Process for the production of 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 trifluoroacetic acid salt, ##STR00014## which comprises the following steps: coupling compound (II) H-Phe-NH2: ##STR00015## with compound (III) Z-Lys(Boc)-OH: ##STR00016## to obtain a compound of formula (IV), Z-Lys-Lys(Boc)-Phe-NH2: ##STR00017## reacting compound (IV) with hydrogen and methanesulfonic acid (V)
MeSO3H(V) in the presence of a catalyst to obtain the free amine salt (VI) MeSO3H.H-Lys(Boc)-Phe-NH2: ##STR00018## reacting salt (VI) with the protected amino acid Z-Dmt(Boc)-OH (VII) ##STR00019## to obtain the protected tripeptide Z-Dmt(Boc)-Lys(Boc)-Phe-NH2 (VIII): ##STR00020## treating compound (VIII) with hydrogen and methanesulfonic acid (V) to obtain the corresponding salt MeSO3.H-Dmt(Boc)-Lys(Boc)-Phe-NH2 (IX): ##STR00021## coupling the acid salt (IX) with Z-D-Arg-ONa (X) ##STR00022## to form the protected tetrapeptide Boc-D-Arg-Dmt(Boc)-Lys(Boc)-Phe-NH2 (XI): ##STR00023## deprotecting compound (XI) to obtain the tetrapeptide H-D-Arg-Dmt-Lys-Phe-NH2 (I) and further salifying it with trifluoroacetic acid in solvents.

2. A process according to claim 1 wherein the deprotection is performed by simultaneous acidolysis of the three Boc groups with organic acids.

3. A process according to claim 1 wherein the deprotection is performed by simultaneously acidolysis of the three Boc group with trifluoroacetic acid, without use of Pd catalysts.

4. A process according the claim 1 wherein the acidolysis can be performed with other acids such as HCl or HBr, leading to the corresponding salts.

5. A process according to claim 1 wherein the final product (I) is obtained in solid crystalline form of the trifluoroacetate salt after simple crystallization without any need of HPLC purification or any freeze-drying.

6. A process according the claim 1 wherein the coupling between (II) and (III) is performed in the presence of N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uranium tetrafluoroborate (known as TBTU) and polar solvents such as NMP, DMF, acetonitrile, DMSO and THF.

7. A process according the claim 1 wherein coupling between (II) and (III) is performed in a temperature range between 10 C. and 50 C.

8. A process according the claim 1 wherein formation of methanesulfonic salt (VI) is obtained in methanol, NMP, acetonitrile or THF as solvent and crystallized from the same solvent.

9. A process according the claim 1 wherein the coupling reaction between compound (IX) and compound (X) is performed in the presence of N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uranium tetrafluoroborate (TBTU) and polar solvents selected from NMP, DMF, acetonitrile, DMSO and THF.

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

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

12. A compound of formula (XI) Boc-D-Arg-(2,6-dimethyl)Tyr(Boc)-Lys(Boc)-Phe-NH2: ##STR00026##

Description

DETAILED DESCRIPTION

(1) The present invention provides, in a first aspect, a novel, efficient process that provides 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 trifluoroacetic acid salt

(3) ##STR00003## which comprises the steps of: coupling compound (II) H-Phe-NH.sub.2:

(4) ##STR00004## with compound (III) Z-Lys(Boc)-OH:

(5) ##STR00005## to obtain a compound of formula (IV), Z-Lys-Lys(Boc)-Phe-NH.sub.2:

(6) ##STR00006## and reacting the compound (IV) with hydrogen and methanesulfonic acid (V)
MeSO.sub.3H(V) in the presence of a catalyst to obtain the free amine salt (VI) MeSO.sub.3H.H-Lys(Boc)-Phe-NH.sub.2:

(7) ##STR00007##

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

(9) ##STR00008## to obtain the protected tripeptide Z-Dmt(Boc)-Lys(Boc)-Phe-NH.sub.2 (VIII):

(10) ##STR00009##

(11) which is treated with hydrogen and methanesulfonic acid (V) to obtain the corresponding salt MeSO.sub.3.H-Dmt(Boc)-Lys(Boc)-Phe-NH.sub.2 (IX):

(12) ##STR00010##

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

(14) ##STR00011##

(15) to form the protected tetrapeptide Boc-D-Arg-Dmt(Boc)-Lys(Boc)-Phe-NH.sub.2 (XI):

(16) ##STR00012##

(17) The tetrapeptide H-D-Arg-Dmt-Lys-Phe-NH.sub.2 (I) is obtained by deprotection and further salt formation with trifluoroacetic acid in solvents of (XI). Deprotection is performed by simple acidolysis of the three Boc groups without use of Pd catalysts which can cause the presence of Pd in the final compound.

(18) The same acidolysis can be performed with other acids such as HCl or HBr, leading to the corresponding salts. This process allows obtaining the peptide as a solid which can be used in formulation as such or can be converted to any other salt if required. The purity of the crude final compound thus obtained is 97% without any additional crystallization and can be easily improved to 99% by selection of the appropriate crystallization mixture.

(19) 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)uranium tetrafluoroborate (known as TBTU) and an organic base belonging to the class of tertiary amines such as NMM, triethylamine and diisopropylamine as well as polar solvents as NMM, DMF, acetonitrile, tetrahydrofuran (THF), 2-methyl-tetrahydrofuran (2-Me-THF), etc.

(20) 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.

(21) The hydrogenation of (IV) can be accomplished with various techniques such as homogeneous catalytic hydrogenation, heterogeneous catalytic hydrogenation or catalytic transfer hydrogenation. In a particular embodiment, hydrogenation is performed with hydrogen gas and Pd on carbon as catalyst.

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

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

(24) 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)uranium tetrafluoroborate (TBTU) and an organic base belonging to the class of tertiary amine such as NMM, triethylamine and diisopropylamine as well as polar solvents as NMM, DMF, acetonitrile, THF, 2-Me-THF, etc.

(25) In another embodiment, the hydrogenation of (VIII) can be accomplished with various techniques as homogeneous catalytic hydrogenation, heterogeneous catalytic hydrogenation or catalytic transfer hydrogenation. In a particular embodiment, the hydrogenation is performed with hydrogen gas and Pd on carbon as catalyst.

(26) In another embodiment, the formation of methanesulfonic salt (IX) is obtained with methanesulfonic acid (V) in dimethylformamide as solvent and crystallized from the same solvent. Other solvents for crystallization are THF, ethyl acetate and acetonitrile.

(27) 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)uranium tetrafluoroborate (known as TBTU) and an organic base belonging to the class of tertiary amine such as NMM, triethylamine and diisopropylamine as well as polar solvents as NMM, DMF, acetonitrile, THF, 2-Me-THF, etc. In one embodiment the deprotection of (XI) is performed with trifluoroacetic acid and solvents. The most appropriate solvents are heptanes, IPA, etc.

(28) 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%.

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

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

(31) ##STR00013##

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

(33) In another preferred aspect the final deprotection is performed by reaction with trifluoroacetic acid and allows to obtain the final product in crystalline solid form of trifluoroacetate salt after simple crystallization without any need of HPLC purification or any freeze-drying, which are purification and isolation processes extremely expensive but commonly used in the manufacture of peptide as drug.

(34) In a preferred aspect, the process allows 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 final compound thus obtained is 99% and each impurity is approx. 0.2% or below.

EXAMPLES

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

(35) Charge 100 mL of DMF, 9.5 g of H-Phe-NH.sub.2, 20 g of Z-Lys(Boc)-OH and 10.6 g of N-methylmorpholine in a flask. Stir the mixture at 20-22 C. for 15 min. Add 18.6 g of TBTU and stir the mixture at room temperature overnight. Add 200 mL of methanol and 250 mL of water into the mixture and stir the mixture at room temperature for 1 h. Filter the mixture to isolate the solid product. Transfer the filter cake into a flask containing 200 mL of methanol. Heat the mixture at refluxing for 1 h, and then cool down to room temperature. Filter the mixture to isolate the solid product. Dry the filter cake at 35-40 C. and under vacuum to obtain 20.5 g of the white solid product.

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

(36) Charge 100 mL of DMF, 12.5 g of 5% Pd/C (60% water content), 10 g of Z-Lys(Boc)-Phe-NH.sub.2 and 1.83 g of methanesulfonic acid into a flask. Change the atmosphere of the flask with hydrogen. Stir the mixture at 20-25 C. and under 1 atm hydrogen for 3 h. HPLC analysis shows that all the Z-Lys(Boc)-Phe-NH.sub.2 was converted. The resultant mixture was directly used in the next step.

Example 3: Preparation of Z-Dmt(Boc)-Lys(Boc)-Phe-NH2 (VIII)

(37) Charge 8.5 g of Z-Dmt(Boc)-OH and 6.1 g of TBTU into the DMF solution of MeSO.sub.3H.H-Lys(Boc)-Phe-NH.sub.2 obtained in last step. Stir the mixture at room temperature for 15 min. Cool down the mixture to 10-15 C. Add 5.8 g of N-methylmorpholine slowly while keeping the temperature below 20 C. Stir the mixture at 10-15 C. for 18 h. Filter the mixture to remove the Pd/C catalyst and other precipitates and wash the filter cake with DMF. Combine the filtrate and the washing solution and add 32 mL of methylene chloride into the filtrate. Add the resultant solution into a flask containing 500 mL of water, and then stir the mixture for 20 min. Filter the mixture to isolate the solid product and wash the filter cake with water three times. Transfer the filter cake into a flask containing 450 mL of acetone and heat the mixture to refluxing under stirring. At refluxing and under stirring add 200 mL of hexanes and cool down the mixture to room temperature. Filter the mixture to isolate the solid product. Repeat the previous work-up operations. Dry the resultant filter cake at 40 C. and under vacuum to give 12.4 g of the product.

Example 4: Preparation of MeSO3H.H-Dmt(Boc)-Lys(Boc)-Phe-NH2 (IX)

(38) Charge 12.4 g of Z-Dmt(Boc)-Lys(Boc)-Phe-NH.sub.2, 100 mL of DMF, 12.4 g of 5% Pd/C (60% water content) and 1.46 g of methanesulfonic acid into a flask. Change the atmosphere of the flask with hydrogen. Stir the mixture at 20-25 C. and under 1 atm hydrogen for 3 h. HPLC analysis shows that all the Z-Lys(Boc)-Phe-NH.sub.2 was converted (HPLC purity 98.1%). The resultant mixture is directly used in the next step.

Example 5: Preparation of Boc-D-Arg-Dmt(Boc)-Lys(Boc)-Phe-NH2 (XI)

(39) Charge 8.5 g of Boc-D-Arg-OH, 8.84 g of TBTU and 9.7 g of N-methylmorpholine into the DMF mixture of H-Dmt(Boc)-Lys(Boc)-Phe-NHe.MeSO.sub.3H at room temperature. Stir the mixture at room temperature for 40 h. Filter the mixture to remove the Pd/C and other precipitates. Add 250 mL of ethyl acetate into the filtrate. Wash the organic solution with water (200 mL4). Concentrate the organic solution at 40-45 C. and under vacuum to remove most of the solvent. Add 100 mL of MTBE to the residue and stir the mixture vigorously at room temperature for 1 h. Filter the mixture to isolate the solid product. Transfer the filter cake into a flask containing 200 mL of ethyl acetate. Heat the mixture at 50-55 C. and under stirring for 3 h, and then cool down to room temperature. Filter the mixture to isolate the solid product. Repeat the previous work-up operations. Dry the filter cake at 40-45 C. and under vacuum to obtain 10.7 g of white solid product. HPLC purity 98.2%.

Example 6: Preparation of H-D-Arg-Dmt-Lys-Phe-NH2 (I), (MTP-131)

(40) Charge 1.0 g and 10 mL TFA in a flask. Stir the mixture at room temperature for 20 min. Add 10 mL of IPA to the reaction mixture. Add the resultant mixture slowly into a flask containing 40 mL of heptane under vigorous stirring. Filter the mixture to isolate the solid product and wash the filter cake with IPA. Dry the filter cake at 40-45 C. and under vacuum to obtain 0.5 g of solid product. Analytical Data: HPLC purity: 96.6%.