JP6703669B2 - Method for producing leuprorelin - Google Patents

Description

The present invention relates to a novel method for producing leuprorelin.

Leuprorelin acetate is a LH-RH (GnRH) agonist and is a drug applied to endometriosis, uterine fibroids, premenopausal breast cancer, prostate cancer, central precocious puberty and the like.

As a method for producing a peptide or a salt thereof which is a derivative of LH-RH, Japanese Patent Application Laid-Open No. 50-59370 (corresponding to US Pat. No. 4,008,209) (Patent Document 1) describes a general formula ( Pyr)Glu-His-Trp-Ser-Tyr (or Phe)-X-Leu (or Ile or Nle)-Arg-Pro-NH-R [wherein, amino acids represent L-form, unless otherwise specified, X Is D-Leu, D-Nle, D-NVal, D-Ser, D-Abu, D-Phg, D-Phe or α-Aibu, and R is an alkyl group which may have a hydroxyl group] The following liquid phase synthesis method is described as a method for producing the peptide.
formula,

[The symbols in the formulas have the same meanings as above].

Moreover, in WO97/48726 (Patent Document 2), a general formula: 5-oxo-Pro-R1-Trp-Ser-R2-R3-OH(II) [wherein, R1 is His, Tyr, Trp or p-NH ₂ -Phe, R 2 represents Tyr or Phe, and R 3 represents a Gly or α-D-amino acid residue which may have a substituent. ] The peptide or its salt represented by these, and general formula: H-R4-R5-Pro-R6 (III) [In formula, R4 is Leu, Ile or Nle, R5 is protected Arg, R6 is a formula Gly. -NH-R7 (in the formula, R7 represents a hydrogen atom or an alkyl group which may have a hydroxyl group) or formula NH-R8 (in the formula, R8 is a hydrogen atom or an alkyl group which may have a hydroxyl group) or ureido group a (-NH-CO-NH ₂₎ is reacted with peptide or its salt represented by a group] represented by each shown), the general formula 5-oxo-Pro-R1- Trp-Ser-R2- A peptide represented by R3-R4-R5-Pro-R6(I') [wherein the symbols have the same meaning as defined above] or a salt thereof is obtained, and then the obtained peptide (I') is deprotected. In the general formula 5-oxo-Pro-R1-Trp-Ser-R2-R3-R4-Arg-Pro-R6(I) characterized by being subjected to a reaction, the symbols in the formula have the same meanings as described above. Processes for making the represented peptides or salts thereof are described.

Furthermore, Non-Patent Document 1 describes a liquid phase synthesis method of PyroGlu-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (Compound I), which comprises the following steps.

In addition, Chinese Patent Publication CN106146622 (Patent Document 3) discloses a method for producing leuprorelin by a solid-phase synthesis method using a para(oxymethyl)phenylacetamide resin (PAM resin). ..

In the above liquid phase synthesis method, in addition to the problem that the production period becomes long due to the long reaction time of the unit step, the reduction reaction is carried out using a metal catalyst such as SnCl ₂ and Pd/C. When used as, it is necessary to strictly control the residual amount of elemental impurities.
Further, the yield up to leuprorelin is low, which is as low as 56.8% by the method described in Patent Document 1 and 20.3% by the method described in Non-Patent Document 1. The purity of the crude leuprorelin product obtained by the method described in Non-Patent Document 1 is as low as 63.3%.

On the other hand, in the solid phase synthesis method, the reaction time per unit step is shorter than that in the liquid phase synthesis. In the method of Patent Document 3, the yield up to the step of condensing and cutting out from the resin is as high as 92.5% in terms of purity, but the total yield including the purification step is low at 36%, and the crude product It is described that the recovery rate during purification is low. This suggests that it is difficult to remove impurities contained in the crude product, and in order to obtain high-purity leuprorelin, it is necessary to discard the eluate containing the target substance during purification.
As described above, in the existing methods for producing leuprorelin, there has not been any that can be produced in a short time and can obtain the target product with high purity and high yield.

Japanese Patent Laid-Open No. 50-59370 WO97/48726 CN106146622 publication

A.N.Balaev et al., Pharmaceutical Chemistry Journal, Vol. 48, No. 3, June, 2014.

An object of the present invention is to provide a method for synthesizing a liquid phase peptide of leuprorelin, which can provide a crude product with a short production period and a high purity as compared with a conventional production method.

The present inventors have conducted extensive studies in view of the above problems, and as a result, a method for peptide synthesis in a liquid phase, in the Fmoc method using a carrier (Tag), a specific carrier was used and a Tag-peptide component was used. 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 operations), and completed the present invention. The present invention includes the following.

[1] The following sequence: a method for producing leuprorelin consisting of H-Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt, which comprises the following steps a to d:
a. In an organic solvent or a mixture of organic solvents, an amino acid or a carrier-protected peptide protected by a carrier for liquid phase peptide synthesis (carrier protected) and an amino group protected by a 9-fluorenylmethyloxycarbonyl group (Fmoc group) Condensing the prepared (N-Fmoc protected) amino acid or N-Fmoc protected peptide to obtain an N-Fmoc-carrier protected peptide,
b. A step of adding a water-soluble amine to the reaction solution after the condensation reaction,
c. Deprotecting the Fmoc group from the protected amino group in the presence of a water soluble amine, and d. A production method including a step of adding an acid to the reaction solution for neutralization, further adding an acidic aqueous solution for washing, separating the layers, removing the aqueous layer, and obtaining an organic layer,
here,
The carrier for liquid phase peptide synthesis is a compound that directly binds to an amino acid or a peptide to make them insoluble in water and has a molecular weight of 300 or more,
The carrier-protected amino acid or peptide is an amino acid or peptide in which the carrier is bound to the carboxyl terminus of the amino acid or peptide, and
The water-soluble amine in step b and step c may be the same or different.
[2] After step d,
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 for washing, and then the organic layer is separated to remove the aqueous layer. The method according to the above [1], which comprises a step of obtaining an organic layer containing a protected peptide.
[3] A compound in which the carrier for liquid phase peptide synthesis has the following structure:

(In the formula, R ₂ , R ₄ and R ₅ are hydrogen atoms, R ₁ and R ₃ are alkoxy groups having 12 to 30, preferably 18 to 22 carbon atoms, and RY is —CH ₂ OH or —CH ₂ NHEt), or a compound having the following structure:

(In the formula, X is —CH ₂ OH or —CH ₂ NHEt; R ² , R ⁴ and R ⁵ are hydrogen atoms, and at least one of R ¹ and R ³ has the following formula:
-O-R ⁶ -Xa-A
And the remaining groups represent 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 group having 1 to 16 carbon atoms. Represents a group, Xa represents O or CONRc (wherein Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms),
A represents any one of formula (1) to formula (11),

(Here, R ⁷ , R ⁸ and R ⁹ may be the same or different and represent 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 Indicates a branched alkylene group)
(Note that each of the above formulas is shown in the state before binding to the carboxyl group of the amino acid or peptide),
The production method according to the above [1], which is a carrier derived from.
[4] The carrier for liquid phase peptide synthesis has the following structure:
General formula (V):

[Wherein, ring A represents an aromatic ring; Y is a hydroxyl group, a bromo group or a chloro group; Ra, Rb and Rc are each independently an organic group having an aliphatic hydrocarbon group, a hydrogen atom or an electron; Is an organic group having an aliphatic hydrocarbon group and at least one of Ra, Rb and Rc being an attracting group; rings A, B and C each independently having an electron withdrawing group; Good] or general formula (V′):

[In the formula, ring A represents an aromatic ring; Y is a hydroxyl group, a bromo group or a chloro group; n is an integer of 1 to 19; Rc' is a divalent organic group having an aliphatic hydrocarbon group. A ring; rings A, B and C may each independently have one or more kinds 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 when a plurality of Y are present may be the same or different; each Rc′ when a plurality of Rc′ is present may be the same or different. The divalent organic group having an aliphatic hydrocarbon group may be represented by the formula (a):

(In the formula, Xa is absent or represents -O-, -S-, -NHCO-, or -CONH-; Rd represents an aliphatic hydrocarbon group having 5 or more carbon atoms; k1 represents ₁ to 10; Represents an integer; each Rd when a plurality of Rd are present may be the same or different; each Xa when a plurality of Xa is present may be the same or different); ,
The organic group having an aliphatic hydrocarbon group is present at the 2-position and/or the 7-position of the fluorene compound, and has the formula (b):

(In the formula, * represents a bonding position; X ₁ is —O—; R ₁ is an aliphatic hydrocarbon group having 5 to 60 carbon atoms; m ₁ is 1),
Formula (c):

(In the formula, * represents a bonding position; X ₂ , X ₂ ′, X ₂ ″ and X ₂ ′″ are —O—; R ₂ and R ₄ each independently have 5 to 60 carbon atoms. 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 And a formula (d):

(In the formula, * represents a bonding position; X ₈ represents —O—; m ₃ is 2 or 3; n ₅ is 1; n ₆ is 3; X ₇ is —O— Yes; m ₃ R ₁₂ 's each independently represent an alkyl group having 4 to 30 carbon atoms), or one or more groups selected from the group consisting of
A fluorene compound represented by
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to the above [1], which is a carrier derived from.
[5] The carrier for liquid phase peptide synthesis has the following structure:
General formula (W)

[In the formula, Y represents a hydroxyl group or an NHEt group; R ^a is
Formula (a):

[In the formula, * represents a bonding position; m ₁ represents an integer of 1 to 10; m ₁ X ₁ 's each independently represent a single bond, or -O-, -S; -, -COO-, -OCONH-, -NHCO- or -CONH- is shown; R ₁ and m ₁ R ₂ are each independently a divalent aliphatic hydrocarbon group having 5 or more carbon atoms. And R ₃ is a hydrogen atom or the formula (W′):

(In the formula, * represents a bonding position; n R ^{b s} are each independently an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or a carbon which may be substituted with one or more halogen atoms. A group represented by the formula 1 to 6; and n represents an integer of 0 to 4].
Formula (b):

(In the formula, * represents a bonding position; m ₂ represents 1 or 2; n ₁ , n ₂ , n ₃ and n ₄ each independently represent 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 are —O—, —S—, —COO—, —OCONH—, Represents —NHCO— or —CONH—; m ₂ R ₄ and m ₂ R ₆ each independently represent an aliphatic hydrocarbon group having 5 or more carbon atoms; R ₅ represents 5 carbon atoms A group represented by the above aliphatic hydrocarbon group);
Formula (c):

(In the formula, * represents a bonding position; m ₃ represents an integer of 0 to 15; n ₅ represents an integer of 0 to 11; n ₆ represents an integer of 0 to 5; m ₃ Xs) ₃ each independently represents a single bond or represents -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 formula (d):

(In the formula, * represents a bonding position; n ₇ X ₄ each independently represents a single bond, or -O-, -S-, -COO-, -OCONH-, -NHCO. - or 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 , Ar represents an integer of 1 to 5; and Ar represents an organic group having an aliphatic hydrocarbon group selected from the group consisting of groups represented by the following formula), and the total number of carbon atoms in the organic group is 30 or more; n R ^b are each independently an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or an alkyl group having 1 to 6 carbon atoms which may be substituted with one or more halogen atoms. Represents a group; and n represents an integer of 0 to 4]
A benzyl compound represented by
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to the above [1], which is a carrier derived from.
[6] The carrier for liquid phase peptide synthesis has the following structure:
General formula (X)

[In the formula, Y represents a hydroxyl group or -NHEt group; k and l each independently represents an integer of 0 to 5, provided that, k + l is not the 0; k-number of R _a and the l R _b of each independently
Formula (a):

(In the formula, * represents a bonding position; m ₁ represents an integer of 1 to 10; m ₁ X ₁ 's each independently do not exist, or are —O—, —S—, — COO—, —OCONH— or —CONH—; m ₁ R ₁ 's each independently represent a divalent aliphatic hydrocarbon group having 5 or more carbon atoms.
Formula (b):

(In the formula, * represents a bonding position; m ₂ represents an integer of _{1 to 2} ; m ₂ , n ₁ , n ₂ , n ₃ and n ₄ each independently represent an integer of 0 to 2. M ₂ X ₂ , m ₂ X ₂ ′, m ₂ X ₂ ′″ and m ₂ X ₂ ″ are each independently absent or —O Represents -, -S-, -COO-, -OCONH- or -CONH-; m ₂ R ₂ and R ₄ are each independently a hydrogen atom, a methyl group or an aliphatic carbon atom having 5 or more carbon atoms. Represents a hydrogen group; R ₃ represents an aliphatic hydrocarbon group having 5 or more carbon atoms), and a formula (e):

(In the formula, * represents a bonding position; m ₃ represents an integer of 0 to 15; n ₅ represents an integer of 0 to 11; n ₆ represents an integer of 0 to 5; is X ₂ absent? , Or —O—, —S—, —NHCO— or —CONH—; m ₃ X _{7 s} are independently absent, or respectively —O—, —S—, —COO—, Represents -OCONH-, -NHCO- or -CONH-; m ₃ R ₁₂ 's each independently represent a hydrogen atom, a methyl group or an aliphatic hydrocarbon group having 5 or more carbon atoms). Shows an organic group having an aliphatic hydrocarbon group having 5 or more carbon atoms selected from the group consisting of groups, in which the total of all aliphatic hydrocarbon groups in the organic group having k+1 aliphatic hydrocarbon groups Has 16 or more carbon atoms;
Ring A may have a substituent in addition to R _a ;
Ring B may have a substituent in addition to R _b ]
A diphenylmethane compound represented by:
Or
General formula (Y)

[In the formula, each of the k Q's independently represents a single bond, or is -O-, -S-, -C(=O)O-, -C(=O)NH- or -NH. -Indicates k; _Ra independently has at least one aliphatic hydrocarbon group having at least one branched chain, has a total number of branched chains of 3 or more, and has a total carbon number of 14; Represents an organic group of 300 or more; k represents an integer of 1 to 4; R ₁ represents a hydrogen atom, or when Z represents a group represented by the following formula (a), Together with R ₂ , a single bond may be shown to form a fluorene ring with ring B; ring A may be R ₁ , k QR _a , and C(X)(Y)Z. And further selected from the group consisting of a halogen atom, 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. It may have one or more substituents; X represents a hydrogen atom or a phenyl group;
Y represents a hydroxyl group or a -NHEt group; and Z is a hydrogen atom or the formula (a):

(In the formula, * represents a bonding position; m represents an integer of 0 to 4; m Qs represent the same meaning as described above; m _Rb 's each independently represent a branched chain. An organic group having at least one aliphatic hydrocarbon group having one or more, a total number of branched chains of 3 or more, and a total carbon number of 14 or more and 300 or less; R ₂ represents a hydrogen atom , Or R ₁ together with R ₁ to form a single bond to form a fluorene ring with Ring A; and Ring B contains m QR _b and R ₂ in addition to a halogen atom, 1 One or more substituents selected from the group consisting of a C1-6 alkyl group optionally substituted with one or more halogen atoms and a C1-6 alkoxy group optionally substituted with one or more halogen atoms. A group represented by
An organic group having at least one aliphatic hydrocarbon group having one or more branched chains in R _a and R _b , a total number of branched chains of 3 or more, and a total carbon number of 14 or more and 300 or less, Formula (b):

(In the formula, * represents a bonding position with an adjacent atom; R ₃ and R ₄ each independently represents a hydrogen atom or a C1-4 alkyl group; X ₁ represents a single bond, C1-4 alkylene. Represents a group or an oxygen atom, provided that R ₃ and R ₄ are not both hydrogen atoms) and has 3 or more of the same or different divalent groups. ]
A branched chain-containing aromatic compound represented by:
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to the above [1], which is a carrier derived from.
[7] A compound in which the carrier for liquid phase peptide synthesis has the following structure:

(In the formula, R ₂ , R ₄ and R ₅ are hydrogen atoms, R ₁ and R ₃ are alkoxy groups having 12 to 30, preferably 18 to 22 carbon atoms, and RY is —CH ₂ It is OH.)
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The production method according to the above [1], which is a carrier derived from.
[8] A compound in which the carrier for liquid phase peptide synthesis has the following structure:

(2,4-didocosyloxybenzyl alcohol),
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The production method according to the above [7], which is a carrier derived from.
[9] The liquid phase peptide synthesis carrier is

(2,4-di(11'-triisopropylsilyloxyundecyloxy)benzyl alcohol),

(2,4-di(11'-t-butyldiphenylsilyloxyundecyloxy)benzyl alcohol),

(In the formula, X is F or Cl),

(2-(3',4',5'-trioctadecyloxybenzyl)-4-methoxybenzyl alcohol), and

(2,4-di(2',3'-dihydrophytyloxy)benzyl alcohol)
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to [1] above, which is a carrier derived from a compound selected from the group consisting of:
[10] A carrier-protected peptide containing a sequence consisting of Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg (SEQ ID NO: 2) is produced by repeating the above steps, and the above-mentioned [7] to [9]. The manufacturing method according to any one of 1.
[11] 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 then the side chain protecting group is deprotected to produce leuprorelin, wherein the production method according to the above [10].
[12] A compound in which the carrier for liquid phase peptide synthesis has the following structure:

(In the formula, R ₂ , R ₄ and R ₅ are hydrogen atoms, R ₁ and R ₃ are alkoxy groups having 12 to 30, preferably 18 to 22 carbon atoms, and RY is —CH ₂ NHEt)
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The production method according to the above [1] or [2], which is a carrier derived from.
[13] The liquid phase peptide synthesis carrier is

(N-ethyl-2,4-didocosyloxybenzylamine),

(N-ethyl-2,4-di(11'-triisopropylsilyloxyundecyloxy)benzylamine),

(N-ethyl-2,4-di(11'-t-butyldiphenylsilyloxyundecyloxy)benzylamine),

(N-ethyl-2-(3',4',5'-trioctadecyloxybenzyloxy)-4-methoxybenzylamine),

(N-ethyl-bis(4-docosyloxyphenyl)methylamine),

(N-ethyl-2,4-di(2',3'-dihydrophytyloxy)benzylamine), and

(N-ethyl-bis[4-(2',3'-dihydrophytyloxy)phenyl]methylamine)
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to [1] or [2] above, which is a carrier derived from a compound selected from the group consisting of:
[14] The above-mentioned [12] or [12] or [12], in which the carrier-protected peptide containing a sequence consisting of Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro (SEQ ID NO: 3) is produced by repeating the above steps. 13] The production method according to [13].
[15] A carrier and a side chain protecting group are deprotected from a carrier-protected peptide containing a sequence consisting of the Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro (SEQ ID NO: 3) to give leuprorelin. The manufacturing method according to the above [14], which is manufactured.
[16] The water-soluble amine is a divalent or higher-valent water-soluble amine having at least one primary or secondary amino group, preferably ethylenediamine, 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triamino. Any one of the above [1] to [15] 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 according to one.
[17] 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 production method according to any one of the above [1] to [16], which is an amount.
[18] 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) with respect to the amount of Fmoc groups present in the system. The production method according to any one of [1] to [17].
[19] The production method according to any one of the above [1] to [18], wherein the acidic aqueous solution in step d has a pH of 1 to 5 (preferably 1 to 4, more preferably 1 to 3).
[20] The manufacturing method according to any one of the above [1] to [19], wherein the step is performed in one pot.

According to the method for producing leuprorelin of the present invention, the peptide extension step can be carried out in a short time, and a high-purity crude product can be obtained. Therefore, a production technique for leuprorelin which is more economical than the prior art Can be provided.

The invention will now be described by way of example of exemplary embodiments, along with preferred methods and materials that can be used in the practice of the invention.
Unless otherwise specified in the text, all technical and scientific terms used herein have the same meanings as commonly understood by a person skilled in the art to which the present invention belongs. Also, any materials and methods equivalent or similar to those described herein can be used in the practice of the present invention as well.
In addition, all publications and patents cited herein in relation to the invention described herein are, for example, indicative of methods, materials, or the like that can be used in the invention. It constitutes a part.

In this specification, when the expressions "X to Y" are used, X is used as a lower limit and Y is included as an upper limit. In this specification, "about" is used to mean ±10%.

The abbreviations of amino acids that are the constituent units of leuprorelin synthesized by the method of the present invention have the following meanings.
Pyr:L-pyroglutamic acid His:L-histidine Trp:L-tryptophan Ser:L-serine Tyr:L-tyrosine dLeu:D-leucine D-Leu:D-leucine Leu:L-leucine Arg:L-arginine Pro- NHEt:L-proline ethylamide

The manufacturing method of the present invention will be described below.
1. N-Fmoc protected amino acid 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid whose amino group is protected with a group (N-Fmoc protected amino acid) means that the amino acid α-amino group is protected with an Fmoc group, On the other hand, a carboxyl group means an amino acid that is not protected and is reactive. When the amino acid has one or more amino groups, it is sufficient that the α-position amino group is protected by the Fmoc group and the other amino groups are protected by other protecting groups.
When the N-Fmoc protected amino acid has a highly functional group such as a hydroxyl group, an amino group other than the α-position, a guanidyl group, a carboxyl group other than the α-position, a thiol group, an indole group, an imidazole group, etc. It is preferable to introduce a general protecting group used in peptide synthesis into the functional group, and by removing the protecting group at any point after the reaction, preferably in the final step of the production of leuprorelin. Can be obtained.
Examples of the hydroxyl-protecting group include tBu group, Trt group, Bz group, acetyl group, silyl group and the like, preferably tBu group, and the amino-protecting groups other than the α-position include Boc group, Fmoc group, Cbz group, Trt group, Mmt group, ivDde group and the like, and preferably Boc group or Trt group, and the guanidyl group protecting group includes Pbf group, Pmc group, nitro group and the like, preferably A Pbf group, a carboxyl group protecting group includes a tBu group, a methyl group, an ethyl group, a Bz group, and the like, and a thiol group protecting group includes a Trt group, an Acm group, a tBu group, an S-tBu group, and the like. Examples of the protective group for the indole group include a Boc group, and examples of the protective group for the imidazole group include a Boc group, a Bom group, a Bum group, and a Trt group.

2. Carrier-protected amino acid, peptide and amino acid amide An amino acid protected by a carrier (carrier-protected amino acid) or a carrier-protected peptide means that the carboxyl terminus of the amino acid or peptide is protected by the carrier described below, and the amino terminus of the amino acid or peptide is Is a reactive amino acid or peptide.
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.
When the carrier-protected amino acid or the carrier-protected peptide has a functional group having high reactivity such as a hydroxyl group, an amino group other than the α-position, a guanidyl group, a carboxyl group, a thiol group, an indole group, and an imidazole group, these functional groups A general protecting group used in peptide synthesis may be introduced in, and the target compound can be obtained by removing the protecting group as needed after completion of the reaction.
Examples of the hydroxyl-protecting group include tBu group, Trt group, Bz group, acetyl group, silyl group and the like, preferably tBu group, and the amino-group protecting group includes Boc group, Fmoc group, Cbz group, Trt. Group, Mmt group, ivDde group and the like, preferably Boc group or Trt group, and the guanidyl group protecting group includes Pbf group, Pmc group, nitro group and the like, preferably Pbf group. The carboxyl protecting group includes tBu group, methyl group, ethyl group, Bz group and the like, and the thiol protecting group includes Trt group, Acm group, tBu group, S-tBu group and the like, 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.

3. Carrier for Liquid Phase Peptide Synthesis The carrier used in the production method of the present invention is a compound having a molecular weight of 300 or more, which is a compound that is directly bound to an amino acid or a peptide to make them insoluble in water. The carrier used in the present invention is a compound having the property of reversibly changing between a dissolved state and an insolubilized (crystallized or oiled) state due to a change in the composition of the solvent in which the carrier is dissolved. In the embodiment of the production method of the present invention, it is not essential that the carrier is insolubilized, and that the composition of the solvent is changed so that the carrier is insolubilized. Examples of such a carrier include, for example, a benzyl compound into which a long chain fatty acid has been introduced, which has been proposed by the present inventors (JP 2003-183298 A, JP 2004-059509 A, WO 2007/034812 JP, WO 2007/ 122847), and a benzyl compound (WO2017/038650) in which silicon is introduced at the end of a long-chain fatty acid to improve the solubility in an organic solvent, or a fluorene compound into which a long-chain fatty acid is introduced (WO2010/104169). Gazette), a diphenylmethane compound into which a long chain fatty acid is introduced (WO2010/113939), a benzyl type compound (WO2011/078295), and a branched type compound (WO2012/029794).
Although not limited to this, 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 may be insolubilized (crystallized or oiled), and In the step of recovering the carrier-protected peptide dissolved in the organic layer (layer of organic solvent or mixture of organic solvents) obtained in the final step of the production method, the carrier-protected peptide may be insolubilized (crystallized or oiled). .
The carrier used in the present invention is derived from the carrier compounds 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.

3-1: Carrier compound A
A compound having the following structure (sometimes referred to herein as "Kb"):

(In the formula, 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. In the formula, RY is —CH ₂ OH. Or —CH ₂ NHEt, which is a group bonded to the carboxyl group of an amino acid or peptide).
In the above formula, R ₁ and R ₃ are preferably an alkoxy group having 18 to 22 carbon atoms.
Of the specific compounds included in the above formula, preferred one is 2,4-didocosyloxybenzyl alcohol represented by the following formula,

Alternatively, it is N-ethyl-2,4-didocosyloxybenzylamine represented by the following formula.

3-2: Carrier compound B
A compound having the following structure (sometimes referred to herein as "KS"):

(In the formula, X is —CH ₂ OH or —CH ₂ NHEt; R ² , R ⁴ and R ⁵ are hydrogen atoms, and at least one of R ¹ and R ³ has the following formula:
-O-R ⁶ -Xa-A
And the remaining groups represent 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 group having 1 to 16 carbon atoms. Represents a group, Xa represents O or CONRc (wherein Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms),
A represents any one of formula (1) to formula (11),

(Here, R ⁷ , R ⁸ and R ⁹ may be the same or different and represent 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 straight chain or branched chain 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 Shows a branched alkylene group).
Of the specific compounds included in the above formula, preferred ones are represented by the following formula,

(N-ethyl-2,4-di(11'-triisopropylsilyloxyundecyloxy)benzylamine),

(2,4-di(11'-triisopropylsilyloxyundecyloxy)benzyl alcohol),

N-ethyl-2,4-di(11'-t-butyldiphenylsilyloxyundecyloxy)benzylamine, and

(2,4-di(11'-t-butyldiphenylsilyloxyundecyloxy)benzyl alcohol).

3-3: Carrier compound C
A compound having the following structure (may be referred to as “KJ1” in the present specification):
General formula (V):

[Wherein, ring A represents an aromatic ring; Y is a hydroxyl group, a bromo group or a chloro group; Ra, Rb and Rc are each independently an organic group having an aliphatic hydrocarbon group, a hydrogen atom or an electron; Is an organic group having an aliphatic hydrocarbon group and at least one of Ra, Rb and Rc being an attracting group; rings A, B and C each independently having an electron withdrawing group; Good] or general formula (V′):

[In the formula, ring A represents an aromatic ring; Y is a hydroxyl group, a bromo group or a chloro group; n is an integer of 1 to 19; Rc' is a divalent organic group having an aliphatic hydrocarbon group. A ring; rings A, B and C may each independently have one or more kinds 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 when a plurality of Y are present may be the same or different; each Rc′ when a plurality of Rc′ is present may be the same or different. The divalent organic group having an aliphatic hydrocarbon group may be represented by the formula (a):

(In the formula, Xa is absent 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; And each Rd when a plurality of Rd are present may be the same or different; and each Xa when a plurality of Xa is present may be the same or different). ,
The organic group having an aliphatic hydrocarbon group is present at the 2-position and/or the 7-position of the fluorene compound, and has the formula (b):

(In the formula, * represents a bonding position; X ₁ is —O—; R ₁ is an aliphatic hydrocarbon group having 5 to 60 carbon atoms; m ₁ is 1),
Formula (c):

(In the formula, * represents a bonding position; X ₂ , X ₂ ′, X ₂ ″ and X ₂ ′″ are —O—; R ₂ and R ₄ each independently have 5 to 60 carbon atoms. 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 And a formula (d):

(In the formula, * represents a bonding position; X ₈ represents —O—; m ₃ is 2 or 3; n ₅ is 1; n ₆ is 3; X ₇ is —O— Yes; m ₃ R ₁₂ 's each independently represent an alkyl group having 4 to 30 carbon atoms), or one or more groups selected from the group consisting of
A fluorene compound represented by.

3-4: Carrier compound D
A compound having the following structure (sometimes referred to as "KJ2" in the present specification):
General formula (W):

[In the formula, Y represents a hydroxyl group or a -NHEt group; ^Ra is
Formula (a):

[In the formula, * represents a bonding position; m ₁ represents an integer of 1 to 10; m ₁ X ₁ 's each independently represent a single bond, or -O-, -S; -, -COO-, -OCONH-, -NHCO- or -CONH- is shown; R ₁ and m ₁ R ₂ are each independently a divalent aliphatic hydrocarbon group having 5 or more carbon atoms. And R ₃ is a hydrogen atom or the formula (W′):

(In the formula, * represents a bonding position; n R ^{b s} are each independently an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or a carbon which may be substituted with one or more halogen atoms. A group represented by formulas 1 to 6; and n represents an integer of 0 to 4). ] A group represented by;
Formula (b):

(In the formula, * represents a bonding position; m ₂ represents 1 or 2; n ₁ , n ₂ , n ₃ and n ₄ each independently represent 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 are —O—, —S—, —COO—, —OCONH—, Represents —NHCO— or —CONH—; m ₂ R ₄ and m ₂ R ₆ each independently represent an aliphatic hydrocarbon group having 5 or more carbon atoms; R ₅ represents 5 carbon atoms The groups represented by the above aliphatic hydrocarbon groups);
Formula (c):

(In the formula, * represents a bonding position; m ₃ represents an integer of 0 to 15; n ₅ represents an integer of 0 to 11; n ₆ represents an integer of 0 to 5; m ₃ Xs) ₃ each independently represents a single bond or represents -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 formula (d):

[In the formula, * represents a bonding position; n ₇ X ₄ each independently represents a single bond, or -O-, -S-, -COO-, -OCONH-, -NHCO. - or 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 , And an integer of 1 to 5; and Ar represents an arylene group. ) Represents an organic group having an aliphatic hydrocarbon group selected from the group consisting of groups represented by the following formula, wherein the total number of carbon atoms in the organic group is 30 or more; A C1-6 alkoxy group, a halogen atom, or a C1-6 alkyl group which may be substituted with one or more halogen atoms; and n represents an integer of 0-4]
The benzyl compound represented by.
Of the specific compounds included in the above formula, preferred ones are represented by the following formula,

(N-ethyl-2-(3',4',5'-trioctadecyloxybenzyloxy)-4-methoxybenzylamine), and

((2-(3',4',5'-trioctadecyloxybenzyl)-4-methoxybenzyl alcohol)).

3-5: Carrier compound E
A compound having the following structure (sometimes referred to as "KJ3" in the present specification):

(In the formula, Y represents a hydroxyl group or a -NHEt group; k and l each independently represent an integer of 0 to 5, provided that k+l is not 0; k R _a and l R R _b of each independently
Formula (a):

(In the formula, * represents a bonding position; m ₁ represents an integer of 1 to 10; m ₁ X ₁ 's each independently do not exist, or are —O—, —S—, — COO-, -OCONH- or -CONH- is shown; m ₁ R ₁ 's each independently represent a divalent aliphatic hydrocarbon group having 5 or more carbon atoms;
Formula (b):

(In the formula, * represents a bonding position; m ₂ represents an integer of _{1 to 2} ; m ₂ , n ₁ , n ₂ , n ₃ and n ₄ each independently represent an integer of 0 to 2. M ₂ X ₂ , m ₂ X ₂ ′, m ₂ X ₂ ′″ and m ₂ X ₂ ″ are each independently absent or —O Represents -, -S-, -COO-, -OCONH- or -CONH-; m ₂ R ₂ and R ₄ are each independently a hydrogen atom, a methyl group or an aliphatic carbon atom having 5 or more carbon atoms. Represents a hydrogen group; R ₃ represents an aliphatic hydrocarbon group having 5 or more carbon atoms), and a formula (e):

(In the formula, * represents a bonding position; m ₃ represents an integer of 0 to 15; n ₅ represents an integer of 0 to 11; n ₆ represents an integer of 0 to 5; is X ₂ absent? , Or —O—, —S—, —NHCO— or —CONH—; m ₃ X ₇ are each independently absent, or —O—, —S—, —COO—, Represents -OCONH-, -NHCO- or -CONH-; m ₃ R ₁₂ 's each independently represent a hydrogen atom, a methyl group or an aliphatic hydrocarbon group having 5 or more carbon atoms). Represents an organic group having an aliphatic hydrocarbon group having 5 or more carbon atoms selected from the group, wherein the total carbon number of all aliphatic hydrocarbon groups in the organic group having k+1 aliphatic hydrocarbon groups is 16 or more;
Ring A may have a substituent in addition to R _a ;
Ring B may have a substituent in addition to R _b ]
A diphenylmethane compound represented by.
When the compound represented by the above formula (X) is used as a carrier, N-[α-(p-cetyloxyphenyl)-benzyl]-1-hydroxy-4-is obtained from the compound represented by the above formula (X). Those excluding fluorosulfonyl-2-naphthamide and tetraundecylbenzhydrol-3,3′,4,4′-tetracarboxylate may be selected and used.
A specific compound included in the above formula is preferably N-ethyl-bis(4-docosyloxyphenyl)methylamine represented by the following formula.

3-5: Carrier compound F
A compound having the following structure (sometimes referred to as "KJ4" in the present specification):

[In the formula, each of the k Q's independently represents a single bond, or is -O-, -S-, -C(=O)O-, -C(=O)NH- or -NH. -Indicates k; _Ra independently has at least one aliphatic hydrocarbon group having at least one branched chain, has a total number of branched chains of 3 or more, and has a total carbon number of 14; Represents an organic group of 300 or more; k represents an integer of 1 to 4; R ₁ represents a hydrogen atom, or when Z represents a group represented by the following formula (a), Together with R ₂ , a single bond may be shown to form a fluorene ring with ring B; ring A may be R ₁ , k QR _a , and C(X)(Y)Z. And further selected from the group consisting of a halogen atom, 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. It may have one or more substituents; X represents a hydrogen atom or a phenyl group;
Y represents a hydroxyl group or a -NHEt group; and Z is a hydrogen atom or the formula (a):

(In the formula, * represents a bonding position; m represents an integer of 0 to 4; m Qs represent the same meaning as described above; m _Rb 's each independently represent a branched chain. An organic group having at least one aliphatic hydrocarbon group having one or more, a total number of branched chains of 3 or more, and a total carbon number of 14 or more and 300 or less; R ₂ represents a hydrogen atom , Or R ₁ together with R ₁ to form a single bond to form a fluorene ring with Ring A; and Ring B contains m QR _b and R ₂ in addition to a halogen atom, 1 One or more substituents selected from the group consisting of a C1-6 alkyl group optionally substituted with one or more halogen atoms and a C1-6 alkoxy group optionally substituted with one or more halogen atoms. A group represented by
An organic group having at least one aliphatic hydrocarbon group having one or more branched chains in R _a and R _b , a total number of branched chains of 3 or more, and a total carbon number of 14 or more and 300 or less, Formula (b):

(In the formula, * represents a bonding position with an adjacent atom; R ₃ and R ₄ each independently represent a hydrogen atom or a C1-4 alkyl group; X ₁ represents a single bond, C1-4 alkylene. Represents a group or an oxygen atom, provided that R ₃ and R ₄ are not both hydrogen atoms) and has three or more divalent groups which are the same or different from each other]
A branched chain-containing aromatic compound represented by.
When the compound represented by the above formula (Y) is used as a carrier, 4-(3,7,11-trimethyldodecyloxy)benzyl alcohol or 3,4,5-tris[4-(3,7,11-trimethyl) Those excluding dodecyloxy)benzyloxy]benzyl alcohol may be selected and used.
Of the specific compounds included in the above formula, preferred ones are represented by the following formula,

(N-ethyl-2,4-di(2',3'-dihydrophytyloxy)benzylamine),

(2,4-di(2',3'-dihydrophytyloxy)benzyl alcohol), and

(N-ethyl-bis[4-(2′,3′-dihydrophytyloxy)phenyl]methylamine).

For binding the carrier compound A, the carrier compound B, the carrier compound C, the carrier compound D, the carrier compound E and the carrier compound F to the carboxyl group, a method generally used in peptide synthesis can be used without limitation in the present invention. For example, it can be performed 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 the 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-methylTHF, 4-methylTHP, isopropyl acetate, chloroform, dichloromethane, and N-methylpyrrolidone. Are THF, DMF, cyclohexane, CPME, MTBE, 2-methylTHF, 4-methylTHP, isopropyl acetate, and N-methylpyrrolidone. Furthermore, a mixed solvent of two or more of the above solvents may be used.

For the preparation of the starting material used in the production method of the present invention, or for the final recovery of the carrier-protected peptide produced using the production method of the present invention, the carrier-protected peptide is insolubilized (crystallized or oiled). If so, a polar solvent is used. Examples of the polar solvent to be used include methanol, ethanol, isopropanol, acetonitrile, propionitrile, DMF, dimethylacetamide, dimethylsulfoxide, water and the like, and mixed solvents of two or more thereof. Among them, methanol or acetonitrile is preferably used.

6. Elongation reaction in the production method of the present invention The production method of the present invention comprises a series of steps including a condensation step, a Fmoc group deprotection step, and a carrier-protected peptide recovery step, which are continuously performed without performing solid-liquid separation operation. Moreover, impurities generated in the synthesis step can be reduced or removed by separation separation. Therefore, peptide production can be continuously performed in the production of leuprorelin.
The peptide synthesis by the method for producing leuprorelin of the present invention includes the following steps.
(A) Condensation reaction step In a solvent mixture of an organic solvent or an organic solvent, an amino acid protected by a carrier (hereinafter referred to as “carrier protected”), a carrier protected amino acid amide or a carrier protected peptide, and 9-fluorenylmethyloxy. Step (b) Scavenging reaction step of condensing an amino acid whose amino group is protected by a carbonyl group (hereinafter referred to as “N-Fmoc protection”) to obtain an N-Fmoc-carrier-protected peptide In the reaction solution after the condensation reaction A step of adding a water-soluble amine (hereinafter sometimes referred to as amine scavenger) to form a scavenged body of an amino acid active ester,
(C) De-Fmoc step De-protecting the Fmoc group from the protected N-terminus in the presence of a water-soluble amine,
as well as,
(D) Acidic aqueous solution washing step A step of adding an acid to the reaction solution for neutralization, further adding an acidic aqueous solution for washing, separating the solution, and removing the aqueous layer.
In the peptide synthesis of the present invention, the following steps can be optionally added after the step (d).
(E) Basic aqueous solution washing step A step of adding an weakly basic aqueous solution for washing, separating the layers, removing the aqueous layer, and obtaining an organic layer.

The above step does not require a solid-liquid separation operation involving insolubilization (crystallization or oil formation) of the carrier-protected peptide, and thus can be performed in one pot.
Furthermore, by carrying out the above steps, the carrier-protected peptide having an amino acid added can be recovered in a state of being dissolved in the organic layer, as compared with the start of the synthetic reaction. Further, 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 continuously performed in that state.
Therefore, one aspect of the production method of the present invention is a method for producing leuprorelin, which comprises repeating the steps (a) to (d) a required number of times. The step of continuously repeating can also be performed in one pot.
In the production method of the present invention, the step (e) may be added in each synthesis cycle.
Further, in the method for producing leuprorelin, which comprises repeating the above steps (a) to (d) a required number of times, in the final step, the N-terminal amino acid Pyr is condensed in the same manner as in step (a). After that, without carrying out the step (c) corresponding to the Fmoc removing step, the same acidic aqueous solution washing as in the step (d) can be carried out to obtain a carrier-protected peptide, and 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 produced by appropriately referring to known methods used in peptide synthesis. Although not limited thereto, Arg-OKb using the carrier compound Kb in which RY is —CH ₂ —OH can be prepared as follows. For example, a carrier compound is dissolved in a solvent such as THF, N-Fmoc-protected amino acid and a condensing agent such as DIPCI are added to carry out condensation, and N-Fmoc which is an intermediate in which the carrier is bound to the carboxyl group of the amino acid. -Can make carrier protected amino acids.

The produced N-Fmoc-carrier protected amino acid can be obtained with high purity, preferably by crystallizing and recovering. For example, without limitation, the reaction solution containing the carrier-protected amino acid is distilled off under reduced pressure, and then the residue is a solvent for solidifying (crystallizing) N-Fmoc-carrier-protected amino acid, for example, Methanol or acetonitrile may be added to cause precipitation, and the precipitate may be filtered, suspended and washed with a solvent, and the obtained solid may be dried to obtain the final product.

The N-Fmoc-carrier-protected amino acid thus obtained can be used to prepare a carrier-protected amino acid by removing the N-terminal protecting group by appropriately referring to known methods used in peptide synthesis. For example, but not limited to this, it can be prepared by dissolving an N-Fmoc-supported 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 also comprises using a carrier compound in place of the carrier-protected amino acid in step (a) of the production method of the present invention, steps (a) to (d), In some cases, it can be obtained by further performing the step (e). For example, in step (a), RY is using a carrier compound H-EtN-Kb and Fmoc-Pro-OH is -CH ₂ -NHEt, by performing each step, H-Pro- a carrier protected amino acid amide 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 in the production method of the present invention.
Therefore, as one aspect of the production method of the present invention, a peptide synthesis method including a step of preparing a carrier-protected peptide before the step (a) can be mentioned.

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, without limitation, the organic layer containing the obtained carrier-protected peptide is evaporated under reduced pressure, and then a poor solvent, for example, cold acetonitrile is added to the residue for precipitation to precipitate the precipitate. After filtration, suspension washing is performed with a solvent, and the obtained solid matter is dried to obtain a synthetic carrier-protected peptide.
Therefore, as one embodiment of the production method of the present invention, a production method including a step of crystallizing/separating a carrier-protected peptide after the steps (a) to (d) and optionally step (e) is mentioned. You can

Each step will be described below.

6-1. Condensation reaction step In this step, the carrier-protected amino acid, amino acid amide or peptide, N-Fmoc-protected amino acid, and a condensing agent (preferably a condensing agent and an activator) are mixed to give an amino acid residue. An extended number of N-Fmoc-carrier protected peptides are obtained.
The method and order of addition of each component can be carried out without particular limitation, and a method usually used in the condensation step in peptide synthesis can be used.

The amount of the N-Fmoc protected amino acid used with respect to the carrier-protected amino acid, amino acid amide or peptide is usually 1.01 to 4 equivalents, preferably 1.03 to 4 equivalents, with respect to the carrier protected amino acid, amino acid amide or peptide. It is preferably 1.05 to 2 equivalents, more preferably 1.1 to 1.5 equivalents. If the amount is less than this range, unreacted carrier-protected peptide is likely to remain and amino acid deficiency is likely to occur. In the production method of the present invention, an unreacted active ester of an amino acid can be inactivated by scavenging (capturing) with a water-soluble amine added thereafter. Therefore, even if a larger amount of N-Fmoc protected amino acid is used, the residual problem does not occur as compared with the conventional method.

As the condensing agent, a condensing agent generally used in peptide synthesis can be used in the present invention without any limitation, and the condensing agent is not limited to this. For example, 4-(4,6-dimethoxy-1,3,5) -Triazin-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) , O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1, 1,3,3-Tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU ), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) ) Dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC). Among them, DMT-MM, HBTU, HATU, or COMU is preferable. The amount of the condensing agent used is usually 1 to 4 equivalents, preferably 1 to 2 equivalents, and more preferably 1.05 to 1.3 equivalents, relative to the carrier-protected peptide.

An activator may be added in the condensation step. Here, the activator is a reagent which, when coexisting with a condensing agent, guides an amino acid to a corresponding active ester, symmetrical acid anhydride or the like to facilitate the formation of a peptide bond (amide bond). As the activator, an activator generally used in peptide synthesis can be used in the present invention without any limitation, and for example, HOBt, HOCt, HOAt, HOOBt, HOSu, HOPht, HONb, pentafluorophenol, Examples thereof include ethyl cyano(hydroxyimino)acetate (Oxyma), and the like, with HOBt, HOOBt, HOCt, HOAt, HONb, HOSu, and Oxyma being preferred. The amount of activator used is usually 1 to 4 equivalents, preferably 1 to 2 equivalents, and more preferably 1.05 to 1.3 equivalents, relative to the carrier-protected peptide.
As the solvent used in the condensation step, a solvent generally used in peptide synthesis can be used without limitation in the present invention, and the solvent is not limited to this, and examples thereof include the above-mentioned solvents. The amount of the solvent used is such 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.

Regarding the reaction temperature, the temperature generally used in peptide synthesis is also used in the present invention and is, for example, usually in the range of -20 to 40°C, preferably 0 to 30°C. The reaction time (1 cycle time) is usually 0.5 to 30 hours.

6-2. Scavenging Reaction Step In the production method of the present invention, a water-soluble amine is added to the reaction system after the amino acid condensation reaction step to scavenge (capture) unreacted amino acid active ester. The water-soluble amine binds with the amino acid active ester to form a scavenging body, and inactivates the active ester. In the present specification, 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-valent water-soluble amine having at least one primary or secondary amino group, for example, 1-methylpiperazine, 4- Aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine and piperazine can be mentioned, preferably 1-methylpiperazine, 4-aminopiperidine and diethylenetriamine, and more preferably , 1-methylpiperazine.
The amount of the water-soluble amine added in step (b) is usually 1 to 10 equivalents, preferably 1 to 6 equivalents, and more preferably 1 to 4 equivalents based on the theoretically remaining amino acid equivalents. If the amount of amine added is less than this range, the scavenging (capturing) of the amino acid active ester will be insufficient, and a double hit will occur in which the remaining amino acid active ester reacts with the amino group regenerated in the next step (c), On the other hand, if the amount exceeds the above range, the Fmoc-removing reaction proceeds at the same time, and a double hit occurs in which the remaining amino acid active ester reacts with the regenerated amino group, resulting in a decrease in the purity and yield. Let
In the production method of the present invention, in the next step, the removal of the Fmoc group from the N-Fmoc-supported peptide is carried out by scavenging the amino acid active ester in the reaction system with an amine scavenger to form a scavenged product. Do after. As a result, the amino acid active ester in the reaction solution is inactivated during the Fmoc removal reaction, and double hits of amino acids can be prevented during deprotection without removing them from the reaction system. Moreover, the amino acid active ester captured by the water-soluble amine can be easily removed in the subsequent washing step.

6-3. Fmoc removal step In the production method of the present invention, the Fmoc group is removed from the N-Fmoc-carrier-protected peptide in the presence of a water-soluble amine. In this step, 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 more water-soluble amine having at least one primary or secondary amino group, and examples thereof include 1-methylpiperazine, 4-aminopiperidine, Examples thereof include diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine and piperazine, preferably 1-methylpiperazine, 4-aminopiperidine and diethylenetriamine, and more preferably 1- It is methylpiperazine. The type of water-soluble amine in this step may be the same as or different from the water-soluble amine added in the scavenging reaction step of step (b).
The equivalent of the water-soluble amine added in this step (c) is 5 to 30 equivalents, preferably 5 to 20 equivalents, and more preferably 10 to 20 equivalents, relative to the amount of Fmoc groups present in the system. If the amount of amine added is less than this range, the scavenging (capturing) of DBF generated by the Fmoc-removing 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 summing increases, and a side reaction (decomposition, racemization) occurs due to the accompanying neutralization step, which causes reduction in purity and reduction in yield.
In this step, the Fmoc-removing reaction is carried out in the presence of a water-soluble amine, but when the water-soluble amine present in the system has a function as a Fmoc-removing reagent, it is not necessary to add another Fmoc-removing reagent to the system. .. On the other hand, another Fmoc-removing reagent may be added to the system in order to efficiently perform the Fmoc-eliminating reaction. Examples of the water-soluble amine having a function as a Fmoc removing agent include the water-soluble amines exemplified above. Preferably, in this step, the Fmoc-removing reagent is added together with the water-soluble amine.
When the Fmoc-removing reagent and the Fmoc-removing reagent are added to the reaction system along with the water-soluble amine in this step, the water-soluble amine and the Fmoc-removing reagent may be added to the system at the same time, or , Fmoc-removing reagent may be added. The term "simultaneous" as used herein is meant to include addition before and after within the range considered to be simultaneous in the reaction in the present technical field. When the Fmoc-removing reagent is added after adding the water-soluble amine to the system, the time of the addition interval can be appropriately adjusted in consideration of the operation and other factors.
The removal of the Fmoc group from the N-terminal can be carried out in the present invention by appropriately changing the removal method generally used in peptide synthesis, if necessary. Examples of the Fmoc-removing reagent that can be used in the present invention include, but are not limited to, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) and 1,5-diazabicyclo[4.3]. .0]-5-nonene (DBN), 1,4-diazabicyclo[2.2.2]-octane (DABCO), triethylamine and tributylamine can be mentioned, and DBU is preferable.
Dibenzofulvene (DBF) is produced during the Fmoc-free reaction, but the water-soluble amine added in this step can scavenge (scaveng) these impurities. The DBF captured by the water-soluble amine can be easily removed in the subsequent acidic aqueous solution washing step.

6-4. The neutralizing step of the acidic aqueous solution washing step (d) can neutralize the organic base present in the system. 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. For example, when hydrochloric acid is used, although not limited thereto, 1N to 12N, preferably 2N to 12N, and more preferably 5N to 12N hydrochloric acid is added.
The neutralization here means that the reaction liquid has a neutral pH, and the pH may be 7.0 or less.
In the step (d), an acidic aqueous solution is further added to the reaction neutralization liquid neutralized with acid to wash, and then liquid separation is performed, the aqueous layer is discarded, and the organic layer is recovered. As a result, impurities that are soluble in the acidic aqueous solution can be removed.
The acidic aqueous solution used is not particularly limited, but 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, and more preferably 1 to 3.
The addition amount of the acidic aqueous solution used for washing is not particularly limited as long as it shows a washing effect, but is 0.1 to 3 times, preferably 0.5 to 2 times, and more preferably 0. It can be used in an 8- to 1.5-fold amount.
The steps of washing, separating, and discarding the aqueous layer are not limited in number, and may be performed once or multiple times. The number of times is appropriately selected depending on the type of compound in the reaction system, the amount of impurities, and the purpose.

In the present invention, impurities can be removed by washing the acidic aqueous solution in step (d). By separating the 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 impurities can be removed, and a carrier-protected peptide dissolved in the reaction system in which the impurities are reduced or removed can be obtained.
Moreover, the liquid separation operation using an aqueous solution is simple and contributes to shortening the process time. Furthermore, the solid-liquid separation operation is not required, and the use of a poor solvent for solidifying the carrier can be reduced.
In the continuous peptide synthesis using the method of the present invention, in the final cycle, after the Fmoc removing step, after neutralizing with an acid, the carrier-protected peptide is solidified (crystallized) to perform a solid-liquid separation operation. Although the carrier-protected peptide may be recovered by using it, washing with an acidic aqueous solution is preferable from the viewpoint of more complete removal of impurities.

6-5. Basic aqueous solution washing step The peptide synthesis of the present invention can optionally include the following steps.
In step (e), a weakly basic aqueous solution is added, washed, and then separated, the aqueous layer is discarded, and the organic layer is collected. As a result, impurities that are soluble in the weakly basic aqueous solution can be removed.
The weakly basic aqueous solution used is not particularly limited, but examples thereof include an aqueous sodium hydrogen carbonate solution, an aqueous sodium carbonate solution, and an aqueous potassium carbonate solution, and preferably an aqueous sodium hydrogen carbonate solution. The pH of the weakly basic aqueous solution is 8 to 12, preferably 8 to 10.
The amount of the weakly basic aqueous solution used for washing is not particularly limited as long as it exhibits a washing effect, but it is 0.1 to 3 times, preferably 0.5 to 2 times, and more preferably 0 times the reaction liquid. It can be used in an amount of 0.8 to 1.5 times.
The steps of washing, separating, and discarding the aqueous layer are not limited in number, and may be performed once or multiple times. The number of times is appropriately selected depending on the type of compound in the reaction system, the amount of impurities, and the purpose.
By washing the weakly basic aqueous solution in step (e), impurities that are soluble in the weakly basic aqueous solution can be removed. In the continuous peptide synthesis in the method of the present invention, 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 by a solid-liquid separation operation.

After the step (d) or the step (e), a water removing step of the step (f) may be added if necessary. In the water removal step, there are a method of causing a salt effect by adding a salt solution and removing water efficiently, and a method of removing water using a dehydrating agent. Salt solutions include, but are not limited to, saline, for example. The dehydrating agent may include, but is not limited to, anhydrous sodium sulfate.

7. Crystallization/separation step of carrier-protected peptide The carrier-protected peptide synthesized by the method of the present invention is insolubilized (for example, crystallized) and separated after step (d), step (e) or step (f). can do. The insolubilization is performed by appropriately referring to a known method in the field of peptide synthesis using a carrier having a property that the dissolved state and the insolubilized (crystallized) state are reversibly changed by the composition change of the solvent in which the carrier is dissolved. It can be carried out, for example, by changing the composition of the solution in which the carrier-protected peptide is dissolved. The conditions for insolubilization can be appropriately selected depending on the type of carrier used and the type and length of the synthesized carrier-protected peptide. For example, the following means for changing the solvent composition can be used, although not limited thereto.

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-protected peptide is dissolved. Preferred means for changing the solution composition include, for example, means for crystallization as it is in a solution in which the carrier-protected peptide is dissolved, or after the solvent of the solution is concentrated and then a poor solvent is added. Here, "concentration" means distilling off a part or all of the solvent, for example, distilling off the solvent under reduced pressure. Then, the precipitated crystals can be separated by, for example, filtration or centrifugation. The separated crystals can be removed from the crystallized carrier-protected peptide, preferably by removing the impurities and the like optionally separated with the crystals by washing with an organic solvent. Although the carrier-protected peptide synthesized by the method of the present invention has impurities sufficiently removed, the impurities can be further removed by further performing these operations.
The poor solvent in the present invention means a solvent in which the carrier-protected peptide is poorly dissolved, that is, the solvent in which the carrier-protected peptide is difficult or insoluble. The carrier-protected peptide is difficult to dissolve or does not dissolve as long as it is a solvent that is liquid at room temperature such that the solubility of the carrier-protected peptide is less than 1% by mass at 25°C. It is preferable to use water-containing methanol or water.
In the last cycle of continuous peptide synthesis using the method of the present invention, the main crystallization/separation step can be performed after step (d), step (e) or step (f), whereby the synthesis The carrier-protected peptide can be recovered.

8. Carrier Deprotection Step The carrier deprotection of the carrier-protected peptide synthesized by the method of the present invention can be carried out by removing (deprotecting) the carrier bound 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 acid treatment is preferable. For example, a deprotection method using TFA can be used, and more specifically, 1-100% trifluoroacetic acid when Kb is used and 1-100% trifluoroacetic acid when KS is used. , 1 to 100% trifluoroacetic acid when the carrier KJ1 and KJ2 are used, 95 to 100% trifluoroacetic acid when the carrier KJ3 is used, and 1 to 100% trifluoroacetic acid when the carrier KJ4 is used. It is preferable to deprotect with.

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. For example, the target peptide can be isolated and purified by extracting and washing the reaction mixture, crystallization, chromatography and the like.

The following abbreviations are used in the present specification and in the following Examples and Comparative Examples.
AAs : any amino acid residue of 1 or more
AAx : arbitrary 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-methylmorphonium chloride
DTT : Dithiothreitol
Fmoc : 9-fluorenylmethyloxycarbonyl
Kb : 2,4-didocosyloxybenzyl
MTBE : methyl tert-butyl ether
Oxyma : ethyl cyano(hydroxyimino)acetate
Pbf : 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
Pyr : Pyroglutamic acid
tBu : tert-butyl
TFA : trifluoroacetic acid
TFE : 2,2,2-trifluoroethanol
THF : tetrahydrofuran
THP : tetrahydropyran
TIS : Triisopropylsilane
Trt : triphenylmethyl

The general synthetic method used in this example is shown below. Although the case where Kb is used as the carrier compound is described as an example, KS, KJ1, KJ2, KJ3 and KJ4 can be similarly used. Further, the addition amount of each reagent is merely an example and is not limited to this.
(1) General method for removing Fmoc

The starting material was dissolved in a mixed solution of THF:DMF (9/1) at 18 v/w, piperidine (1.5 equiv) and DBU (1.0 equiv) were added, and the mixture was stirred at room temperature for 10 minutes. 6N hydrochloric acid (3.50 equiv) was added while cooling with ice, and the solvent was evaporated under reduced pressure. Acetonitrile was added to the residue, and the deposited precipitate was filtered and further subjected to acetonitrile suspension washing, and the obtained solid was dried under reduced pressure to obtain a Fmoc-free body.

The condensation deprotection method used in the method of the present invention is shown below. Although the case where Kb-OH is used as the carrier compound is described as an example, Kb-NHEt, KS, KJ1, KJ2, KJ3 and KJ4 can be used similarly. Further, the addition amount of each reagent is merely an example and is not limited to this.
(2) 1- pot condensation deprotection method using amine scavenger (water-soluble amine)

The starting material was dissolved in a mixed solution of THF:DMF (9/1) at 18 v/w to obtain Fmoc-AAx-OH (1.30 equiv), COMU (1.25 equiv), and DIPEA (2.30 equiv). Was added and the mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (0.45 equiv) was added as a water-soluble amine, and the mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (20.0 equiv) and DBU (7.0 equiv) were added, and the mixture was stirred at room temperature for 10 minutes. THF (0.8 v/w) and 0.1 N hydrochloric acid (18 v/w) were added to the reaction solution containing 6N hydrochloric acid (49.50 equiv), which was washed and separated, and the aqueous layer was discarded. Furthermore, a 0.5N sodium hydrogen carbonate aqueous solution (18 v/w) was added, washed and separated, the aqueous layer was discarded, and an amino acid condensate was obtained as a solution.

The method for deprotecting (separating) the carrier used in the method of the present invention from the peptide is shown below. Although the case where Kb is used as the carrier compound is described as an example, KS, KJ1, KJ2, KJ3 and KJ4 can be similarly used. Further, the addition amount of each reagent is merely an example and is not limited to this.
(3) Kb protecting group general deprotection method

The raw material was dissolved in a mixed solution of DCM:TFE:TFA (90/9/1) at 19.75 v/w, and the mixture was 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, and the mixture was washed, separated, and the aqueous layer was discarded. Diisopropyl ether was added to the obtained organic layer, and the mixture was evaporated under reduced pressure. Diisopropyl ether was added to the residue and the deposited precipitate was suspended and washed, and the obtained solid was dried under reduced pressure to obtain a deprotected Kb body.

Hereinafter, peptide synthesis using the method of the present invention will be shown.
The expression "v/w" in the following description of the specification indicates the amount of solvent to be added in the series of peptide synthesis reactions, based on the starting material.
Example 1: Synthesis of H-Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (SEQ ID NO: 1) using Kb-OH.
Synthesis of compound 1

2,4-didocosyloxybenzyl alcohol (denoted as "Kb-OH") (10.0 g, 13.20 mmol) was dissolved in dehydrated THF (132 mL), and Fmoc-Arg(Pbf)-OH (12. 85 g, 19.80 mmol, 1.50 equiv), DIPCI (3.06 ml, 19.80 mmol, 1.5 equiv), and DMAP (80.7 mg, 0.66 mmol, 0.05 equiv) were added, and the mixture was stirred at room temperature for 30 minutes. . The reaction solution was evaporated under reduced pressure. Methanol was added to the residue and the deposited precipitate was filtered, suspended in methanol, and suspended in acetonitrile twice. The obtained solid was dried under reduced pressure to obtain compound 1 (18.33 g, quant).

Synthesis of compound 2

Compound 1 (19.85 g, quant) was obtained by performing Fmoc-free general synthetic method on Compound 1.

Synthesis of compound 3 (H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-OKb: SEQ ID NO: 4)

The following amino acids were introduced into compound 2 by a method of repeating 1-pot condensation deprotection method using an amine scavenger (water-soluble amine) to obtain compound 3 in a solution.
1st residue: Fmoc-Leu-OH
Second residue: Fmoc-dLeu-OH
Third residue: Fmoc-Tyr(tBu)-OH
4th residue: Fmoc-Ser(tBu)-OH
Fifth residue: Fmoc-Trp(Boc)-OH
Sixth residue: Fmoc-His(Trt)-OH

Synthesis of Compound 4 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-OKb: SEQ ID NO: 5)

DMF was added to the obtained solution of compound 3 so that the theoretical yield was 1.8 v/w. H-Pyr-OH (3.30 g, 25.55 mmol), COMU (7.0 g, 16.26 mmol, 1.25 equiv), DIPEA (5.10 ml, 29.28 mmol, 2.25 equiv) were added. The resulting mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (0.45 equiv) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 5 minutes. 0.01N hydrochloric acid (18 v/w) was added, and the mixture was washed, separated, and the aqueous layer was discarded. The solvent of the obtained organic layer was distilled off under reduced pressure. Acetonitrile is added to the residue, and the deposited precipitate is filtered, washed with acetone and suspended, and the obtained solid is dried under reduced pressure to give compound 4 (28.78 g, 87.50%). ) Got.

Synthesis of Compound 5 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-OH: SEQ ID NO: 6)

Compound 4 (20.0 g, 7.90 mmol) was subjected to a Kb protecting group general deprotection method to obtain compound 5 (14.39 g, quant).

Synthesis of Compound 6 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-Pro-NHEt: SEQ ID NO:7)

Compound 5 (6.20 g, 3.45 mmol) was dissolved in THF to 16.2 v/w, and H-Pro-NHEt.HCl (0.80 g, 4.5 mmol) dissolved in DMF 1.8 v/w. , 1.3 equiv) was added. _{DMT-MM · 2H 2 O (} 1.84g, 6.05mmol, 1.75equiv), DIPEA (2.41ml, 13.83mmol, 4.0equiv) was added. The resulting mixed liquid was stirred for 60 minutes while cooling with ice. Cyclohexane (18 v/w) and 0.01 N hydrochloric acid (18 v/w) were added to the obtained reaction solution, which was washed and separated, and the aqueous layer was discarded. The solvent of the obtained organic layer was distilled off under reduced pressure. Diisopropyl ether was added to the residue, the deposited precipitate was filtered, washed with diisopropyl ether by suspension, and the obtained solid was dried under reduced pressure to obtain compound 6 (5.33 g, 80.51%).

Synthesis of compound 7 (Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt: SEQ ID NO: 1)

Compound 6 (5.33 g, 2.78 mmol) was dissolved in 92 ml of a mixed solution of TFA/TIS/water (90/1/9), DTT (3.70 g, 40 mg/ml) was added, and the mixture was stirred at room temperature for 3 hours. did. The reaction solution was filtered using Celite, the filter residue was washed with methanol and filtered, toluene (mixed liquid amount) was added to the obtained filtrate, and the solvent was distilled off under reduced pressure. Cold MTBE was added to the residue, the deposited precipitate was filtered, further MTBE-suspended and hexane-suspended, and the obtained solid was dried under reduced pressure to give compound 7 (4.21 g, quant, HPLC purity 76.28). %) was obtained.

Example 2: Synthesis of H-Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (SEQ ID NO: 1) using Kb-NHEt carrier.
Synthesis of compound 8

N-Ethyl-2,4-didocosyloxybenzylamine (denoted as "EtNKb") (12.5 g, 15.23 mmol) was dissolved in THF/DMF (9/1) at 18 v/w. , Fmoc-Pro-OH/H ₂ O (7.04 g, 19.80 mmol, 1.30 equiv), COMU (8.15 g, 19.04 mmol, 1.25 equiv), and DIPEA (8.62 ml, 49.49 mmol, 3.30 equiv) was added and the mixture was stirred at room temperature for 15 minutes. 1-Methylpiperazine (0.45 equiv) was added, and the mixture was stirred at room temperature for 10 minutes. 1-Methylpiperazine (20.0 equiv) and DBU (7.0 equiv) were added, and the mixture was stirred at room temperature for 10 minutes. Cyclohexane (4.5 v/w) and 0.1 N hydrochloric acid (18 v/w) were added to the reaction solution containing 6 N hydrochloric acid (50.70 equiv) while cooling with ice, and the mixture was washed, separated, and the aqueous layer was discarded. Further, 0.5N sodium hydrogen carbonate aqueous solution (18 v/w) was added, washed and separated, the aqueous layer was discarded, and compound 8 was obtained as a solution.

Synthesis of Compound 9 (H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-Pro-EtNKb: SEQ ID NO:8)

The following amino acids were introduced into compound 8 by repeating the 1-pot condensation deprotection method using an amine scavenger (water-soluble amine) to obtain compound 9 in a solution.
1st residue: Fmoc-Arg(Pbf)-OH
Second residue: Fmoc-Leu-OH
Third residue: Fmoc-dLeu-OH
4th residue: Fmoc-Tyr(tBu)-OH
Fifth residue: Fmoc-Ser(tBu)-OH
Sixth residue: Fmoc-Trp(Boc)-OH
7th residue: Fmoc-His(Trt)-OH

Synthesis of Compound 10 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-Pro-EtNKb: SEQ ID NO: 9)

H-Pyr-OH (2.95 g, 22.84 mmol, 1.50 equiv), COMU (9.46 g, 22.08 mmol, 1.45 equiv), DIPEA (6.50 ml, 37.31 mmol) was added to the obtained compound 9 solution. , 2.50 equiv) was added. The resulting mixture was stirred at room temperature for 15 minutes. 1-Methylpiperazine (0.65 equiv) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 10 minutes. While cooling with ice, 6N hydrochloric acid (3.10 equiv) was added for neutralization, and then 0.1N hydrochloric acid (18 v/w) was added, followed by washing, liquid separation, and discarding the aqueous layer. Further, 0.5N sodium hydrogen carbonate aqueous solution (18 v/w) was added, and the mixture was washed and separated, and the aqueous layer was discarded. The solvent of the obtained organic layer was distilled off under reduced pressure. Cold acetyl chloride was added to the residue, and the deposited precipitate was filtered, washed with acetyl chloride and dried under reduced pressure to obtain Compound 10 (36.93 g, 90.83). %) was obtained.

Synthesis of compound 7 (Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt: SEQ ID NO: 1)

Compound 10 (5.00 g) was dissolved in 93.6 ml of a mixed solution of TFA/TIS/water (90/1/9), DTT (1.87 g, 20 mg/ml) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was filtered through Celite, the filter residue was washed with methanol and filtered, cold MTBE was added to the concentrated residue of the obtained filtrate, and the deposited precipitate was filtered, and MTBE was washed twice with hexane. Washing was performed, and the obtained solid was dried under reduced pressure to obtain compound 7 (2.45 g, 93.0%, HPLC purity 95.18%).

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, the peptide having the above sequence can be synthesized.
The synthetic scheme is shown below.

The specific method reported in Non-Patent Document 1 will be described below.
Synthesis of Compound IV (H-Arg(ωNO ₂ )-Pro-NHEt) Boc-Arg(ωNO ₂ )-OH (298 g, 1.67 mol), HCl.H-Pro-NHEt (260 g, 1.7 mol), 1 -Hydroxybenzotriazole monohydrate (260 g, 1.7 mol) and N-methylmorpholine (213 g, 2.11 mol) were dissolved in THF (3 L) with stirring. The reaction solution was cooled to −5° C., and dicyclohexylcarbodiimide (400 g, 1.94 mol) was added. 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, and the mixture was washed twice with a saturated saline solution containing 5% sodium carbonate (2 L) and once with a saturated saline solution containing 5% citric acid (2 L). The organic layer was dried over sodium sulfate and then concentrated to dryness. The obtained residue was dissolved in 1,4-dioxane (1.5 L) and cooled to 10°C. 12% HCl-1,4-dioxane solution (5 L) was added with stirring. After stirring at 15°C for 30 minutes, the mixture was stirred at room temperature for 1.5 hours. The precipitate was filtered, washed twice with diethyl ether (1 L) and twice with acetone (1 L), and dried under reduced pressure. Compound IV was obtained as a white powder in an amount of 440 g (yield: 63.6%).

Synthesis of compound VI (Boc-D-Leu-Leu-Arg(ωNO ₂ )-Pro-NHEt: SEQ ID NO: 10) Compound IV (225 g, 0.54 mol) and Boc-D-Leu-Leu-OH (190 g, 0) 0.55 mol), 1-hydroxybenzotriazole monohydrate (113 g, 0.74 mol) and N-methylmorpholine (169 g, 1.67 mol) were dissolved in DMF (1.5 L) with stirring. The reaction solution was cooled to 0-3° C. and dicyclohexylcarbodiimide (134 g, 0.65 mol) was added. The reaction was carried out at 0° C. for 1 hour and 5° C. for 215 hours, and the completion of the reaction was confirmed by TLC. The reaction solution was diluted with water (1.5 L). The precipitate was filtered off and washed with ethyl acetate (1 L). Ethyl acetate (2 L) and saturated sodium carbonate solution (3 L) were added to the filtered solution. After liquid separation, the organic layer was washed with saturated sodium carbonate solution (1 L), 5% aqueous sodium carbonate solution (1 L), 5% aqueous citric acid solution (1 L), and water (1 L). Diethyl ether (3 L) was added to the organic layer, decantation was performed, diethyl ether (1 L) was added, and the mixture was left standing at 5° C. for 12 hours. The precipitate was filtered and then dried under reduced pressure. Compound VI was obtained as a white powder in an amount of 283 g (yield: 78.2%).

Synthesis of compound VII (HD-Leu-Leu-Arg(ωNO ₂ )-Pro-NHET: SEQ ID NO: 11) Compound VI (215 g, 0.32 mol) was stirred at 0° C. with 12% HCl-1, Added to 4-dioxane solution (600 mL). The reaction solution was warmed to room temperature over 30 minutes. The solution layer was removed by decantation, 12% HCl-1,4-dioxane solution (300 mL) was added, and the mixture was stirred for 30 minutes. The solution layer was removed, diethyl ether was added to the residue, and crystallization by decantation was repeated 3 times, acetone (800 ml) was added, decantation was performed, and the same amount of acetone was added again. The precipitate was collected by filtration, washed twice with diethyl ether (400 ml), and dried under reduced pressure. 68 g of Compound VII (yield 81.4%, HPLC purity 97.7%) was obtained as white fine crystals.

Synthesis of Compound IX (Z-Ser-Tyr-D-Leu-Leu-Arg(ωNO ₂ )-Pro-NHET: SEQ ID NO: 12) Compound VII (168 g, 0.26 mol) and Z-Ser-Tyr-OH (105 g , 0.26 mol). 1-Hydroxybenzotriazole monohydrate (53 g, 0.35 mol) and N-methylmorpholine (78 g, 0.77 mol) were sequentially dissolved in DMF (0.8 L) with stirring. After confirming complete dissolution, the reaction solution was cooled to 0° C., and dicyclohexylcarbodiimide (65 g, 0.35 mol) was added. The reaction was carried out at 0°C for 1 hour and 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, the organic layer was isolated, and the organic layer was washed twice with a saturated sodium carbonate solution (1.5 L) twice with a 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 as 221 g (yield 89%, HPLC purity 97.9%) as a white powder.

Compound X (H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt: SEQ ID NO: 13) Compound IX (160 g, 0.17 mol) was dissolved in glacial acetic acid (1.2 L). 20% Palladium hydroxide hydrate (1.5 g) was added and stirred, and hydrogenation was carried out for 45 to 48 hours until the reaction was completed. The hydrogenation catalyst was removed by filtration and concentrated under reduced pressure. Ethyl acetate (2 L) was added to the residue and the suspension was washed to remove the solvent. Methanol (800 ml) and 12% HCl-1,4-dioxane solution (200 mL) were added to the residue. The solution was concentrated and acetone (1 L) was added. The precipitate was collected by filtration, washed twice with acetone (300 ml), and dried under reduced pressure. 105 g (yield 75%, HPLC purity 89.1%) of compound X was obtained as a white amorphous solid.

Synthetic compound X (85 g, 0.1 mol) of the final compound (leuprorelin: Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt: SEQ ID NO: 1 ) and Pyr-His-Trp. -OH (20 g, 0.13 mol), 1-hydroxybenzotriazole monohydrate (20 g, 0.13 mol) and N-methylmorpholine (20 g, 0.2 mol) were sequentially stirred at room temperature while stirring in DMF (350 mL). It was dissolved in. After confirming complete dissolution, a solution of dicyclohexylcarbodiimide (20 g, 0.1 mol) dissolved in DMF (100 ml) was added dropwise over 5 hours. The reaction was carried out for 2 hours until completion of the reaction was confirmed by TLC, and water (100 ml) was added. The reaction solution was stirred for 30 minutes, the precipitate was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate (1 L) was treated three times until it solidified to a residue. The precipitate was collected by filtration, washed twice with ethyl acetate (300 ml), and dried under reduced pressure. The final compound was obtained as a gray powder in 129 g (76.3% yield, 63.3% HPLC purity).

In the conventional synthesis method reported in Non-Patent Document 1 which is a comparative example, a step of solidifying a carrier-protected peptide, filtering and drying is required, and the operation is complicated, and a large amount of an organic solvent is required, Furthermore, the drying process increases the process time.

2,4-di(11'-triisopropylsilyloxyundecyloxy)benzyl alcohol (hereinafter, 2,4-di(11'-triisopropylsilyloxyundecyloxy)benzyl group may be referred to as KS1) Of compound 13 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-KS1: SEQ ID NO: 14)

Example 3: Synthesis of compound 11 (H-Arg(Pbf)-O-KS1)

2,4-di(11'-triisopropylsilyloxyundecyloxybenzyl alcohol (1.00 g, 1.26 mmol) was used in the same manner as in JP 6116782 Example (1-c) (however, Fmoc Fmoc-Arg(Pbf)-OH was used instead of -Gly-OH) to obtain a solution of compound 11. The obtained solution was concentrated under reduced pressure and dried to give compound 11 (1.515g, 1.26mmol). , Quant).

Example 4: Synthesis of compound 12 (H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-KS1 : SEQ ID NO: 15 )

The following amino acids were introduced to the compound 11 obtained in Example 3 by repeating the 1-pot condensation deprotection method using an amine scavenger (water-soluble amine) to obtain the compound 12 in a solution.
1st residue: Fmoc-Leu-OH
Second residue: Fmoc-dLeu-OH
Third residue: Fmoc-Tyr(tBu)-OH
4th residue: Fmoc-Ser(tBu)-OH
Fifth residue: Fmoc-Trp(Boc)-OH
Sixth residue: Fmoc-His(Trt)-OH

Example 5: Synthesis of compound 13 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-KS1 : SEQ ID NO: 14 )

DMF was added to the obtained Compound 12 solution so that the theoretical yield was 1.8 v/w. H-Pyr-OH (0.213 g, 1.64 mmol), COMU (0.675 g, 1.58 mmol), DIPEA (0.505 ml, 2.90 mmol) were added. The resulting mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (0.45 equiv) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 5 minutes. 6N hydrochloric acid was added, and 0.1N hydrochloric acid was further added so that the theoretical yield was 18 v/w, followed by washing, liquid separation, and discarding the aqueous layer. To the obtained organic layer was added 0.5N aqueous sodium hydrogen carbonate solution in the same amount as 0.1N hydrochloric acid, and the organic layer was washed and separated, and the aqueous layer was discarded. The solvent of the obtained organic layer was distilled off under reduced pressure to obtain Compound 13 (1.09 g, 33.7%).

For 2,7-didocosyloxy-9-(3-fluorophenyl)-9-bromofluorene (hereinafter, 2,7-didocosyloxy-9-(3-fluorophenyl)-9-fluorenyl group may be referred to as Fl) Of compound 17 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-Fl: SEQ ID NO: 16)

Example 6: Synthesis of compound 14 (Fmoc-Arg(Pbf)-F1)

2,7-Didocosyloxy-9-(3-fluorophenyl)-9-bromofluorene (0.90 g, 0.91 mmol) was used in the same manner as in Patent No. 6092513 Example 6 (however, Z-Ala-OH Compound 14 (1.03 g, 0.663 mmol, 73.0%) was obtained by using Fmoc-Arg(Pbf)-OH instead of.

Example 7: Synthesis of compound 15 (H-Arg(Pbf)-O-F1)

Compound 14 (0.900 g, 0.578 mmol) was subjected to Fmoc general synthetic method using 1-methylpiperazine instead of piperidine, and washed with hydrochloric acid water and aqueous sodium hydrogen carbonate solution to give a solution of compound 15 Obtained.

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).

The following amino acids were introduced into compound 15 by repeating the 1-pot condensation deprotection method using an amine scavenger (water-soluble amine) to obtain compound 16 in a solution.
1st residue: Fmoc-Leu-OH
Second residue: Fmoc-dLeu-OH
Third residue: Fmoc-Tyr(tBu)-OH
4th residue: Fmoc-Ser(tBu)-OH
Fifth residue: Fmoc-Trp(Boc)-OH
Sixth residue: Fmoc-His(Trt)-OH

Example 9: Synthesis of compound 17 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-Fl: SEQ ID NO: 16).

DMF was added to the obtained Compound 16 solution so that the theoretical yield was 1.8 v/w. H-Pyr-OH (0.097 g, 0.75 mmol), COMU (0.310 g, 0.723 mmol), DIPEA (0.232 ml, 1.33 mmol) were added. The resulting mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (0.45 equiv) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 5 minutes. The solvent of the obtained organic layer was distilled off under reduced pressure. Acetonitrile is added to the residue, the deposited precipitate is filtered, further washed with acetone, and the resulting solid is dried under reduced pressure to give Compound 17 (0.999 g, 0.37 mmol, 64.6%).

2,4-di(2',3'-dihydrophytyloxy)benzyl alcohol (hereinafter, 2,4-di(2',3'-dihydrophytyloxy)benzyl group may be referred to as KJ1) Of compound 21 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-KJ1: SEQ ID NO: 18)

Example 10: Synthesis of compound 18 (Fmoc-Arg(Pbf)-O-KJ1)

2,4-Di(11'-triisopropylsilyloxyundecyloxybenzyl alcohol (1.76 g, 2.22 mmol) was used as in Patent 5929756 Example 23, except that Fmoc-Ser(tBu)- Fmoc-Arg(Pbf)-OH was used instead of OH to obtain a solution of compound 18. The obtained solution was concentrated to obtain compound 18 (1.65 g, 2.08 mmol, 93.7%).

Example 11: Synthesis of compound 19 (H-Arg(Pbf)-O-KJ1)

Compound BD was subjected to Fmoc general synthetic method using 1-methylpiperazine instead of piperidine, and washed with aqueous hydrochloric acid and aqueous sodium hydrogen carbonate solution 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).

The following amino acids were introduced into compound 19 by a method of repeating a 1-pot condensation deprotection method using an amine scavenger (water-soluble amine) to obtain compound 20 in a solution.
1st residue: Fmoc-Leu-OH
Second residue: Fmoc-dLeu-OH
Third residue: Fmoc-Tyr(tBu)-OH
4th residue: Fmoc-Ser(tBu)-OH
Fifth residue: Fmoc-Trp(Boc)-OH
Sixth residue: Fmoc-His(Trt)-OH

Example 13: Synthesis of Compound 21 (Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-O-KJ1: SEQ ID NO: 18).

DMF was added to the obtained compound 20 solution so that the theoretical yield was 1.8 v/w. H-Pyr-OH (3.30 g, 25.55 mmol), COMU (7.0 g, 16.26 mmol, 1.25 equiv), DIPEA (5.10 ml, 29.28 mmol, 2.25 equiv) were added. The resulting mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (0.45 equiv) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 5 minutes. 0.01N hydrochloric acid (18 v/w) was added, and the mixture was washed, separated, and the aqueous layer was discarded. The solvent of the obtained organic layer was distilled off under reduced pressure to obtain Compound 21 (28.78 g, 87.50%).

N-ethyl-(Bis(4-docosyloxyphenyl))methylamine (hereinafter, N-ethyl-(Bis(4-docosyloxyphenyl))methylamino group may be referred to as NEt-KJ3)) Of compound 7 (Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt: SEQ ID NO: 1)

Example 14: Synthesis of compound 22 (Fmoc-Arg(Pbf)-NEt-KJ3)

Fmoc-Pro-OH (0.354 g, 1.05 mmol) was dissolved in 14 mL of toluene, DMF (10.8 μL, 0.14 mmol) and then thionyl chloride (91 μL, 1.26 mmol) were added, and the mixture was heated to 40° C. And stirred for 2 hours. Thionyl chloride (45.5 μL, 0.63 mmol) was further added, and the mixture was stirred for 1 hour. The obtained reaction solution was concentrated under reduced pressure. To the residue, 7 mL of toluene was added and concentrated under reduced pressure, which was repeated twice. The obtained residue was cooled to room temperature, 14 mL of dichloromethane was added and dissolved, and DIPEA (244 μL, 1.4 mmol) was added and dissolved. H-NEt-KJ3 (0.601 g, 0.7 mmol) was added to the obtained mixed liquid, and the mixture was stirred for 1 hour. The obtained reaction liquid was concentrated under reduced pressure, 20 mL of acetonitrile was added, the obtained slurry liquid was filtered, and the obtained filtrate was concentrated to dryness to obtain a crude compound 22. The obtained crude compound 22 was subjected to silica gel column chromatography (mobile phase dichloromethane), the target product fractions were collected and concentrated to obtain compound 22 (0.58 g, 0.53 mmol, 75.1%).

Example 15: Synthesis of compound 23 (H-Pro-NEt-KJ3)

Compound 22 (0.500 g, 0.424 mmol) was subjected to Fmoc general synthetic method using 1-methylpiperazine instead of piperidine, washed with aqueous hydrochloric acid and aqueous sodium hydrogen carbonate solution to give a solution of compound 23. Obtained.

Example 16: Synthesis of compound 24 (H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-dLeu-Leu-Arg(Pbf)-Pro-NEt-KJ3: SEQ ID NO:20).

The following amino acids were introduced into compound 23 by repeating the 1-pot condensation deprotection method using an amine scavenger (water-soluble amine) to obtain compound 24 in a solution.
1st residue: Fmoc-Arg(Pbf)-OH
Second residue: Fmoc-Leu-OH
Third residue: Fmoc-dLeu-OH
4th residue: Fmoc-Tyr(tBu)-OH
Fifth residue: Fmoc-Ser(tBu)-OH
Sixth residue: Fmoc-Trp(Boc)-OH
7th residue: Fmoc-His(Trt)-OH

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.

DMF was added to the obtained solution of the compound 24 so that the theoretical yield was 1.8 v/w. H-Pyr-OH (53 mg, 0.41 mmol, 1.45 eq), COMU (171 mg, 0.40 mmol, 1.40 equiv), DIPEA (0.12 ml, 0.69 mmol, 2.5 equiv) were added. The resulting mixture was stirred at room temperature for 30 minutes. 1-Methylpiperazine (0.6 equiv) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 5 minutes. 0.1N Hydrochloric acid (18 v/w) was added, the mixture was washed and separated, and the aqueous layer was discarded. The solvent of the obtained organic layer was distilled off under reduced pressure. To the residue was added acetyl chloride and the deposited precipitate was filtered, washed with acetyl chloride and dried under reduced pressure to give Compound 25 (0.998 g, 64.6%). , HPLC purity 86.10%) was obtained.

Example 18: Synthesis of compound 7 (Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt: SEQ ID NO: 7)

Compound 25 (0.10 g, 0.037 mmol) was dissolved in 1.23 ml of a mixed solution of TFA/TIS/water (90/1/9), DTT (25 mg, 20 mg/ml) was added, and the mixture was stirred at room temperature for 3 hours. did. The reaction solution was filtered using Celite, the filter residue was washed with methanol and filtered, toluene (mixed liquid amount) was added to the obtained filtrate, and the solvent was distilled off under reduced pressure. Cold MTBE was added to the residue, the deposited precipitate was filtered, further MTBE-suspended and hexane-suspended, the obtained solid was dried under reduced pressure, and compound 7 (19 mg, 47.5%, HPLC purity 94. 93%).

The above detailed description merely illustrates the objectives and objects of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings provided herein without departing from the scope of the appended claims.

Since the production method of the present invention can carry out the peptide extension step in a short time by a simple means and can obtain a crude product of high purity, a production method of leuprorelin which is more economical than the prior art can be obtained. Can be provided. Leuprorelin is useful as a medicine.

Claims (21)

A method for producing leuprorelin consisting of the following sequence: H-Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro-NHEt (SEQ ID NO: 1), which comprises steps a to d:
a. An amino acid protected by a carrier for liquid phase peptide synthesis (carrier protected), a carrier protected amino acid amide or a carrier protected peptide, and a 9-fluorenylmethyloxycarbonyl group (Fmoc group) in an organic solvent or a mixture of organic solvents A step of condensing with an amino acid whose amino group is protected by (N-Fmoc protected) or N-Fmoc protected peptide to obtain an N-Fmoc-carrier protected peptide
b. A step of adding a water-soluble amine having a valence of 2 or more to the reaction solution after the condensation reaction to scavenge the amino acid active ester ,
c. Deprotecting the Fmoc group from the protected amino group by adding a de-Fmoc reagent in the presence of a water-soluble amine having a valence of 2 or more, and d. A production method including a step of adding an acid to the reaction solution for neutralization, further adding an acidic aqueous solution for washing, separating the layers, removing the aqueous layer, and obtaining an organic layer,
here,
The carrier for liquid phase peptide synthesis is a compound that directly binds to an amino acid or a peptide to make 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 bound to the carboxyl terminus of the amino acid or peptide, and
The water-soluble divalent or higher amines in step b and step c may be the same or different. After step d,
Step e: A weak basic aqueous solution having a pH of 8 to 12 is added to the organic layer obtained in Step d to wash, then, liquid separation is performed, the aqueous layer is removed, and an organic layer containing a carrier-protected peptide is obtained. The manufacturing method according to claim 1, including a step of obtaining. The liquid phase peptide synthesis carrier is a compound having the following structure:
(In the formula, R ₂ , R ₄ and R ₅ are hydrogen atoms, R ₁ and R ₃ are alkoxyl groups having 12 to 30 carbon atoms, and RY is —CH ₂ OH or —CH ₂ NHEt. Or a compound having the following structure:
(In the formula, X is —CH ₂ OH or —CH ₂ NHEt; R ² , R ⁴ and R ⁵ are hydrogen atoms, and at least one of R ¹ and R ³ has the following formula:
-O-R ⁶ -Xa-A
And the remaining groups represent 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 group having 1 to 16 carbon atoms. Represents a group, Xa represents O or CONRc (wherein Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms),
A represents any one of formula (1) to formula (11),
(Here, R ⁷ , R ⁸ and R ⁹ may be the same or different and represent 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 Indicates a branched alkylene group)
(Note that each of the above formulas is shown in the state before binding to the carboxyl group of the amino acid or peptide),
The method according to claim 1, which is a carrier derived from. The carrier for liquid phase peptide synthesis has the following structure:
General formula (V):
[Wherein, ring A represents an aromatic ring; Y is a hydroxyl group, a bromo group or a chloro group; Ra, Rb and Rc are each independently an organic group having an aliphatic hydrocarbon group, a hydrogen atom or an electron; Is an organic group having an aliphatic hydrocarbon group and at least one of Ra, Rb and Rc being an attracting group; rings A, B and C each independently having an electron withdrawing group; Good] or general formula (V′):
[Wherein, ring A represents an aromatic ring; Y is a hydroxyl group, a bromo group or a chloro group; n represents an integer of 1 to 19; Rc′ represents a divalent organic group having an aliphatic hydrocarbon group. A ring; 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 when a plurality of Y are present may be the same or different; each Rc′ when a plurality of Rc′ are present may be the same or different; May be used], wherein the divalent organic group having an aliphatic hydrocarbon group has the formula (a):
(In the formula, Xa is absent 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; And each Rd when a plurality of Rd are present may be the same or different; and each Xa when a plurality of Xa is present may be the same or different). ,
The organic group having an aliphatic hydrocarbon group is present at the 2-position and/or the 7-position of the fluorene compound, and has the formula (b):
(In the formula, * represents a bonding position; X ₁ is —O—; R ₁ is an aliphatic hydrocarbon group having 5 to 60 carbon atoms; m ₁ is 1),
Formula (c):
(In the formula, * represents a bonding position; X ₂ , X ₂ ′, X ₂ ″ and X ₂ ′″ are —O—; R ₂ and R ₄ each independently have 5 to 60 carbon atoms. 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 And a formula (d):
(In the formula, * represents a bonding position; X ₈ represents —O—; m ₃ is 2 or 3; n ₅ is 1; n ₆ is 3; X ₇ is —O— And m ₃ R ₁₂ 's each independently represent an alkyl group having 4 to 30 carbon atoms), or one or more groups selected from the group consisting of
A fluorene compound represented by
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The method according to claim 1, which is a carrier derived from. The carrier for liquid phase peptide synthesis has the following structure:
General formula (W)
[In the formula, Y represents a hydroxyl group or a -NHEt group; ^Ra is
Formula (a):
[In the formula, * represents a bonding position; m ₁ represents an integer of 1 to 10; m ₁ X ₁ 's each independently represent a single bond, or -O-, -S; -, -COO-, -OCONH-, -NHCO- or -CONH- is shown; R ₁ and m ₁ R ₂ are each independently a divalent aliphatic hydrocarbon group having 5 or more carbon atoms. And R ₃ is a hydrogen atom or the formula (W′):
(In the formula, Y represents a hydroxyl group or a -NHEt group; * represents a bonding position; n R ^{b s} are each independently an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or 1 The alkyl group having 1 to 6 carbon atoms which may be substituted with the above halogen atom; and n is a group represented by 0).
Formula (b):
(In the formula, * represents a bonding position; m ₂ represents 1 or 2; n ₁ , n ₂ , n ₃ and n ₄ each independently represent 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 are —O—, —S—, —COO—, —OCONH—, Represents —NHCO— or —CONH—; m ₂ R ₄ and m ₂ R ₆ each independently represent an aliphatic hydrocarbon group having 5 or more carbon atoms; R ₅ represents 5 carbon atoms A group represented by the above aliphatic hydrocarbon group);
Formula (c):
(In the formula, * represents a bonding position; m ₃ represents an integer of 0 to 15; n ₅ represents an integer of 0 to 11; n ₆ represents an integer of 0 to 5; m ₃ Xs) ₃ each independently represents a single bond or represents -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 formula (d):
[In the formula, * represents a bonding position; n ₇ X ₄ each independently represents a single bond, or -O-, -S-, -COO-, -OCONH-, -NHCO. - or 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 , And an integer of 1 to 5; and Ar represents an arylene group. ) Represents an organic group having an aliphatic hydrocarbon group selected from the group consisting of groups represented by the total number of carbons in the organic group has at least 30; n number of R ^b are each independently, A C1-C6 alkoxy group, a halogen atom, or a C1-C6 alkyl group which may be substituted with one or more halogen atoms; and n represents an integer of 0-4]
A benzyl compound represented by
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The method according to claim 1, which is a carrier derived from. The carrier for liquid phase peptide synthesis has the following structure:
General formula (X)
[In the formula, Y represents a hydroxyl group or -NHEt group; k and l each independently represents an integer of 0 to 5, provided that, k + l is not the 0; k-number of R _a and the l R _b of each independently
Formula (a):
(In the formula, * represents a bonding position; m ₁ represents an integer of 1 to 10; m ₁ X ₁ 's each independently do not exist, or are —O—, —S—, — COO-, -OCONH- or -CONH- is shown; m ₁ R ₁ 's each independently represent a divalent aliphatic hydrocarbon group having 5 or more carbon atoms;
Formula (b):
(In the formula, * represents a bonding position; m ₂ represents an integer of _{1 to 2} ; m ₂ n ₁ , n ₂ , n ₃ and n ₄ each independently represent an integer of 0 to 2. M ₂ X ₂ , m ₂ X ₂ ′, m ₂ X ₂ ′″ and m ₂ X ₂ ″ each independently do not exist or are —O. -, -S-, -COO-, -OCONH- or -CONH- is shown; m ₂ R ₂ and R ₄ are each independently 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):
(In the formula, * represents a bonding position; m ₃ represents an integer of 0 to 15; n ₅ represents an integer of 0 to 11; n ₆ represents an integer of 0 to 5; is X ₂ absent? , Or —O—, —S—, —NHCO— or —CONH—; m ₃ X _{7 s} are independently absent, or respectively —O—, —S—, —COO—, Represents -OCONH-, -NHCO- or -CONH-; m ₃ R ₁₂ 's each independently represent a hydrogen atom, a methyl group or an aliphatic hydrocarbon group having 5 or more carbon atoms). Shows an organic group having an aliphatic hydrocarbon group having 5 or more carbon atoms selected from the group consisting of groups, in which the total of all aliphatic hydrocarbon groups in the organic group having k+1 aliphatic hydrocarbon groups Has 16 or more carbon atoms;
Ring A may have a substituent in addition to R _a ;
Ring B may have a substituent in addition to R _b . ]
A diphenylmethane compound represented by:
Or
General formula (Y)

[In the formula, each of the k Q's independently represents a single bond, or is -O-, -S-, -C(=O)O-, -C(=O)NH- or -NH. -Indicates k; _Ra independently has at least one aliphatic hydrocarbon group having at least one branched chain, has a total number of branched chains of 3 or more, and has a total carbon number of 14; Represents an organic group of 300 or more; k represents an integer of 1 to 4; R ₁ represents a hydrogen atom, or when Z represents a group represented by the following formula (a), Together with R ₂ , a single bond may be shown to form a fluorene ring with ring B; ring A may be R ₁ , k QR _a , and C(X)(Y)Z. And further selected from the group consisting of a halogen atom, 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. It may have one or more substituents; X represents a hydrogen atom or a phenyl group;
Y represents a hydroxyl group or a -NHEt group; and Z is a hydrogen atom or the formula (a):
(In the formula, * represents a bonding position; m represents an integer of 0 to 4; m Qs each independently represent a single bond, or -O-, -S-, -C; (═O)O—, —C(═O)NH— or —NH— ; m R _{b s} each independently have at least one aliphatic hydrocarbon group having one or more branched chains. Represents an organic group having a total number of branched chains of 3 or more and a total number of carbon atoms of 14 or more and 300 or less; R ₂ represents a hydrogen atom or, together with R ₁ , represents a single bond. May form a fluorene ring together with ring A; and ring B may have, in addition to m QR _b and R ₂ , C1 which may be substituted with a halogen atom or one or more halogen atoms. -6 alkyl group and one or more substituents selected from the group consisting of C1-6 alkoxy groups optionally substituted by one or more halogen atoms) Indicates;
An organic group having at least one aliphatic hydrocarbon group having one or more branched chains in R _a and R _b , a total number of branched chains of 3 or more, and a total carbon number of 14 or more and 300 or less, Formula (b):
(In the formula, * represents a bonding position with an adjacent atom; R ₃ and R ₄ each independently represents a hydrogen atom or a C1-4 alkyl group; X ₁ represents a single bond, C1-4 alkylene. Represents a group or an oxygen atom, provided that R ₃ and R ₄ are not both hydrogen atoms) and has 3 or more of the same or different divalent groups. ]
A branched chain-containing aromatic compound represented by:
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The method according to claim 1, which is a carrier derived from. The liquid phase peptide synthesis carrier is a compound having the following structure:
(In the formula, R ₂ , R ₄ and R ₅ are hydrogen atoms, R ₁ and R ₃ are alkoxyl groups having 12 to 30 carbon atoms, and RY is —CH ₂ OH.)
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The method according to claim 1 or 2, wherein the carrier is derived from. The liquid phase peptide synthesis carrier is a compound having the following structure:
(2,4-didocosyloxybenzyl alcohol),
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The method according to claim 7, which is a carrier derived from. The liquid phase peptide synthesis carrier,
(2,4-di(11'-triisopropylsilyloxyundecyloxy)benzyl alcohol),
(2,4-di(11'-t-butyldiphenylsilyloxyundecyloxy)benzyl alcohol),
(In the formula, X is F or Cl),
(2-(3',4',5'-trioctadecyloxybenzyl)-4-methoxybenzyl alcohol), and
(2,4-di(2',3'-dihydrophytyloxy)benzyl alcohol)
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to claim 1, which is a carrier derived from a compound selected from the group consisting of: A carrier-protected peptide containing a sequence consisting of Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg (SEQ ID NO: 2) is produced by repeating the above steps, and the carrier-protected peptide is produced according to claim 7. The manufacturing method described. A peptide obtained by deprotecting a carrier from a carrier-protected peptide containing the sequence consisting of Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg (SEQ ID NO: 2) and H-Pro-NHEt (L-proline The production method according to claim 10, wherein leuprorelin is produced by deprotecting the side chain protecting group after condensation with ethyl amide). The liquid phase peptide synthesis carrier is a compound having the following structure:
(In the formula, R ₂ , R ₄ and R ₅ are hydrogen atoms, R ₁ and R ₃ are an alkoxyl group having 12 to 30, preferably 18 to 22 carbon atoms, and RY is —CH ₂ NHEt)
(Note that the above formula is shown before binding to the carboxyl group of an amino acid or peptide)
The method according to claim 1 or 2, wherein the carrier is derived from. The liquid phase peptide synthesis carrier,
(N-ethyl-2,4-didocosyloxybenzylamine),
(N-ethyl-2,4-di(11'-triisopropylsilyloxyundecyloxy)benzylamine),
(N-ethyl-2,4-di(11'-t-butyldiphenylsilyloxyundecyloxy)benzylamine),
(N-ethyl-2-(3',4',5'-trioctadecyloxybenzyloxy)-4-methoxybenzylamine),
(N-ethyl-bis(4-docosyloxyphenyl)methylamine),
(N-ethyl-2,4-di(2',3'-dihydrophytyloxy)benzylamine), and
(N-ethyl-bis[4-(2',3'-dihydrophytyloxy)phenyl]methylamine)
(Note that each of the above formulas is shown before being bound to the carboxyl group of the amino acid or peptide)
The production method according to claim 1 or 2, which is a carrier derived from a compound selected from the group consisting of: The production according to claim 12 or 13, wherein a carrier-protected peptide containing a sequence consisting of Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro (SEQ ID NO: 3) is produced by repeating the above steps. Method. Leuprorelin is produced by deprotecting a carrier and a side chain protecting group from a carrier-protected peptide containing a sequence consisting of Pyr-His-Trp-Ser-Tyr-dLeu-Leu-Arg-Pro (SEQ ID NO: 3), The manufacturing method according to claim 14. 16. In step a, the carrier-protected amino acid, carrier-protected peptide or carrier-protected amino acid amide and N-Fmoc-protected amino acid or N-Fmoc-protected peptide are condensed in the presence of a condensing agent. Manufacturing method described in. The water-soluble amine having a valence of 2 or more is selected from the group consisting of 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine and piperazine. The manufacturing method according to any one of 1 to 16 . Amine equivalent of divalent or more water-soluble amine in step b is in an amount of 1 to 10 equivalents to the amino acid equivalent to theoretically remaining after the condensation reaction of step a, any one of claims 1 to 17 The manufacturing method described. Amine equivalent of divalent or more water-soluble amine in step c has, relative to the amount of Fmoc group present in the system, from 5 to 30 equivalents, A process according to any one of claims 1 to 18. A pH of the acidic aqueous solution of step d is 1 to 5, the production method according to any one of claims 1 to 19. Performing said steps in a one-pot process according to any one of claims 1 to 20.