USH1056H - Tripeptide, its preparation and use - Google Patents

Tripeptide, its preparation and use Download PDF

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Publication number
USH1056H
USH1056H US07/527,928 US52792890A USH1056H US H1056 H USH1056 H US H1056H US 52792890 A US52792890 A US 52792890A US H1056 H USH1056 H US H1056H
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US
United States
Prior art keywords
proline
group
arginyl
compound
leucyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US07/527,928
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English (en)
Inventor
Jean-Marie Bernard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rhodia Chimie SAS
Original Assignee
Rhone Poulenc Chimie SA
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Filing date
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Assigned to RHONE-POULENC CHIMIE, SERVICE BREVETS CHIMIE reassignment RHONE-POULENC CHIMIE, SERVICE BREVETS CHIMIE ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: BERNARD, JEAN-MARIE
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Publication of USH1056H publication Critical patent/USH1056H/en
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    • 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
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Definitions

  • the present invention concerns a tripeptide its preparation and use.
  • This tripeptide is an intermediate compound in an important synthesis, i.e., preparation of hormone permitting of the liberation of luteinizing hormone, which is also known by the abbreviation LHRH.
  • the LHRH hypothalamic hormone functions to regulate the secretion of gonadotrophic hormones. Because of the demand for it on a clinical level, numerous derivatives of the latter have been synthesized. The majority of these compounds involve, in the interior of their molecules, the tripeptide of the present invention, which has never, to the inventor's knowledge, previously been synthesized. This tripeptide, which is thus novel and is the object of the present application, has the following formula: ##STR1##
  • the tripeptide of the invention is more commonly known as Leucyl-Arginyl-Proline.
  • the tripeptide can also be protected on at least one of its acid and/or amine functions by one or more conventional protecting groups used in peptide synthesis, particularly such as those described by Gross and Meienhofer (The Peptides, Volume 3, Academic Press 1981), specifically incorporated by reference herein.
  • Representative protecting groups of the amine function include the following groups:
  • Illustrative protecting groups for the C-terminal acid function include:
  • ester groups particularly alkyl esters whose alkyl chains contain from 1 to 20 carbon atoms, more particularly methyl, ethyl, phenyl, benzyl and t-butyl esters;
  • amide groups which may either be primary or substituted on the nitrogen atom.
  • the tripeptide that forms the object of the present invention is prepared by a procedure that comprises:
  • a in a first step (a), putting L-nitroArginine, protected on the amine function thereof into contact with L-Proline protected on the acid function thereof in the presence of an activator and at least one base in a solvent for a time sufficient to obtain L-nitroArginyl-L-Proline dipeptide containing protecting groups;
  • a third step (c) putting the dipeptide resulting from the second step into contact in a solvent with L-Leucine protected on its amine function in the presence of an activator, at least one base and an auxiliary nucleophile for a time sufficient to obtain the compound of claim 1.
  • the protecting group of one of the amine functions of the L-nitroArginine is preferably a benzyloxycarbonyl group, the group protecting the second amine function is preferably a nitro group and the group protecting the acid function of the Proline is preferably a benzyl group.
  • the group protecting the amine function of the L-Leucine is also preferably a benzyloxycarbonyl group.
  • the condensation activator may be selected from among the following compounds:
  • Illustrative condensation activators include:
  • dimethoxytriazine chloride in slight excess with respect to the Arginine can cause the formation of an intermediate protected dimethoxytriazine-proline which is decomposed as the reaction proceeds.
  • dicyclohexylcarbodiimide can result in the appearance of a substantial precipitate of dicyclohexylurea, which can be difficult to separate, and thus is not preferred.
  • the molar ratio of the activating agent and the protected Arginine in step (a) preferably ranges from 1:1 to 1.5:1.
  • the molar ratio of the L-Proline to the L-nitroArginine in step (a) preferably ranges from 1:1 to 2:1.
  • the at least one base for activating and causing condensation in step (a) is preferably selected from tertiary nitrogen bases and alkali metal bases.
  • Preferred bases include: N-methyl-morpholine, triethylamine, sodium carbonate and diisopropylethylamine.
  • the base is preferably selected from amines that are soluble in organic media and that do not present a basicity so high that racemization of the Arginine could occur. It is therefore preferred to use hindered tertiary amine bases such as N-methylmorpholine, diisopropylethylamine and N-methylpiperidine.
  • the molar ratio of the base to the L-nitroArginine in the activation substep in step (a) preferably ranges from 1:1 to 2:1.
  • the hydrochloride of the protected proline is displaced, preferably with a relatively strong base having a pK greater than that of the protected proline. It is therefore preferable to use a base in the condensation substep in step (a) selected from triethylamine and sodium bicarbonate.
  • the molar ratio of the base to the L-Proline in the condensation substep in step (a) preferably ranges from 1:1 to 2:1.
  • the solvent used in the activation and condensation substeps of step (a) is preferably selected from dichloromethane, ethyl acetate, dioxane, tetrahydrofuran, dimethylformamide, t-butanol, toluene and dimethoxyethane. It is preferred to use dichloromethane.
  • reaction temperature used during the activation substep and during the coupling (condensation) substep of step (a) preferably ranges from -30° to 40° C. and more preferably ranges from -15° C. to ambient temperature.
  • the protected arginyl-proline dipeptide may be washed and then separated, for example, by crystallization from ethyl acetate.
  • the second step (b) consists of eliminating the protecting groups carried by the Arginine and the Proline and also in reducing the nitro group carried by the Arginine portion of the dipeptide. This double reaction is carried out by hydrogenation of the Arginyl-Proline dipeptide containing protecting groups in an acidic medium.
  • the hydrogenation of the second step may be carried out in the presence of a catalyst based on a noble metal, i.e., nickel, palladium or platinum, deposited on an inert support and in the presence of a mineral or organic acid.
  • a noble metal i.e., nickel, palladium or platinum
  • the noble metal is preferably palladium deposited on carbon.
  • a quantity by weight of noble metal from 0.5 to 5% with respect to the dipeptide, which corresponds to about 5 to 50% by weight of supported catalyst with respect to the weight of the dipeptide.
  • the hydrogenation is preferably carried out in a solvent made up of a mixture of carboxylic acid and water also containing at least two equivalents of a strong acid.
  • Each equivalent of strong acid can block the presence of guanidine and primary amine functions in the form of salts, thereby avoiding the formation of diketopiperazine.
  • the acid used in step (b) is preferably selected from hydrochloric acid, hydrobromic acid, sulfuric acid, p-toluenesulphonic acid and methanesulphonic acid. It is preferred to use hydrochloric acid.
  • the solvent preferably used in step (b) is made up of a mixture of carboxylic acid and water, and more preferably is a mixture of acetic acid and water containing 90% by volume of acetic acid.
  • the hydrogen pressure used in step (b) advantageously ranges from 1 to 20 bars and more preferably from 1 to 5 bars.
  • the reaction temperature in step (b) preferably ranges from 0° to 100° C. and more preferably from ambient temperature to 50° C.
  • the unprotected Arginyl-Proline dipeptide obtained in step (b) may be purified and separated by crystallization in ethyl acetate.
  • the third step (c) of the procedure according to the invention consists of condensing the Arginyl-Proline dipeptide with Leucine.
  • This condensation may be carried out in three substeps:
  • step (c) The substep of activation in step (c) is carried out by adding an activating agent preferably selected from isobutyl chloroformate and pivaloyl chloride.
  • Activating agents that are too onerous (Castro reagent), do not yield good results (methoxytriazine chloride) or involve the formation of secondary products that are difficult to separate (dicyclohexylcarbodiimide) are not preferred.
  • the preferred isobutyl chloroformate and pivaloyl chloride activators are only slightly onerous compounds and give only small quantities of secondary products, which are easily eliminated by crystallization.
  • the molar ratio of the activating agent to the L-Leucine in the activation substep of step (c) advantageously ranges from 0.5:1 to 1:1.
  • a mixed anhydride (pivaloyl-protected Leucine or isobutyl-protected Leucine) is helped in the activation substep of step (c) by the presence of a weak activating base, preferably selected from hindered tertiary amines such as N-methylmorpholine and diisopropylethylamine.
  • a weak activating base preferably selected from hindered tertiary amines such as N-methylmorpholine and diisopropylethylamine.
  • the molar ratio of the base to the Leucine advantageously ranges from 1:1 to 2:1.
  • the mixed anhydride is displaced by using an auxiliary nucleophile which serves to avoid the formation of a mixed anhydride between the protected Leucine and the Arginyl-Proline dipeptide (Z Leu-Arg-Pro-CO-O-CO-Leu-Z) by attack on the dipeptide Arg-Pro or tripeptide Leu-Arg-Pro carboxylate on the activated and protected Leucine.
  • an auxiliary nucleophile which serves to avoid the formation of a mixed anhydride between the protected Leucine and the Arginyl-Proline dipeptide (Z Leu-Arg-Pro-CO-O-CO-Leu-Z) by attack on the dipeptide Arg-Pro or tripeptide Leu-Arg-Pro carboxylate on the activated and protected Leucine.
  • auxiliary nucleophiles include:
  • hydroxybenzotriazole is preferred.
  • the molecular ratio of the auxiliary nucleophile to the Leucine advantageously ranges from 0.1:1 to 2:1 and preferably from 0.8:1 to 1.2:1.
  • step (c) The activation reaction in step (c) is carried out in the same solvents and under the same conditions as those described for the activation substep of the first step (a) of the process in accordance with the invention. It is preferred, however, to use dichloromethane or dimethylformamide at a temperature of around -15° C.
  • the condensation substep of step (c) may be carried out by introducing, into the medium containing the activated Leucine, the dipeptide in solution in a solvent selected from dimethylformamide, dichloromethane, trifluoroethanol, acetic acid, isopropanol, tetrahydrofuran, acetonitrile and dimethoxyethane. It is preferred to use a solvent made up of dimethylformamide and water.
  • the base used for the condensation substep of step (c) is preferably selected from sodium bicarbonate, triethylamine, tributylamine, and sodium acetate.
  • the molar ratio of the base to the Leucine in the condensation substep of step (c) advantageously ranges from 1:1 to 4:1, and more preferably from 1.5:1 to 2.5:1.
  • the condensation temperature advantageously ranges from -30° to 50° C., and more preferably from 0° C. to ambient temperature.
  • the molar ratio of activated Leucine to dipeptide in the condensation substep of step (c) advantageously ranges from 1.5:1 to 2:1.
  • the tripeptide obtained in step (c) may be extracted, preferably by a mixture of ethyl acetate and butanol, or more generally by a mixture of ester and alcohol that is not miscible with water.
  • the tripeptide may then be concentrated and precipitated by taking the medium up in isopropanol and pouring it into isopropyl ether.
  • reaction mixture is taken to a volume of 300 ml by adding 30 ml of diisopropyl ether and adding methylene chloride. Extraction is then conducted.
  • the organic phase is washed successively with 100 ml of water, 100 ml of 2.5 M potassium bicarbonate, three times with 50 ml of 2.5 M potassium bicarbonate, four times with 50 ml of N hydrochloric acid and finally with water until the pH of the aqueous phase is equal to 5.
  • the organic solution is concentrated to 100 ml, and 400 ml of ethyl acetate is added with slow stirring. After fifteen minutes, crystallization of the dipeptide begins.
  • the product is maintained in the cold for one night and filtered over a No. 3 fritted glass filter. The precipitate is washed with 50 ml of ethyl acetate and then dried under increasing vacuum.
  • the pivaloyl chloride enables the desired product to be obtained in a good yield and at good purity.
  • dichloromethane as a solvent permits a harmonious succession of reaction, extraction and crystallization.
  • the concentration is 20% at the beginning and 30% at the end (the Proline is added in powder form).
  • Ethyl acetate permits a substantially complete crystallization of the product and elimination of the impurities formed.
  • the solution is filtered on two Millipore filters (LC and LS) WP of porosity 10 microns and microns.
  • the palladium is rinsed with 2 ⁇ 25 ml of distilled water, and the solution is concentrated. 100 ml of water and then 3.12 ml of aqueous HCl are added and the mixture is concentrated under vacuum. The residue is taken up once in 100 ml of bidistilled water. Then the residual water is removed by addition of 100 ml of absolute ethanol and codistilled until dryness. This last operation is then repeated an additional time.
  • the residue obtained is taken up in 300 ml of a 50:50 (by volume) solution of methanol and ethyl acetate, then poured into one liter of ethyl acetate to obtain a white precipitate which is filtered over a No. 3 fritted glass filter.
  • the precipitate is washed with 100 ml of ethyl acetate and dried under increasing vacuum over P 2 O 5 at ambient temperature.
  • aqueous solution is left in the cold for one night.
  • a part of the precipitate of hydroxybenzotriazole is removed by filtration.
  • the remainder is extracted by several washings with diisopropyl ether (5 ⁇ 50 ml).
  • the desired product is extracted with a 50:50 mixture (by volume) of ethyl acetate and n-butanol.
  • the organic phase is then washed twice with 10 ml of N HCl, then twice with 15 ml of water.
  • the organic phase is then concentrated under vacuum at 30° C.
  • the viscous oil obtained is taken up in 50 ml of warm isopropanol (50° C.) and poured into 300 ml of isopropyl ether to obtain a white precipitate which is filtered and dried under increasing vacuum.
  • Table 2 contains examples of varying the activating agents, solvents, auxiliary nucleophiles and other conditions, along with the results obtained from each.
  • the process of invention also comprises the step of hydrogenating L-nitroArginyl-L-Proline dipeptide containing protecting groups in an acid medium for a time sufficient to eliminate the protecting groups and to reduce the nitro radical in the L-nitroArginine portion of the dipeptide.
  • the invention is directed to a process comprising the step of hydrogenating an L-nitroArginyl-L-Proline dipeptide - containing aromatic amino acid, wherein said L-nitroArginyl-L-Proline dipeptide contains protecting groups, in an acid medium for a time sufficient to eliminate the protecting groups and to reduce the nitro radical in the L-nitroArginine portion of the dipeptide.
  • the claimed invention can be used in synthesizing polypeptide-containing aromatic amino acids.
  • Illustrative amino acids include dihydroxyphenylalanine, tyrosine, phenylalanine and particularly, tryptophan.
  • an L-nitroArginyl-L-Proline moiety containing protecting groups can be bonded to a compound containing a aromatic amino-acid. Then, in accord with the hydrogenation step explained above, the nitroArginyl portion of the peptide can be reduced for a time sufficient to eliminate the protecting groups on the L-nitroArginyl-L-Proline portion of the molecule and also to reduce the nitro radical in the L-nitroArginyl portion of the molecule.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
US07/527,928 1989-05-24 1990-05-24 Tripeptide, its preparation and use Abandoned USH1056H (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8906777A FR2647453A1 (fr) 1989-05-24 1989-05-24 Nouveau tripeptide, son procede de preparation et son utilisation
FR8906777 1989-05-24

Publications (1)

Publication Number Publication Date
USH1056H true USH1056H (en) 1992-05-05

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US07/527,928 Abandoned USH1056H (en) 1989-05-24 1990-05-24 Tripeptide, its preparation and use

Country Status (5)

Country Link
US (1) USH1056H (fr)
EP (1) EP0399885A1 (fr)
JP (1) JPH035499A (fr)
CA (1) CA2017357A1 (fr)
FR (1) FR2647453A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4242256A (en) 1977-03-15 1980-12-30 Robert Sharpe Synthesis of peptide analogues
US4954616A (en) 1987-06-19 1990-09-04 Solvay & Cie (Societe Anonyme) Use of guanidine-related compounds comprising a substituted tetraphenylborate ion in solution phase peptide synthesis

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3855198A (en) * 1972-09-29 1974-12-17 American Home Prod Novel intermediates for synthesis of l-(5-oxoprolyl)-l-histidy-l-tryptophyl-l-seryl-l-tyrosyl-l-glycyl-l-leucyl-l-arginyl-l-prolyl-glycine amide
CH611878A5 (fr) * 1974-07-04 1979-06-29 Takeda Chemical Industries Ltd
BE847761A (fr) * 1975-10-29 1977-02-14 Nouveaux octapeptides et procedes pour les produire,
HU185238B (en) * 1980-02-12 1984-12-28 Takeda Chemical Industries Ltd Process for preparing peptides containing guanidino-group

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4242256A (en) 1977-03-15 1980-12-30 Robert Sharpe Synthesis of peptide analogues
US4954616A (en) 1987-06-19 1990-09-04 Solvay & Cie (Societe Anonyme) Use of guanidine-related compounds comprising a substituted tetraphenylborate ion in solution phase peptide synthesis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Gross and Meienhofer, "Reversible Blocking of Amino and Carboxyl Groups," The Peptides, vol. 3, Chapter III, pp. 59-118 (Academic Press 1981).
Gross et al., eds., The Peptides, 1980, vol. 2, pp. 101-103, 106-114, 118, and 169-173.

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Publication number Publication date
EP0399885A1 (fr) 1990-11-28
CA2017357A1 (fr) 1990-11-24
FR2647453A1 (fr) 1990-11-30
JPH035499A (ja) 1991-01-11

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