17.3 g of the white solid product.

Example 6 Preparation of H-D-Arg-DMeTyr-Lys-Phe-NH2.3AcOH

(44) Charge 2.0 g of Z-D-Arg-DMeTyr(Bzl)-Lys(Z)-Phe-NH.sub.2, 20 mL of acetic acid and 5% Pd/C catalyst (which is obtained by washing 5.0 g of 5% Pd/C containing 60% of water with 30 mL of acetic acid) in a flask. Change the atmosphere of the flask with hydrogen. Stir the mixture at room temperature and pressure of 1 atm of hydrogen for 2 h. Filter the mixture to remove the Pd/C catalyst and wash the filter cake with 10 mL of acetic acid. Combine the filtrate and washing solution and concentrate the solution at 20 C. and under vacuum to remove most the solvent. Add 100 mL of acetonitrile into the residue and stir the mixture at room temperature for 20 min. Filter the mixture to isolate the solid product. Dry the filter cake at room temperature and under vacuum to give 0.7 g of the white product.