Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1027—Tetrapeptides containing heteroatoms different from O, S, or N

Definitions

• the present invention is directed to a new process for the preparation of a tetrapeptide, more specifically the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe- NH2, or a pharmaceutically acceptable salt thereof.
• the present invention also relates to new intermediates used in process.
• the present invention relates to a new process for the preparation of the peptide H-Tyr-D- Ala-Phe(pF)-Phe-NH2' or a pharmaceutically acceptable salt thereof.
• WO 97/07129 discloses a process for producing inter alia the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH 2 .
• the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH is also disclosed in WO 97/07130.
• Said peptide exhibits peripheral analgesic activity and selectivity for the ⁇ 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 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 compared to methods known in the art. Furthermore, the process of the present application provides the product in a higher yield.
• 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 containing as few reaction steps as possible.
• the present invention provides a new process for large scale preparation of the peptide H- Tyr-D-Ala-Phe(pF)-Phe-NH2, which is a peptide of the formula (I)
• A is an amino protecting group
• R is an activating agent residue group; 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 , wherein R is the ester or amide residue group, in the presence of a solvent, providing a protected dipeptide derivative (TV)
• A is an amino protecting group, and R is an ester or an amide residue group
• R is an ester or an amide residue group
• A is an amino protecting group
• R is an activating agent residue group; previously prepared by a pre-activation step or generated in situ, is reacted with the product of step 1, i.e. the dipeptide derivative (5) in the presence of a solvent, providing the protected tripeptide derivative(VIII)
• A is an amino protecting group
• R is an ester or an amide residue group
• R is an ester or an amide residue group
• A is an amino protecting group
• R is an activating agent residue group
• A is an amino protecting group
• R is an ester or an amide residue group
• the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH2 (I), may if desired be reacted with a pharmaceutically acceptable acid, such as AcOH, H3PO4, citric acid, lactic acid and HCI.
• 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. Pharmaceut. Sci, 66(1977) 1-19.
• Step 2 (i) A coupling step, (ii) A deprotection step
• step 3(ii) could instead be performed after the coupling step (i) in Step 1 or step 2.
• the transformation step is performed after the coupling step (i) in step 1.
• the preferred way of performing the process of the present invention could therefore schematically be described as comprising the following steps;
• Step 1 (i) A coupling step
• Step 2 (i) A coupling step, (ii) A deprotection step
• the N -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.
• 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.l, Eds.: E. Gross & J.
• 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 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 suitable protecting group such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z)
• A is an amino protecting group
• R is an activating agent residue group
• Step 1-3 a variety of powerful solvents may be used, as long as the amino component is essentially soluble and available for immediate reaction with the activated peptide derivative.
• suitable solvents for the coupling step are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP) and EtOAc, or mixtures thereof.
• benzyl-like group denotes any substituted or un-substituted benzyl group that is hydrogenolyzed under similar reaction conditions as the benzyloxycarbonyl group.
• 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, activated esters, azide, or anhydrides.
• Isobutylchloroformiate iBuOCOCl
• isobutylchloroformiate iBuOCOCl
• the activated peptide derivative will have the following structure, exemplified on D-alanine,
• the tertiary amine may be selected from any tertiary amine.
• NMM N-methylmorpholine
• di-isopropylethylamine and triethylamine are preferred.
• a secondary amine which is sterically hindered may also be used.
• 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.
• 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 O 99/47548
• 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.
• 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 0°C to -20°C, and particularly preferred is from -5°C to -15°C.
• the rate of addition is adjusted so that the preferred temperature is maintained.
• 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 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.
• Step 3(ii) is only required if the protected tetrapeptide derivative (XI) 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.
• 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, Vol.l, Eds.: E. Gross & J. Meienhofer, Academic Press, Inc, London 1979, pp. 264-309. 12
• the activation time can be extended to at least 30 min at a temperature about 0 - -15°C, contrary to the recommended 1-2 min at -15°C.
• 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 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°C and the reaction mixture is then allowed to reach 20-30°C in about 30-60 min. or longer before crystallization of the product is initiated directly from the reaction mixture.
• R is an ester or an amide residue group
• target compound (I) is a useful intermediate for the preparation of target compound (I).
• Z-Phe(pF) (compound 1)(1 eq.) is first dissolved in acetone (4.7L/mole) and cooled before 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°C is recommended (from -9°C to -14°C actual) where the reaction occurs immediately upon addition of NMM, yet prevents the mixed anhydride from decomposing to rapidly.
• 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°C (about -10°C actual).
• This slurry is upon completion of the activation added at a rate that maintains the temperature around -10°C (from -8°C to -13°C actual) (about 30 minutes), 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.
• 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°C and for more than 5 hours or 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 °C. Yield 74% calculated from compound 1 (Z-Phe(pF) ) and 100% purity. 19
• Compound 4 prepared in the previous step is mixed with DMF (4.2L/mole) and and a Pd/C 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°C and 3bar H 2 .
• the reaction mixture is then filtered and cooled to about -15°C before the next step. 99.6% purity in solution.
• 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(l-2h actual run) at 20-40°C (20-25°C actual 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 lOL/mole).
• EtOAc typically lOL/mole
• the free base compound I is dissolved in a mixture of water and acetone with one equivalent HCI added and clear filtered ( 146g/mole 25% HCl/H 2 O, 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 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°C.
• the solid is then separated by centrifugation or filtration and washed with MIBK before drying under vacuum at 20-50°C for more than 16 hours or until the solvent levels are lower than specified in the release specifications.
• Z-Phe(pF) (1 eq.) is first dissolved in acetonitrile (EtOAc)(1.7L/mole) and cooled before addition of /-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 - 15°C is recommended (from -8°C to -11°C actual) where the reaction occurs immediately upon addition of N-Methylmorpholine, yet prevents the mixed anhydride from decomposing to rapidly.
• H-Phe- ⁇ H 2 x HCI (0.9-1.3 eq.) (1.04 eq actual) is meanwhile dissolved in DMF (4.0L/mole), neutralized with N-Methylmorpholine (0.9- 1.5 eq.) ( 1.04eq actual) and cooled to 0 - -20°C (about -10°C actual). This slurry is upon completion of the activation added at a rate that maintains the temperature around -10°C (from -6°C to -13°C actual) (about 15 minutes) 8 min actual.
• the product was crystallized from the reaction mixture by 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°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°C. Yield 90% and 99.9% purity.
• Z-Phe(pF)-Phe-NH 2 prepared in the previous step is mixed with DMF (3.5L/mole) and a Pd/C catalyst (5% Pd actual content) is added (0.2-10% w/w / LEF-582) (5% actual) and 28
• the resulting mixture hydrogenated for more than 0.5 hours (1.3h actual) at 25-30°C and about 3bar H .
• the reaction mixture is then filtered and cooled to about -15°C before the next step. 99.6% purity in solution and >99% conversion of starting material.
• 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- ⁇ H 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°C (from -8°C to -11°C actual). After completion of the coupling the product was crystallized from the reaction mixture by slow addition of water
• Z-D-Ala-Phe(pF)-Phe-NH 2 prepared in the previous step is mixed with DMF (2.9L/mole) 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°C and about 3bar H .
• the reaction mixture is then filtered and cooled to about -15°C before the next step. Purity 99.4%. Conversion of starting material >99% 29
• the product was crystallized from the reaction mixture at about 20-45°C by slow addition of acetonitrile and water (3.4L/mole MeCN + 0.9L/mole 15% NH 3 in H 2 O hold 5min and seed, hold 4-24h, then add a total of 13.9L/mole H 2 O in four portions with about 30min or longer hold in between each. Filter or centrifuge and wash first with water and then MeCN before optional drying under vacuum at 20-60°C.
• Z-Tyr-D-Ala-Phe(pF)-Phe-NH 2 is mixed with DMF (2.6L/mole actual run) and a 5 % Pd/C (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°C (20- 25°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 30
• 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% HCl/H 2 O, 2L 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.
• 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.
• 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 27.0° C on a Varian UNITY plus 400 MHz instrument.
• Chemical shift reference for proton spectra was the middle peak of the DMSO-c multiplet taken as 2.49 ppm.
• Reference for carbon spectra was the middle peak of the DMSO-d multiplet taken as 39.5 ppm.
• Atom numbering used in assignment is arbitrary and refers to the figure above.
• PROTON SPECTRA The one dimensional proton spectrum allows groupwise assignment of alpha protons (3.9 4.4 ppm), benzyl-CH2 (2.6-3.1 ppm), amide-NH and phenol-OH (8.2-8.5 ppm) and also specific assignment for Ala-CH3 (I4-CH3) (0.74 ppm).
• the two dimensional DQFCOS Y spectrum allows for groupwise assignment of the spin 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.
• the one dimensional carbon spectrum allows for groupwise assignment of alpha carbons, benzyl-CH2, 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 C-F couplings allows specific assignment of the carbons in the fluoroaromatic ring.
• the two dimensional carbon-proton correlated (HMQC) spectrum 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 correlated (HMBC) spectrum gives a correlation between carbons and protons situated two to three bonds apart. This allows 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-Qf ⁇ .and aryl carbons as well as between aryl protons and benzyl-CH2 allows specific assignment of aryl protons and carbons of the individual aromatic amino acids.

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