WO2019198834A1 - Procédé de production de leuproréline - Google Patents

Procédé de production de leuproréline Download PDF

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Publication number
WO2019198834A1
WO2019198834A1 PCT/JP2019/016069 JP2019016069W WO2019198834A1 WO 2019198834 A1 WO2019198834 A1 WO 2019198834A1 JP 2019016069 W JP2019016069 W JP 2019016069W WO 2019198834 A1 WO2019198834 A1 WO 2019198834A1
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Prior art keywords
group
carrier
formula
carbon atoms
peptide
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PCT/JP2019/016069
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English (en)
Japanese (ja)
Inventor
一郎 嶋
なつみ 岩永
鈴木　康介
秀司 藤田
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Jitsubo株式会社
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Priority to JP2019548093A priority Critical patent/JP6703669B2/ja
Publication of WO2019198834A1 publication Critical patent/WO2019198834A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a novel method for producing leuprorelin.
  • Leuprorelin acetate is an LH-RH (GnRH) agonist and is a drug applied to endometriosis, uterine fibroids, premenopausal breast cancer, prostate cancer, central precocious puberty and the like.
  • GnRH LH-RH
  • Patent Document 1 JP-A-50-59370 (corresponding to US Pat. No. 4,008,209) (Patent Document 1) includes a general formula ( Pyr) Glu-His-Trp-Ser-Tyr (or Phe) -X-Leu (or Ile or Nle) -Arg-Pro-NH-R [wherein amino acids are not specified, indicate L-form, X Represents D-Leu, D-Nle, D-NVal, D-Ser, D-Abu, D-Phg, D-Phe or ⁇ -Aibu, and R represents an alkyl group which may have a hydroxyl group].
  • the following liquid phase synthesis method is described as a method for producing the peptide to be prepared. formula,
  • Patent Document 2 discloses a general formula: 5-oxo-Pro-R1-Trp-Ser-R2-R3-OH (II) [wherein R1 is His, Tyr, Trp or p-NH 2 -Phe, R2 represents Tyr or Phe, and R3 represents an optionally substituted Gly or ⁇ -D-amino acid residue. Or a salt thereof represented by the general formula: H-R4-R5-Pro-R6 (III) [wherein R4 represents Leu, Ile or Nle, R5 represents protected Arg, and R6 represents formula Gly.
  • Non-Patent Document 1 describes a liquid phase synthesis method of PyroGlu-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (Compound I) comprising the following steps.
  • Patent Document 3 discloses a method for producing leuprorelin by a solid phase synthesis method using para (oxymethyl) phenylacetamide resin (PAM resin). .
  • the reaction time of the unit process is shorter than that in the liquid phase synthesis.
  • the yield up to the condensation / resin cutting step is as high as 92.5% in terms of purity, but the total yield including the purification step is as low as 36%, and the crude product It is described that the recovery rate at the time of purification is low. This suggests that impurities contained in the crude product are difficult to remove, and when obtaining high-purity leuprorelin, it is necessary to discard the eluate containing the target product during purification. As described above, there is no existing leuprorelin production method that can be produced in a short time and can obtain the target product with high purity and high yield.
  • An object of the present invention is to provide a liquid phase peptide synthesis method for leuprorelin, which has a shorter production period and can obtain a crude product having a high purity as compared with conventional production methods.
  • the inventors of the present invention have proposed a liquid phase synthesis method for peptides.
  • a specific carrier is used and a Tag-peptide component is synthesized.
  • the inventors have found that the process time can be shortened by removing impurities without performing solid-liquid separation (concentration, solid-liquid separation, and drying operation), and the present invention has been completed.
  • the present invention includes the following.
  • a method for producing leuprorelin comprising the following sequence: H-Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (SEQ ID NO: 1), comprising the following steps a to d: a.
  • An amino acid, carrier-protected amino acid amide or carrier-protected peptide protected with a carrier for liquid phase peptide synthesis in an organic solvent or a mixture of organic solvents, and a 9-fluorenylmethyloxycarbonyl group (Fmoc group)
  • the liquid phase peptide synthesis carrier is a compound that binds directly to amino acids or peptides and renders them insoluble in water and has a molecular weight of 300 or more
  • the carrier-protected amino acid, amino acid amide or peptide is an amino acid or peptide in which the carrier is bonded to the carboxyl terminus of the amino acid or peptide, and
  • the water-soluble amines in step b and step c may be the same or different.
  • Step e A weak basic aqueous solution having a pH of 8 to 12, preferably 8 to 10, is added to the organic layer obtained in Step d, followed by liquid separation, separation of the aqueous layer, and removal of the carrier.
  • the liquid phase peptide synthesis carrier is a compound having the following structure:
  • R 2 , R 4 and R 5 are hydrogen atoms
  • R 1 and R 3 are alkoxy groups having 12 to 30 carbon atoms, preferably 18 to 22 carbon atoms
  • RY is —CH 2 OH or —CH 2 NHEt), or a compound having the following structure:
  • X is —CH 2 OH or —CH 2 NHEt
  • R 2 , R 4 and R 5 are hydrogen atoms
  • at least one of R 1 and R 3 is represented by the following formula: —O—R 6 —Xa-A
  • the remaining group represents an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms
  • R 6 represents a linear or branched alkylene having 1 to 16 carbon atoms
  • Xa represents O or CONRc (where Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms);
  • A represents any one of formulas (1) to (11),
  • R 7 , R 8 and R 9 may be the same or different and each represents a linear or branched alkyl group having 1 to 6 carbon atoms, or an aryl group which may have a substituent
  • R 10 represents a single bond or a linear or branched alkylene group having 1 to 3 carbon atoms
  • R 11 , R 12 and R 13 may be the same or different, and may be a straight chain having 1 to 3 carbon atoms or Represents a branched alkylene group
  • each said formula is shown in the state before couple
  • the production method according to the above [1] which is a carrier derived from the above.
  • the carrier for liquid phase peptide synthesis has the following structure: Formula (V):
  • ring A represents an aromatic ring
  • Y represents a hydroxyl group, a bromo group or a chloro group
  • Ra, Rb and Rc each independently represents an organic group, a hydrogen atom or an electron having an aliphatic hydrocarbon group
  • An attraction group, and at least one of Ra, Rb and Rc is an organic group having an aliphatic hydrocarbon group
  • the rings A, B and C may each independently have an electron withdrawing group; Good], or general formula (V ′):
  • Rings A, B, and C may each independently have one or more selected from an organic group having an aliphatic hydrocarbon group and an electron-withdrawing group;
  • Each ring A may be the same or different;
  • each Y in the case where a plurality of Ys are present may be the same or different;
  • each Rc ′ in the case where a plurality of Rc ′s are present is the same or different.
  • the divalent organic group having an aliphatic hydrocarbon group is represented by the formula (a):
  • Xa does not exist or represents —O—, —S—, —NHCO— or —CONH—;
  • Rd represents an aliphatic hydrocarbon group having 5 or more carbon atoms;
  • k 1 represents 1 to 10
  • the organic group having an aliphatic hydrocarbon group is present at the 2-position and / or the 7-position of the fluorene compound, the formula (b):
  • R 3 is an organic group having an aliphatic hydrocarbon group having 5 to 60 carbon atoms; n 1 , n 2 , n 3 and n 4 are 1; m 2 is 1 A group represented by formula (d):
  • Y represents a hydroxyl group or NHEt group
  • R a is Formula (a):
  • m 1 represents an integer of 1 to 10; and m 1 X 1 s each independently represent a single bond, or —O—, —S -, -COO-, -OCONH-, -NHCO- or -CONH-; each of R 1 and m 1 R 2 independently represents a divalent aliphatic hydrocarbon group having 5 or more carbon atoms; And R 3 is a hydrogen atom or the formula (W ′):
  • n R b s are each independently a carbon atom having 1 to 6 carbon atoms, a halogen atom, or a carbon atom that may be substituted with one or more halogen atoms
  • n 1 , n 2 , n 3 and n 4 each independently represents an integer of 0 to 2; m 2 X 2 , m 2 X 2 ′ and m 2 X 2 ′′ each independently represent a single bond, or —O—, —S—, —COO—, —OCONH—, —NHCO— or —CONH—; each of m 2 R 4 and m 2 R 6 independently represents an aliphatic hydrocarbon group having 5 or more carbon atoms; R 5 represents 5 carbon atoms; A group represented by the above) Formula (c):
  • n 3 is from 0 to an integer of 15; n 5 is an integer of 0 to 11; n 6 represents an integer of 0 to 5; m 3 amino X 3 each independently represents a single bond, or —O—, —S—, —COO—, —OCONH—, —NHCO— or —CONH—; and m 3 R 7 are Each independently represents a hydrogen atom, a methyl group or an aliphatic hydrocarbon group having 5 or more carbon atoms; and the formula (d):
  • n 7 X 4 s each independently represent a single bond, or —O—, —S—, —COO—, —OCONH—, —NHCO) - or it shows a -CONH-;
  • R 8 represents a divalent aliphatic hydrocarbon group;
  • n 7 amino R 9 are each independently a monovalent aliphatic hydrocarbon group;
  • n 7 is 1 represents an integer of 1 to 5; and
  • Ar represents an arylene group), and represents an organic group having an aliphatic hydrocarbon group selected from the group consisting of: 30 or more; each of n R b s is independently an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or an alkyl having 1 to 6 carbon atoms which may be substituted with one or more halogen atoms.
  • n an integer of 0 to 4.
  • the liquid phase peptide synthesis carrier has the following structure: Formula (X)
  • Y represents a hydroxyl group or —NHEt group; k and l each independently represents an integer of 0 to 5; provided that k + 1 is not 0; k Ra and l R b of each independently Formula (a):
  • M 2 X 2 , m 2 X 2 ′, m 2 X 2 ′ ′′ and m 2 X 2 ′′ are each independently absent or —O -, -S-, -COO-, -OCONH- or -CONH-; each of m 2 R 2 and R 4 independently represents a hydrogen atom, a methyl group or an aliphatic carbon atom having 5 or more carbon atoms A hydrogen group; R 3 represents an aliphatic hydrocarbon group having 5 or more carbon atoms), and a group represented by formula (e):
  • k Q's each independently represent a single bond, or —O—, —S—, —C ( ⁇ O) O—, —C ( ⁇ O) NH— or —NH
  • k R a each independently has at least one aliphatic hydrocarbon group having one or more branched chains, the total number of branched chains is 3 or more, and the total number of carbon atoms is 14
  • k represents an integer of 1 to 4
  • R 1 is a hydrogen atom, or when Z is a group represented by the following formula (a):
  • R 2 together with R 2 may form a fluorene ring with ring B;
  • ring A is in addition to R 1 , k QR a , and C (X) (Y) Z And further substituted with a halogen atom, a C1-6 alkyl group optionally substituted by one or more halogen atoms, and one or more halogen atoms.
  • X represents a hydrogen atom or a phenyl group
  • Y represents a hydroxyl group or —NHEt group
  • Z represents a hydrogen atom or formula (a):
  • ring B may further contain a halogen atom, 1
  • substituents selected from the group consisting of a C1-6 alkyl group optionally substituted by one or more halogen atoms and a C1-6 alkoxy group optionally substituted by one or more halogen atoms
  • the liquid phase peptide synthesis carrier is a compound having the following structure:
  • R 2 , R 4 and R 5 are hydrogen atoms, R 1 and R 3 are alkoxy groups having 12 to 30 carbon atoms, preferably 18 to 22 carbon atoms, and RY is —CH 2 OH.
  • the above formula is shown in a state before binding to the carboxyl group of amino acid or peptide
  • the liquid phase peptide synthesis carrier is a compound having the following structure:
  • the liquid phase peptide synthesis carrier comprises
  • a peptide obtained by deprotecting a carrier from a carrier-protected peptide containing the sequence consisting of the Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg (SEQ ID NO: 2), and H-Pro-NHEt ( L-proline ethylamide) is condensed, and the side chain protecting group is deprotected to produce leuprorelin.
  • the liquid phase peptide synthesis carrier is a compound having the following structure:
  • R 2 , R 4 and R 5 are hydrogen atoms
  • R 1 and R 3 are alkoxy groups having 12 to 30 carbon atoms, preferably 18 to 22 carbon atoms
  • RY is —CH 2 NHEt
  • the water-soluble amine is a divalent or higher water-soluble amine having at least one primary or secondary amino group, preferably ethylenediamine, 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triamino Any one of [1] to [15] above, selected from the group consisting of ethylamine, 1-ethylpiperazine, N, N-dimethylethylenediamine and piperazine, more preferably 1-methylpiperazine, 4-aminopiperidine, and diethylenetriamine.
  • the manufacturing method as described in one.
  • the amine equivalent of the water-soluble amine in step b is 1 to 10 equivalents (preferably 1 to 6 equivalents, more preferably 1 to 4 equivalents) with respect to the amino acid equivalent theoretically remaining after the condensation reaction in step a.
  • the amine equivalent of the water-soluble amine in step c is 5 to 30 equivalents (preferably 5 to 20 equivalents, more preferably 10 to 20 equivalents) relative to the amount of Fmoc group present in the system.
  • the peptide elongation step can be performed in a short time, and a high-purity crude product can be obtained. Therefore, the production technology for leuprorelin is more economical than the prior art. Can be provided.
  • N-Fmoc protected amino acid 9-Fluorenylmethyloxycarbonyl (Fmoc) amino acid protected amino group is an amino acid ⁇ -amino group is protected with Fmoc group
  • a carboxyl group means an amino acid that is not protected and is reactive.
  • the N-Fmoc protected amino acid has a functional group rich in reactivity, such as hydroxyl group, amino group other than ⁇ -position, guanidyl group, carboxyl group other than ⁇ -position, thiol group, indole group, imidazole group, etc. It is preferable to introduce a general protecting group used in peptide synthesis to the functional group, and at any time after the completion of the reaction, preferably by removing the protecting group in the final step of the production of leuprorelin, Can be obtained.
  • a functional group rich in reactivity such as hydroxyl group, amino group other than ⁇ -position, guanidyl group, carboxyl group other than ⁇ -position, thiol group, indole group, imidazole group, etc.
  • Examples of the protecting group for the hydroxyl group include tBu group, Trt group, Bz group, acetyl group, silyl group, etc., preferably tBu group, and the protecting group for amino group other than ⁇ -position includes Boc group, Fmoc group, Cbz group, Trt group, Mmt group, ivDde group and the like can be mentioned, preferably Boc group or Trt group, and the protecting group for guanidyl group includes Pbf group, Pmc group, nitro group, etc.
  • carboxyl group protecting group includes tBu group, methyl group, ethyl group, Bz group and the like
  • thiol group protecting group includes Trt group, Acm group, tBu group, S-tBu group and the like.
  • the protecting group for the indole group include a Boc group
  • examples of the protecting group for the imidazole group include a Boc group, a Bom group, a Bum group, and a Trt group.
  • An amino acid protected with a carrier-protected amino acid, a peptide and an amino acid amide carrier (carrier-protected amino acid) or a carrier-protected peptide is an amino acid or peptide whose amino terminus is protected with the carrier described below. Refers to an amino acid or peptide in a reactive state.
  • the carrier-protected amino acid amide refers to an amino acid amide in which at least one amide group of the amino acid amide is protected by the carrier for liquid phase peptide synthesis described below, and at least one amino group is not protected and is reactive.
  • the carrier-protected amino acid or the carrier-protected peptide has a functional group rich in reactivity, such as a hydroxyl group, an amino group other than the ⁇ -position, a guanidyl group, a carboxyl group, a thiol group, an indole group, an imidazole group, these functional groups
  • a general protecting group used in peptide synthesis may be introduced, and the target compound can be obtained by removing the protecting group as necessary after the completion of the reaction.
  • Examples of the protecting group for the hydroxyl group include tBu group, Trt group, Bz group, acetyl group, silyl group, etc., preferably tBu group, and the protecting group for amino group includes Boc group, Fmoc group, Cbz group, Trt group. Group, Mmt group, ivDde group, etc., preferably Boc group or Trt group, and the protecting group for guanidyl group includes Pbf group, Pmc group, nitro group, etc., preferably Pbf group
  • examples of the protecting group for the carboxyl group include a tBu group, a methyl group, an ethyl group, and a Bz group.
  • Examples of the protecting group for the thiol group include a Trt group, an Acm group, a tBu group, and an S-tBu group.
  • Examples of the protecting group for the indole group include a Boc group, and examples of the protecting group for the imidazole group include a Boc group, a Bom group, a Bum group, and a Trt group.
  • the carrier used in the production method of the present invention is a compound that binds directly to amino acids or peptides and renders them insoluble in water and has a molecular weight of 300 or more.
  • the carrier used in the present invention is a compound having the characteristic that the dissolved state and the insolubilized (crystallized or oiled) state are reversibly changed by the composition change of the solvent in which the carrier is dissolved.
  • Such a carrier is, for example, a benzyl compound into which a long-chain fatty acid proposed by the present inventors has been introduced (Japanese Patent Application Laid-Open No. 2003-183298, Japanese Patent Application Laid-Open No. 2004-059509, WO 2007/034812, WO 2007 / 122847), a benzyl compound (WO2017 / 038650) in which silicon is further introduced into the terminal of the long chain fatty acid to improve solubility in an organic solvent, or a fluorene compound (WO2010 / 104169) into which a long chain fatty acid is introduced.
  • the carrier may be insolubilized (crystallized or oiled) in the step of preparing the carrier-protected amino acid or carrier-protected peptide used in step a of the production method of the present invention.
  • the carrier-protected peptide may be insolubilized (crystallized or oiled).
  • the carrier used in the present invention is derived from the carrier compound described below. Hereinafter, the structure of the compound from which the carrier used in the present invention is derived will be described in the state before binding to the carboxyl group of an amino acid or peptide.
  • Carrier compound A A compound having the following structure (sometimes referred to as “Kb” in the present specification):
  • R 2 , R 4 and R 5 are hydrogen atoms, and R 1 and R 3 are alkoxy groups having 12 to 30 carbon atoms.
  • RY represents —CH 2 OH. Or —CH 2 NHEt, which is a group bonded to the carboxyl group of an amino acid or peptide).
  • R 1 and R 3 are preferably alkoxy groups having 18 to 22 carbon atoms.
  • 2,4-didocosyloxybenzyl alcohol represented by the following formula:
  • N-ethyl-2,4-didocosyloxybenzylamine represented by the following formula.
  • Carrier compound B A compound having the following structure (sometimes referred to as “KS” in the present specification):
  • X is —CH 2 OH or —CH 2 NHEt
  • R 2 , R 4 and R 5 are hydrogen atoms
  • at least one of R 1 and R 3 is represented by the following formula: —O—R 6 —Xa-A
  • the remaining group represents an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms
  • R 6 represents a linear or branched alkylene having 1 to 16 carbon atoms
  • Xa represents O or CONRc (where Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms);
  • A represents any one of formulas (1) to (11),
  • R 7 , R 8 and R 9 may be the same or different and each represents a linear or branched alkyl group having 1 to 6 carbon atoms or an aryl group which may have a substituent;
  • R 10 represents a single bond or a linear or branched alkylene group having 1 to 3 carbon atoms, and
  • R 11 , R 12 and R 13 may be the same or different, and may be a straight chain having 1 to 3 carbon atoms or Represents a branched alkylene group).
  • preferred ones are represented by the following formula:
  • Carrier compound C A compound having the following structure (sometimes referred to as “KJ1” in the present specification): Formula (V):
  • ring A represents an aromatic ring
  • Y represents a hydroxyl group, a bromo group or a chloro group
  • Ra, Rb and Rc each independently represents an organic group, a hydrogen atom or an electron having an aliphatic hydrocarbon group
  • An attraction group, and at least one of Ra, Rb and Rc is an organic group having an aliphatic hydrocarbon group
  • the rings A, B and C may each independently have an electron withdrawing group; Good], or general formula (V ′):
  • Rings A, B, and C may each independently have one or more selected from an organic group having an aliphatic hydrocarbon group and an electron-withdrawing group;
  • Each ring A may be the same or different;
  • each Y in the case where a plurality of Ys are present may be the same or different;
  • each Rc ′ in the case where a plurality of Rc ′s are present is the same or different.
  • the divalent organic group having an aliphatic hydrocarbon group is represented by the formula (a):
  • Xa does not exist or represents —O—, —S—, —NHCO— or —CONH—;
  • Rd represents an aliphatic hydrocarbon group having 5 or more carbon atoms;
  • K 1 represents 1 to 10
  • the organic group having an aliphatic hydrocarbon group is present at the 2-position and / or the 7-position of the fluorene compound, the formula (b):
  • R 3 is an organic group having an aliphatic hydrocarbon group having 5 to 60 carbon atoms; n 1 , n 2 , n 3 and n 4 are 1; m 2 is 1 A group represented by formula (d):
  • m 3 R 12 s are each independently an alkyl group having 4 to 30 carbon atoms) and are one or more groups selected from the group consisting of: The fluorene compound represented by these.
  • Carrier compound D A compound having the following structure (sometimes referred to as “KJ2” in the present specification): General formula (W):
  • Y represents a hydroxyl group or -NHEt group
  • R a is Formula (a):
  • m 1 represents an integer of 1 to 10; and m 1 X 1 s each independently represent a single bond, or —O—, —S -, -COO-, -OCONH-, -NHCO- or -CONH-; each of R 1 and m 1 R 2 independently represents a divalent aliphatic hydrocarbon group having 5 or more carbon atoms; And R 3 is a hydrogen atom or the formula (W ′):
  • n R b s are each independently a C 1-6 alkoxy group, a halogen atom, or a carbon that may be substituted with one or more halogen atoms.
  • m 2 represents 1 or 2
  • n 1 , n 2 , n 3 and n 4 each independently represents an integer of 0 to 2
  • m 2 X 2 , m 2 X 2 ′ and m 2 X 2 ′′ each independently represent a single bond, or —O—, —S—, —COO—, —OCONH—, —NHCO— or —CONH—
  • each of m 2 R 4 and m 2 R 6 independently represents an aliphatic hydrocarbon group having 5 or more carbon atoms
  • R 5 represents 5 carbon atoms
  • n 3 is from 0 to an integer of 15; n 5 is an integer of 0 to 11; n 6 represents an integer of 0 to 5; m 3 amino X 3 each independently represents a single bond, or —O—, —S—, —COO—, —OCONH—, —NHCO— or —CONH—; and m 3 R 7 are Each independently represents a hydrogen atom, a methyl group or an aliphatic hydrocarbon group having 5 or more carbon atoms; and the formula (d):
  • n 7 X 4 s each independently represent a single bond, or —O—, —S—, —COO—, —OCONH—, —NHCO; - or it shows a -CONH-;
  • R 8 represents a divalent aliphatic hydrocarbon group;
  • n 7 amino R 9 are each independently a monovalent aliphatic hydrocarbon group;
  • n 7 is 1 represents an integer of 1 to 5; and
  • Ar represents an arylene group.
  • Carrier compound E A compound having the following structure (sometimes referred to as “KJ3” in the present specification):
  • Y represents a hydroxyl group or —NHEt group
  • k and l each independently represents an integer of 0 to 5, provided that k + 1 is not 0
  • m 1 represents an integer of 1 to 10; m 1 X 1 s each independently do not exist, or —O—, —S—, — COO—, —OCONH— or —CONH—; each m 1 R 1 independently represents a divalent aliphatic hydrocarbon group having 5 or more carbon atoms, Formula (b):
  • M 2 X 2 , m 2 X 2 ′, m 2 X 2 ′ ′′ and m 2 X 2 ′′ are each independently absent or —O -, -S-, -COO-, -OCONH- or -CONH-; each of m 2 R 2 and R 4 independently represents a hydrogen atom, a methyl group or an aliphatic carbon atom having 5 or more carbon atoms A hydrogen group; R 3 represents an aliphatic hydrocarbon group having 5 or more carbon atoms), and a group represented by formula (e):
  • Carrier compound F A compound having the following structure (sometimes referred to as “KJ4” in the present specification):
  • k Q's each independently represent a single bond, or —O—, —S—, —C ( ⁇ O) O—, —C ( ⁇ O) NH— or —NH
  • k R a each independently has at least one aliphatic hydrocarbon group having one or more branched chains, the total number of branched chains is 3 or more, and the total number of carbon atoms is 14
  • k represents an integer of 1 to 4
  • R 1 is a hydrogen atom, or when Z is a group represented by the following formula (a):
  • R 2 together with R 2 may form a fluorene ring with ring B;
  • ring A is in addition to R 1 , k QR a , and C (X) (Y) Z And further substituted with a halogen atom, a C1-6 alkyl group optionally substituted by one or more halogen atoms, and one or more halogen atoms.
  • X represents a hydrogen atom or a phenyl group
  • Y represents a hydroxyl group or —NHEt group
  • Z represents a hydrogen atom or formula (a):
  • ring B may further contain a halogen atom, 1
  • substituents selected from the group consisting of a C1-6 alkyl group optionally substituted by one or more halogen atoms and a C1-6 alkoxy group optionally substituted by one or more halogen atoms
  • Y 4- (3,7,11-trimethyldodecyloxy) benzyl alcohol or 3,4,5-tris [4- (3,7,11-trimethyl) Dodecyloxy) benzyloxy] benzyl alcohol may be selected and used.
  • preferred ones are represented by the following formula:
  • the carrier compound A, carrier compound B, carrier compound C, carrier compound D, carrier compound E, and carrier compound F may be bound to the carboxyl group using a method generally used in peptide synthesis without any limitation in the present invention. For example, it can be carried out by amidation using COMU or esterification using DIPCI. 4).
  • Solvent The solvent used in the production method of the present invention is not particularly limited, and a solvent used in liquid phase peptide synthesis can be used. Examples of the solvent include, but are not limited to, THF, DMF, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, chloroform, dichloromethane, and N-methylpyrrolidone.
  • the carrier-protected peptide is insolubilized (crystallized or oiled) for the preparation of the starting material used in the production method of the present invention or for the last recovery of the carrier-protected peptide produced using the production method of the present invention. If so, a polar solvent is used.
  • the polar solvent to be used include methanol, ethanol, isopropanol, acetonitrile, propionitrile, DMF, dimethylacetamide, dimethyl sulfoxide, water, and a mixed solvent of two or more of these. Of these, methanol or acetonitrile is preferably used.
  • Elongation reaction in the production method of the present invention comprises a series of steps up to a condensation step, a Fmoc group deprotection step, and a carrier-protected peptide recovery step without performing a solid-liquid separation operation. Further, impurities generated in the synthesis step can be reduced or removed by liquid separation. Therefore, in the production of leuprorelin, peptide elongation can be performed continuously.
  • Peptide synthesis by the method for producing leuprorelin of the present invention includes the following steps.
  • A) Condensation reaction step An amino acid, carrier-protected amino acid amide or carrier-protected peptide protected with a carrier (hereinafter referred to as “carrier protection”) in an organic solvent or a mixture of organic solvents, and 9-fluorenylmethyloxy
  • a step of condensing an amino group whose amino group is protected with a carbonyl group (hereinafter referred to as “N-Fmoc protection”) to obtain an N-Fmoc-carrier-protected peptide
  • N-Fmoc protection a scavenge reaction step
  • a water-soluble amine hereinafter sometimes referred to as an amine scavenger
  • De-Fmoc step Deprotecting the Fmoc group from the N-terminal protected in the presence of a water-soluble amine, as well as,
  • D) Acidic aqueous solution washing step A step of adding an acid
  • step (E) Basic aqueous solution washing step A step in which a weak basic aqueous solution is added and washed, followed by liquid separation, and removal of the aqueous layer to obtain an organic layer.
  • the above process does not require a solid-liquid separation operation involving insolubilization (crystallization or oiling) of the carrier-protected peptide, and can be performed in one pot. Furthermore, by carrying out the above steps, the carrier-protected peptide to which an amino acid has been added can be recovered in a state dissolved in the organic layer as compared with the start of the synthesis reaction. Moreover, since impurities are reduced or removed from the organic layer in which the carrier-protected peptide is dissolved, the next peptide synthesis reaction (peptide extension reaction) can be carried out as it is.
  • one aspect of the production method of the present invention is a method for producing leuprorelin, which comprises repeating the steps (a) to (d) as many times as necessary.
  • the process of repeating repeatedly can also be performed in one pot.
  • the step (e) may be added in each synthesis cycle.
  • the N-terminal amino acid Pyr is condensed in the same manner as in step (a).
  • step (c) corresponding to the de-Fmoc step is not performed, and the aqueous acidic solution washing similar to the step (d) can be performed to obtain the carrier-protected peptide.
  • Such an embodiment is also included in the present invention.
  • the carrier-protected amino acid in step (a) of the production method of the present invention can be prepared by appropriately referring to known methods used in peptide synthesis.
  • Arg-OKb using a carrier compound Kb in which RY is —CH 2 —OH can be prepared as follows.
  • the carrier compound is dissolved in a solvent such as THF, N-Fmoc protected amino acid and a condensing agent such as DIPCI are added for condensation, and N-Fmoc is an intermediate in which the carrier is bound to the carboxyl group of the amino acid.
  • -A carrier-protected amino acid can be produced.
  • the produced N-Fmoc-carrier-protected amino acid can be obtained with high purity, preferably by crystallization and recovery.
  • the reaction solution containing the carrier-protected amino acid is distilled off under reduced pressure, and then the residue is solidified (crystallized) with N-Fmoc-carrier-protected amino acid, for example, Methanol or acetonitrile is added for precipitation, and the precipitate is filtered, then washed with a solvent, and the obtained solid is dried to obtain a final product.
  • N-Fmoc-carrier-protected amino acid is used in peptide synthesis, and a carrier-protected amino acid can be prepared by removing the N-terminal protecting group with appropriate reference to known methods.
  • a carrier-protected amino acid can be prepared by dissolving N-Fmoc-carrier-protected amino acid in a solvent such as THF and adding a de-Fmoc reagent such as DBU or piperazine to perform a de-Fmoc reaction.
  • the carrier-protected amino acid amide in step (a) of the production method of the present invention is also replaced with a carrier compound in place of the carrier-protected amino acid in step (a) of the production method of the present invention, and steps (a) to (d), In some cases, it can also be obtained by carrying out step (e).
  • the carrier compound H-EtN—Kb and Fmoc-Pro—OH, in which RY is —CH 2 —NHEt are used to perform each step, whereby the carrier-protected amino acid amide H-Pro— EtN-Kb can be obtained.
  • the carrier-protected amino acid or carrier-protected amino acid amide thus obtained can be used as a starting material for the production method of the present invention. Therefore, as one embodiment of the production method of the present invention, a peptide synthesis method including a carrier protective peptide preparation step can be mentioned before the step (a).
  • the carrier-protected peptide obtained using the production method of the present invention can be recovered using a known method used in the field of peptide synthesis. For example, it can be recovered from the solvent by crystallization. For example, but not limited to this, the organic layer containing the obtained carrier-protected peptide is distilled off under reduced pressure, and then the residue is precipitated by adding a poor solvent, for example, cold acetonitrile. After filtration, it is washed with a solvent, and the resulting solid is dried to obtain a synthesized carrier-protected peptide.
  • a poor solvent for example, cold acetonitrile
  • a production method including the steps of crystallizing and separating the carrier-protected peptide after the above steps (a) to (d), and optionally further after step (e) may be mentioned.
  • Condensation reaction step In this step, an amino acid residue is obtained by mixing a carrier-protected amino acid, amino acid amide or peptide, an N-Fmoc-protected amino acid, and a condensing agent (preferably a condensing agent and an activator) in a solvent. An N-Fmoc-carrier protected peptide with an extended number is obtained.
  • the method and order of addition of each component can be carried out without any particular limitation, and a method usually used in the condensation step in peptide synthesis can be used.
  • the amount of N-Fmoc-protected amino acid to be used for the carrier-protected amino acid, amino acid amide or peptide is usually 1.01 to 4 equivalents, preferably 1.03 to 4 equivalents, relative to the carrier-protected amino acid, amino acid amide or peptide.
  • the amount is preferably 1.05 to 2 equivalents, more preferably 1.1 to 1.5 equivalents. If it is less than this range, unreacted carrier-protected peptide tends to remain, and amino acid loss tends to occur.
  • an active ester of an unreacted amino acid can be inactivated by scavenging (capturing) with a water-soluble amine added thereafter. Therefore, even if more N-Fmoc-protected amino acid is used, the remaining problem does not occur as compared with the conventional method.
  • a condensing agent generally used in peptide synthesis can be used without limitation in the present invention.
  • DMT-MM 4- (4,6-dimethoxy-1,3,5 -Triazin-2-yl) -4-methylmorphonium chloride
  • HBTU O- (benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
  • HATU 7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
  • HBTU 7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
  • HBTU (6-chlorobenzotriazol-1-yl) -1, 1,3,3-tetramethyluronium hexafluorophosphate
  • TBTU O- (6-chlorobenzotriazol
  • an activator may be added.
  • the activator is a reagent that facilitates the formation of a peptide bond (amide bond) by introducing an amino acid to a corresponding active ester, a symmetric acid anhydride, or the like by coexistence with a condensing agent.
  • an activator generally used in peptide synthesis can be used without limitation in the present invention.
  • HOBt, HOCt, HOAt, HOOBt, HOSu, HOPht, HONb, pentafluorophenol examples thereof include ethyl cyano (hydroxyimino) acetate (Oxyma) and the like, and preferably HOBt, HOOBt, HOCt, HOAt, HONb, HOSu, and Oxyma.
  • the amount of the activator used is usually 1 to 4 equivalents, preferably 1 to 2 equivalents, more preferably 1.05 to 1.3 equivalents, relative to the carrier-protected peptide.
  • a solvent that is generally used in peptide synthesis can be used without limitation in the present invention, and is not limited thereto, and examples thereof include the solvents described above.
  • the amount of the solvent used is such an amount that the concentration in which the carrier-protected peptide or the like is dissolved is usually 0.1 mM to 1 M, preferably 1 mM to 0.5 M.
  • reaction temperature a temperature generally used in peptide synthesis is also used in the present invention.
  • it is usually in the range of ⁇ 20 to 40 ° C., preferably 0 to 30 ° C.
  • the reaction time (one cycle time) is usually 0.5 to 30 hours.
  • a water-soluble amine is added to the reaction system to scavenge (capture) the unreacted amino acid active ester.
  • Water-soluble amines combine with amino acid active esters to form scavenges and inactivate active esters.
  • the water-soluble amine used in the present invention may be referred to as an amine scavenger.
  • the water-soluble amine as a scavenger that can be used in the present invention is preferably a divalent or higher water-soluble amine having at least one primary or secondary amino group, such as 1-methylpiperazine, 4- Examples include aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N, N-dimethylethylenediamine, ethylenediamine and piperazine, preferably 1-methylpiperazine, 4-aminopiperidine and diethylenetriamine, more preferably 1-methylpiperazine.
  • the amount of the water-soluble amine added in the step (b) is usually 1 to 10 equivalents, preferably 1 to 6 equivalents, more preferably 1 to 4 equivalents with respect to the theoretically remaining amino acid equivalent.
  • the Fmoc group is removed from the N-Fmoc-carrier-protected peptide, which is the next step, by scavenging (capturing) the amino acid active ester in the reaction system with an amine scavenger to form a scavenge body. After that.
  • the amino acid active ester in the reaction solution is inactivated, and it is possible to prevent amino acid double hits during deprotection without removing them from the reaction system.
  • the amino acid active ester captured by the water-soluble amine can be easily removed in a subsequent washing step.
  • the Fmoc group is removed from the N-Fmoc-carrier-protected peptide in the presence of a water-soluble amine.
  • a water-soluble amine is additionally added to the reaction system.
  • the water-soluble amine that can be used in this step is preferably a divalent or higher water-soluble amine having at least one primary or secondary amino group, such as 1-methylpiperazine, 4-aminopiperidine, Examples include diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N, N-dimethylethylenediamine, ethylenediamine, and piperazine, preferably 1-methylpiperazine, 4-aminopiperidine, and diethylenetriamine, more preferably 1-methylpiperazine. Methyl piperazine.
  • the kind of water-soluble amine in this step may be the same as or different from the water-soluble amine added in the scavenge reaction step of step (b).
  • the equivalent amount of the water-soluble amine added in this step (c) is 5 to 30 equivalents, preferably 5 to 20 equivalents, more preferably 10 to 20 equivalents, relative to the amount of Fmoc group present in the system. If the amount of amine added is less than this range, scavenging (capturing) of DBF caused by the de-Fmoc reaction will be insufficient, and it will be difficult to remove impurities in the subsequent acidic aqueous solution washing step. The amount of acid required for the sum is increased, and a side reaction (decomposition, racemization) occurs due to the accompanying neutralization step, which causes a decrease in purity and a decrease in yield.
  • the de-Fmoc reaction is carried out in the presence of a water-soluble amine, but if the water-soluble amine present in the system has a function as a de-Fmoc reagent, it is not necessary to add another de-Fmoc reagent to the system .
  • another de-Fmoc reagent may be added to the system in order to efficiently perform the de-Fmoc reaction.
  • the water-soluble amine having a function as a de-Fmoc reagent include the water-soluble amines exemplified above.
  • the de-Fmoc reagent is added together with the water-soluble amine.
  • the de-Fmoc reagent when the de-Fmoc reagent is added to the reaction system together with the water-soluble amine, the water-soluble amine and the de-Fmoc reagent may be added to the system at the same time, or after the water-soluble amine is added to the system.
  • a de-Fmoc reagent may be added.
  • the term “simultaneous” as used herein means that the addition is performed before and after the reaction is considered to be simultaneous in the reaction in this technical field.
  • the time of an addition interval can be suitably adjusted in consideration of operation and other factors.
  • the removal of the Fmoc group from the N-terminal can be used in the present invention by appropriately changing the removal method generally used in peptide synthesis as necessary.
  • the de-Fmoc reagent that can be used in the present invention include, but are not limited to, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), 1,5-diazabicyclo [4.3. 0.0] -5-nonene (DBN), 1,4-diazabicyclo [2.2.2] -octane (DABCO), triethylamine, and tributylamine, and DBU is preferable.
  • DBU 1,8-diazabicyclo [5.4.0] -7-undecene
  • DBN 1,5-diazabicyclo [4.3. 0.0] -5-nonene
  • DABCO 1,4-diazabicyclo [2.2.2] -octane
  • triethylamine and tributylamine
  • dibenzofulvene (DBF) is generated, but the water-soluble amine added in this step can scavenge (capture) these impurities.
  • DBF dibenzofulvene
  • the DBF trapped by the water-soluble amine can be easily removed in the subsequent acidic aqueous solution washing step.
  • the organic base present in the system can be neutralized by the neutralization step of the acidic aqueous solution washing step (d).
  • the acid used for neutralization is not particularly limited as long as it can neutralize the base in the reaction solution, and examples thereof include aqueous solutions of hydrogen chloride, phosphoric acid, acetic acid, sulfuric acid and the like.
  • hydrochloric acid although not limited thereto, 1N to 12N, preferably 2N to 12N, more preferably 5N to 12N hydrochloric acid is added.
  • neutralization means that the reaction solution has a neutral pH, and the pH may be 7.0 or less.
  • an acidic aqueous solution is further added to the reaction neutralized solution neutralized with an acid, washed, and then separated, the aqueous layer is discarded, and the organic layer is recovered. Thereby, impurities soluble in the acidic aqueous solution can be removed.
  • the acidic aqueous solution to be used is not particularly limited, and examples thereof include dilute hydrochloric acid, dilute sulfuric acid, phosphoric acid aqueous solution, and acetic acid aqueous solution, and dilute hydrochloric acid is preferable.
  • the pH of the acidic aqueous solution is 1 to 5, preferably 1 to 4, more preferably 1 to 3.
  • the amount of the acidic aqueous solution used for washing is not particularly limited as long as it shows a washing effect, but it is 0.1 to 3 times, preferably 0.5 to 2 times, more preferably 0.8 times the amount of the reaction solution. It can be used in an amount of 8 to 1.5 times.
  • the washing, liquid separation, and aqueous layer disposal steps are not limited in number and may be performed once or a plurality of times. The number of times is appropriately selected according to the type of compound in the reaction system, the amount of impurities, and the purpose.
  • impurities can be removed by washing the acidic aqueous solution in step (d).
  • acidic aqueous solution for example, H 2 N-AAx-amine (scavenger) conjugate, condensate decomposition product, pH adjusting base, DBF-amine (scavenger) conjugate, amine (scavenger), de-Fmoc reagent, etc.
  • the carrier-protected peptide dissolved in the reaction system in which the impurities are reduced or removed can be obtained.
  • the liquid separation operation using an aqueous solution is simple and contributes to shortening of the process time.
  • a solid-liquid separation operation is not necessary, and the use of a poor solvent for solidifying the carrier can be reduced.
  • the carrier-protected peptide in the last cycle, after the de-Fmoc step, after neutralization with acid, the carrier-protected peptide is solidified (crystallized), and solid-liquid separation operation is performed.
  • the carrier-protected peptide may be recovered by using, but it is preferable to perform washing with an acidic aqueous solution from the viewpoint of more complete removal of impurities.
  • step (e) The peptide synthesis of the present invention can optionally further include the following steps.
  • step (e) a weakly basic aqueous solution is added and washed, followed by liquid separation, the aqueous layer is discarded, and the organic layer is recovered. Thereby, impurities soluble in the weakly basic aqueous solution can be removed.
  • the weak basic aqueous solution to be used is not specifically limited, For example, sodium hydrogencarbonate aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution is mentioned, Preferably, it is sodium hydrogencarbonate aqueous solution.
  • the pH of the weakly basic aqueous solution is 8 to 12, preferably 8 to 10.
  • the addition amount of the weakly basic aqueous solution used for washing is not particularly limited as long as it shows a washing effect, but it is 0.1 to 3 times, preferably 0.5 to 2 times, more preferably 0 to the reaction solution. .8 to 1.5 times the amount can be used.
  • the washing, liquid separation, and aqueous layer disposal steps are not limited in number and may be performed once or a plurality of times. The number of times is appropriately selected according to the type of compound in the reaction system, the amount of impurities, and the purpose.
  • step (d) in the last cycle, in step (d) Thereafter, without carrying out step (e), the carrier-protected peptide dissolved in the obtained organic layer may be solidified (crystallized), and the carrier-protected peptide may be recovered using a solid-liquid separation operation.
  • a water removal step of step (f) may be added as necessary.
  • the water removal step includes a method of producing a salt effect by adding a salt solution and efficiently removing water, and a method of removing water using a dehydrating agent.
  • the salt solution include, but are not limited to, saline.
  • anhydrous sodium sulfate can be mentioned.
  • Crystallization / separation step of carrier-protected peptide The carrier-protected peptide synthesized by the method of the present invention is insolubilized (eg, crystallized) and separated after step (d), step (e) or step (f). can do.
  • Insolubilization is performed by referring to known methods as appropriate in the field of peptide synthesis using a carrier that has a property that the dissolved state and the insolubilized (crystallized) state reversibly change depending on the composition of the solvent in which the carrier is dissolved. For example, it can be performed by changing the composition of the solution in which the carrier protective peptide is dissolved.
  • the conditions for insolubilization can be appropriately selected according to the type of carrier used and the type and length of the synthesized carrier protective peptide. For example, although not limited to this, the following solvent composition change means can be mentioned.
  • the means for changing the solution composition is not particularly limited as long as it can change the composition of the solution in which the carrier protective peptide is dissolved.
  • Preferable means for changing the solution composition include, for example, a means for crystallization by adding a poor solvent as it is to a solution in which the carrier-protected peptide is dissolved or after concentrating the solvent of the solution.
  • the concentration means that a part or all of the solvent is distilled off, for example, the solvent is distilled off under reduced pressure.
  • the precipitated crystals can be separated by, for example, filtration or centrifugation. The separated crystals are preferably washed with an organic solvent to remove impurities separated together with the crystals from the crystallized carrier-protected peptide.
  • the poor solvent in the present invention refers to a solvent in which the carrier-protected peptide is poorly soluble, that is, the carrier-protected peptide is difficult to dissolve or does not dissolve.
  • the carrier-protecting peptide is difficult to dissolve or does not dissolve as long as it is a liquid solvent at room temperature at which the solubility of the carrier-protecting peptide is less than 1% by mass at 25 ° C., and acetonitrile, water-containing acetonitrile, methanol, any proportion Water-containing methanol and water are preferred.
  • this crystallization / separation step can be performed after step (d), step (e), or step (f), thereby synthesizing.
  • the carrier-protected peptide can be recovered.
  • Carrier deprotection of the carrier-protected peptide synthesized by the method of the present invention can be performed by removing (deprotecting) the carrier bonded to the carboxyl group of the peptide.
  • the method for removing the carrier is not particularly limited, and a known deprotection method may be used, but it is preferably performed by acid treatment.
  • a deprotection method using TFA can be used. More specifically, 1 to 100% trifluoroacetic acid is used when Kb is used, and 1 to 100% trifluoroacetic acid is used when KS is used.
  • the peptide (leuprorelin) obtained using the production method of the present invention can be isolated and purified according to a method commonly used in peptide synthesis.
  • the target peptide can be isolated and purified from the reaction mixture by extraction washing, crystallization, chromatography and the like.
  • AAs any amino acid residue of 1 or more AAx : any amino acid residue
  • Boc tert-butoxycarbonyl
  • COMU (1-cyano-2-ethoxy-2- oxoethylideneaminooxy ) dimethylamino-morpholino-carbenium hexafluorophosphate
  • CPME cyclopentyl methyl ether
  • DBU 1,8-diazabicyclo [5.4.0] -7-undecene
  • DCM dichloromethane
  • DIPCI Diisopropylcarbodiimide DIPEA : N, N-diisopropylethylamine
  • DMAP N, N-dimethyl-4-aminopyridine
  • DMF N, N-dimethylformamide
  • DMT-MM 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-
  • the starting material was dissolved in a mixed solution of THF: DMF (9/1) to 18 v / w, piperidine (1.5 equiv) and DBU (1.0 eqiv) were added, and the mixture was stirred at room temperature for 10 minutes. 6N hydrochloric acid (3.50equiv) was added while cooling with ice, and the solvent was distilled off under reduced pressure. Acetonitrile was added to the residue and the deposited precipitate was filtered, further washed with acetonitrile, and the obtained solid was dried under reduced pressure to obtain a de-Fmoc form.
  • condensation deprotection method used in the method of the present invention is shown below. Although the case where Kb-OH is used as a carrier compound is described as an example, Kb-NHEt, KS, KJ1, KJ2, KJ3 and KJ4 can be used similarly. Moreover, the addition amount of each reagent is only an example, and is not limited thereto.
  • the starting material was dissolved in a mixture of THF: DMF (9/1) to 18 v / w, Fmoc-AAx-OH (1.30equiv), COMU (1.25equiv), and DIPEA (2.30equiv) And stirred at room temperature for 30 minutes.
  • THF THF
  • Fmoc-AAx-OH 1.30equiv
  • COMU 1.25equiv
  • DIPEA 2.30equiv
  • 1-methylpiperazine (0.45 equiv) was added and stirred at room temperature for 30 minutes.
  • 1-Methylpiperazine (20.0equiv) and DBU (7.0equiv) were added and stirred at room temperature for 10 minutes.
  • the raw material was dissolved in a mixed solution of DCM: TFE: TFA (90/9/1) to 19.75 v / w and stirred at room temperature for 30 minutes.
  • the precipitate was filtered, DIPEA was added to the filtrate so as to be 1.0 equiv of TFA, 0.01N hydrochloric acid (18 v / w) was added, washed and separated, and the aqueous layer was discarded.
  • Diisopropyl ether was added to the resulting organic layer, and the mixture was distilled off under reduced pressure.
  • Diisopropyl ether was added to the residue, and the deposited precipitate was washed, and the obtained solid was dried under reduced pressure to obtain a Kb-protected product.
  • Compound 1 (19.85 g, quant) was obtained by subjecting compound 1 to de-Fmoc general synthesis.
  • EtNKb N-ethyl-2,4-didocosyloxybenzylamine
  • Comparative Example 1 Synthesis of H-Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (SEQ ID NO: 1) described in Non-Patent Document 1 (Phermaceutical Chemistry Journal Vol48 No. 3 June 2014) According to the method, peptides having the above sequences can be synthesized. A synthesis scheme is shown below.
  • the reaction was carried out at room temperature for 16 hours, and the completion of the reaction was confirmed by TLC.
  • the precipitate was filtered off and washed twice with THF (300 ml).
  • the THF solutions were combined and concentrated to dryness.
  • Chloroform (4.5 L) was added to the residue, washed twice with a saturated saline solution (2 L) containing 5% sodium carbonate, and washed once with a saturated saline solution (2 L) containing 5% citric acid.
  • the organic layer was dehydrated with sodium sulfate and concentrated to dryness.
  • the resulting residue was dissolved in 1,4-dioxane (1.5 L) and cooled to 10 ° C.
  • the reaction was carried out at 0 ° C. for 1 hour and at 5 ° C. for 24 hours, and the completion of the reaction was confirmed by TLC.
  • the reaction solution was added to water (3 L) and ethyl acetate (3 L), and the pH was adjusted to 8 with sodium hydrogen carbonate.
  • the precipitate was filtered off and the organic layer was isolated, and then the organic layer was washed twice with a saturated sodium carbonate solution (1.5 L), 5% aqueous citric acid solution (1 L) and water (1 L).
  • the organic layer was concentrated to 0.7 L and diethyl ether (3 L) was added.
  • the precipitate was collected by filtration, washed twice with diethyl ether (300 ml), and dried under reduced pressure.
  • Compound IX was obtained in 221 g (yield 89%, HPLC purity 97.9%) as a white powder.
  • Synthetic compound X (85 g, 0.1 mol) and Pyr-His-Trp of the final compound (leuprorelin: Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt: SEQ ID NO: 1)
  • leuprorelin Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt: SEQ ID NO: 1
  • DMF 350 mL
  • a solution of dicyclohexylcarbodiimide (20 g, 0.1 mol) dissolved in DMF (100 ml) was added dropwise over 5 hours.
  • Non-patent Document 1 which is a comparative example, the steps of solidifying the carrier-protected peptide, filtration, and drying are required, and the operation is complicated and requires a large amount of organic solvent. Furthermore, the process time is increased due to the drying process.
  • 2,4-di (11′-triisopropylsilyloxyundecyloxy) benzyl alcohol (hereinafter, 2,4-di (11′-triisopropylsilyloxyundecyloxy) benzyl group may be referred to as KS1) is used.
  • Synthesis of Compound 13 (Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -dLeu-Leu-Arg (Pbf) -O-KS1: SEQ ID NO: 14)
  • 2,7-didocosyloxy-9- (3-fluorophenyl) -9-bromofluorene (hereinafter 2,7-didocosyloxy-9- (3-fluorophenyl) -9-fluorenyl group may be referred to as Fl) Synthesis of Compound 17 (Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -dLeu-Leu-Arg (Pbf) -O-Fl: SEQ ID NO: 16)
  • Patent No. 6092513 Example 6 using 2,7-didocosyloxy-9- (3-fluorophenyl) -9-bromofluorene (0.90 g, 0.91 mmol) except that Z-Ala-OH (Fmoc-Arg (Pbf) -OH was used instead of) to give compound 14 (1.03 g, 0.663 mmol, 73.0%).
  • Example 8 Synthesis of Compound 16 (H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -dLeu-Leu-Arg (Pbf) -O-Fl: SEQ ID NO: 17)
  • 2,4-di (2 ′, 3′-dihydrophytyloxy) benzyl alcohol (hereinafter, 2,4-di (2 ′, 3′-dihydrophytyloxy) benzyl group may be referred to as KJ1) Synthesis of Compound 21 (Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -dLeu-Leu-Arg (Pbf) -O-KJ1: SEQ ID NO: 18)
  • the compound BD was subjected to general de-Fmoc synthesis using 1-methylpiperazine instead of piperidine, and washed with aqueous hydrochloric acid and aqueous sodium hydrogencarbonate to obtain a solution of compound 19.
  • Example 12 Synthesis of compound 20 (H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -dLeu-Leu-Arg (Pbf) -O-KJ1: SEQ ID NO: 19)
  • N-ethyl- (Bis (4-docosyloxyphenyl)) methylamine (hereinafter, N-ethyl- (Bis (4-docosyloxyphenyl)) methylamino group may be referred to as NEt-KJ3)) Synthesis of Compound 7 (Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt: 1)
  • Fmoc-Pro-OH (0.354 g, 1.05 mmol) was dissolved in 14 mL of toluene, DMF (10.8 ⁇ L, 0.14 mmol) was added, and thionyl chloride (91 ⁇ L, 1.26 mmol) was added, and the mixture was heated to 40 ° C. And stirred for 2 hours. Further, thionyl chloride (45.5 ⁇ L, 0.63 mmol) was added and stirred for 1 hour. The resulting reaction solution was concentrated under reduced pressure. The residue was added with 7 mL of toluene and concentrated under reduced pressure twice.
  • the compound 22 (0.500 g, 0.424 mmol) was subjected to general de-Fmoc synthesis using 1-methylpiperazine instead of piperidine, washed with aqueous hydrochloric acid and aqueous sodium hydrogen carbonate solution to obtain a solution of compound 23. Obtained.
  • Example 17 Synthesis of compound 25 (Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -dLeu-Leu-Arg (Pbf) -Pro-NEt-KJ3: SEQ ID NO: 21)
  • Example 18 Synthesis of Compound 7 (Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt: SEQ ID NO: 7)
  • the production method of the present invention since the peptide elongation step can be performed in a short time by a simple means, and a high-purity crude product is obtained, a production technique for leuprorelin that is more economical than the conventional technique is used. Can be provided. Leuprorelin is useful as a medicament.

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Abstract

Le but de la présente invention est de fournir un procédé de production de leuproréline permettant d'obtenir un produit brut de pureté élevée en un temps de production plus court, par comparaison avec des procédés de production classiques. L'invention concerne un procédé de production de leuproréline, comprenant les étapes suivantes de a à d : étape a consistant à obtenir un peptide protégé par N-Fmoc par condensation d'un acide aminé ou d'un peptide protégé et d'un acide aminé protégé par N-Fmoc dans un solvant organique ou un mélange de solvants organiques ; étape b consistant à ajouter une amine soluble dans l'eau à la solution de réaction à la suite de la réaction de condensation ; étape c consistant à déprotéger le groupe Fmoc du groupe amino protégé en présence de l'amine soluble dans l'eau ; et l'étape d consistant à obtenir une couche organique par ajout d'acide pour neutraliser la solution de réaction, ajouter en outre une solution aqueuse acide pour laver le produit, effectuer une séparation liquide, puis retirer la couche aqueuse.
PCT/JP2019/016069 2018-04-13 2019-04-13 Procédé de production de leuproréline WO2019198834A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020186840A1 (fr) * 2019-03-19 2020-09-24 广州同隽医药科技有限公司 Composé contenant une structure cyclique de fluorène et utilisation associée
CN114276222A (zh) * 2021-12-31 2022-04-05 杭州澳赛诺生物科技有限公司 一种作为多肽液相合成载体的二芳基苯甲醇类化合物及其制备方法与应用
JP7154513B1 (ja) 2021-07-02 2022-10-18 ペプチスター株式会社 液相ペプチド製造方法
US11814367B2 (en) 2021-03-15 2023-11-14 Maze Therapeutics, Inc. Inhibitors of glycogen synthase 1 (GYS1) and methods of use thereof
US11827660B2 (en) 2019-02-01 2023-11-28 Sederma Synthesis strategy for gap protecting group
US12024537B2 (en) 2018-05-31 2024-07-02 Sederma Compositions and methods for chemical synthesis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099656A1 (fr) * 2006-03-01 2007-09-07 Kaneka Corporation Procede de production d'un peptide
US20130060004A1 (en) * 2010-05-07 2013-03-07 Ananda Kuppanna Novel Process For The Preparation Of Leuprolide And Its Pharmaceutically Acceptable Salts Thereof
WO2016140232A1 (fr) * 2015-03-04 2016-09-09 Jitsubo株式会社 Procédé de synthèse peptidique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099656A1 (fr) * 2006-03-01 2007-09-07 Kaneka Corporation Procede de production d'un peptide
US20130060004A1 (en) * 2010-05-07 2013-03-07 Ananda Kuppanna Novel Process For The Preparation Of Leuprolide And Its Pharmaceutically Acceptable Salts Thereof
WO2016140232A1 (fr) * 2015-03-04 2016-09-09 Jitsubo株式会社 Procédé de synthèse peptidique

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12024537B2 (en) 2018-05-31 2024-07-02 Sederma Compositions and methods for chemical synthesis
US11827660B2 (en) 2019-02-01 2023-11-28 Sederma Synthesis strategy for gap protecting group
WO2020186840A1 (fr) * 2019-03-19 2020-09-24 广州同隽医药科技有限公司 Composé contenant une structure cyclique de fluorène et utilisation associée
US11814367B2 (en) 2021-03-15 2023-11-14 Maze Therapeutics, Inc. Inhibitors of glycogen synthase 1 (GYS1) and methods of use thereof
JP7154513B1 (ja) 2021-07-02 2022-10-18 ペプチスター株式会社 液相ペプチド製造方法
JP2023007948A (ja) * 2021-07-02 2023-01-19 ペプチスター株式会社 液相ペプチド製造方法
CN114276222A (zh) * 2021-12-31 2022-04-05 杭州澳赛诺生物科技有限公司 一种作为多肽液相合成载体的二芳基苯甲醇类化合物及其制备方法与应用
CN114276222B (zh) * 2021-12-31 2024-04-09 杭州澳赛诺生物科技有限公司 一种作为多肽液相合成载体的二芳基苯甲醇类化合物及其制备方法与应用

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