JPH1149798A - Production of peptide derivative and its intermediate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a peptide derivative exhibiting an antioxytocin action and useful for preventing premature miscarriage, etc., by removing a protecting group from a specific carboxyl group-containing group-bound peptide derivative, and forming a cyclic structure with an amide bond in the molecule. SOLUTION: This method for producing a peptide derivative comprises oxidizing a compound (salt) of formula I (R<3> is a protecting group for the amino group of Ile; Acm is acetamide methyl) with a peroxide, reacting the product with a compound of formula II (MBz1 is 4-methoxybenzyl) in the presence of a sulfide compound to form a disulfide bond and remove the protecting group R<3> , reacting the obtained compound of formula III with a compound of the formula: R<2> -D-Trp(-R<1> )-OR<4> (R<1> is a protecting group for indolyl group of the formula) (D-Trp, H; R<2> is a protecting group for the α-amino group of D-Trp, R<4> is a protecting group for the carboxyl group of D-Trp), removing the protecting group R<2> from the obtained compound of formula IV and forming a cyclic structure with an intermolecular amide bond to obtain the peptide derivative of formula V.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a peptide derivative having a physiological activity, and more particularly, to a method for producing a peptide derivative which is used for prevention of premature miscarriage and has a small anti-vasopressin action and exerts an anti-oxytocin action. The present invention relates to a method and an intermediate thereof and a method for producing the intermediate.

[0002]

2. Description of the Related Art One of the most important problems in obstetrics is
Preterm birth treatments are included. Many pregnant women beyond the 20th week of gestation experience preterm and premature births, which are a major cause of neonatal morbidity and mortality. Therefore,
Even today, where neonatal care has made great strides,
It is considered preferable to keep the fetus in the mother's body until the last month.

[0003] In general, uterine muscle relaxants are used for the treatment of preterm babies, but in recent years, oxytocin antagonists have been proposed as ideal uterine muscle relaxants for suppressing preterm babies. This is because evidence has been provided to support one of the peptide hormones, oxytocin, as a physiological labor initiator. Based on this concept, the following compound <I>, which is a peptide derivative, was reported as a novel oxytocin antagonist (International Patent Publication, WO94 / 2).
5485).

[0004]

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[0005] Oxytocin is one of the posterior pituitary hormones and consists of 9 amino acid residues. On the other hand, vasopressin, which is also one of the posterior pituitary hormones, is composed of nine amino acid residues, and differs from oxytocin only in two kinds of amino acid residues. However, although their structures are similar but their physiological actions are completely different, oxytocin contracts smooth muscle including uterine muscle, while vasopressin has peripheral vasoconstriction and blood pressure increasing action. For this reason, conventionally developed anti-oxytocin drugs often have an anti-vasopressin action as a side effect. However, the compound <I>, which is a peptide derivative, has a potent oxytocin antagonist, but does not exhibit the vasopressin antagonism that oxytocin antagonists usually have as a side effect.
For this reason, compound <I> is considered as one of ideal uterine muscle relaxants.

[0006]

However, the above-mentioned WO 94
/ 25485 discloses a method for producing compound <I>, which is a method for producing a peptide derivative by a solid phase method using a resin carrier, and it is difficult to produce a large amount of the present compound in high yield and high purity. .

[0007] For example, in the solid phase method, peptide synthesis can be automatically performed using a commercially available automatic peptide synthesizer or the like.
The steps are long because they are linked by a condensation reaction. In addition, in each step, purification after the condensation reaction cannot be performed, and a peptide in which amino acids are partially deleted is mixed. In order to reduce the number of defective peptides, the reaction is carried out using an excess amount of amino acid derivative that is 2 to 6 times the amino group content of the carrier resin during the condensation reaction. However, it is generally difficult to prevent the generation of defective peptides. Also, the above publication (WO94 / 25485)
In the method disclosed in (1), a liquid ammonia solution in which metallic sodium is dissolved is used to separate the target peptide from the carrier resin, but this method is difficult for industrial implementation. In addition, in the method disclosed in the above publication, a compound in which the protecting group for the thiol group is a 4-methylbenzyl group is used as an intermediate in the synthesis of compound (I), so that the intramolecular S
In forming the S bond, a liquid ammonia solution in which sodium metal is dissolved is used for removing the protecting group, and a high degree of caution is required in terms of safety and the like. Also, in the step of separating the required peptide portion from the carrier resin, degradation products of the carrier resin and impurities in which one or more amino acids have been deleted due to the inability to purify intermediates on the way are mixed. Therefore,
In order to obtain a high-purity target compound, the purification step becomes complicated. Accordingly, an object of the present invention is to provide a method for industrially and efficiently producing compound <I> with high yield and high purity.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on the method for producing the compound <I>, which is a peptide derivative, and as a result, have constructed a synthesis scheme by a fragment condensation method and have an intramolecular disulfide bond. The inventors have found that the above object can be achieved by intramolecular dehydration condensation of a compound, and have completed the present invention.

That is, the present invention provides a compound of the formula <I> characterized in that the protecting group R ² is removed from the compound <II> or a salt thereof and a cyclic structure is formed by an amide bond between an amino group and a carboxyl group in the molecule. The present invention provides a method for producing a peptide derivative or a salt thereof represented by the formula: Further, the present invention provides compound <III>, which is an intermediate of compound <I>, or a salt thereof. In addition, the present invention also provides a method for producing compound <II> or a salt thereof, which is an intermediate of compound <I>.

[0010]

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[Abbreviations and the like] (Optical isomers) When amino acids and the like used in the present specification have optical isomers, "D-" is attached only when the D-isomer is used, and is not particularly specified. In the case, L-form is shown. (Amino acid residue) Arg: arginine, Asn: asparagine, Ile: isoleucine, Gln: glutamine,
Gly: glycine, Pro: proline, Trp: tryptophan

(Protecting group) Acm: acetamidomethyl,
Boc: t-butoxycarbonyl, Z: benzyloxycarbonyl, Cl-Z: 2-chlorobenzyloxycarbonyl, Fmoc: 9-fluorenylmethoxycarbonyl, MBzl: 4-methoxybenzyl, Mtr: 4-methoxy-2,3 , 6-Trimethylbenzenesulfonyl

(Dehydration condensation coupling reagent) HOBt:
1-hydroxybenzotriazole, HOSu: N-hydroxysuccinimide, HOOBt: 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine, HONB: N-hydroxy-5-norbornene- 2,3-dicarboxylic imide, DCC: dicyclohexylcarbodiimide, WSC: water-soluble carbodiimide, CDI: carbodiimidazole, EEDQ: 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline, BOP: 1-ethoxycarbonyl- 2-isobutoxy-1-yl-oxytris (dimethylamino) phosphonium-hexafluorophosphate, DPPA: diphenylphosphoryl azide, HB
TU: O-benzotriazole-N, N, N ', N'-
Tetramethyluronium-hexafluorophosphate, EDC: 1-ethyl-3- (3-diethylaminopropyl) carbodiimide

(Solvent) DOX: dioxane, DMF: dimethylformamide, TFA: trifluoroacetic acid, NM
M: N-methylmorpholine, NMP: N-methylpyrrolidone

(Removal of Protecting Group) In the present invention, the method for removing the protecting group can be carried out by a conventional method used in peptide synthesis. For example, a removal method by catalytic reduction, a removal method under strong basic conditions such as ammonia compounds and hydrazine, a removal method under acidic conditions using TFA, hydrogen fluoride or trifluoromethanesulfonic acid, or a removal method using iodine, etc. A method for removing the protecting group can be used. In the method using TFA, in addition to the use of TFA alone, a mixture of TFA and mercaptans or phenols, or a mixture of these and further added methanesulfonic acid or the like may be used.

(Dehydration Condensation Method) The dehydration condensation method for individual peptide derivatives or amino acids in the present invention can be carried out by a general peptide synthesis method. For example, a C-terminal activation method including an active ester method and a symmetric acid anhydride method,
Alternatively, a coupling method using a coupling reagent can be exemplified. (1) Active esters in the active ester method include alkyl esters such as cyanomethyl ester; thiophenyl ester, p-nitrophenyl thioester, p-methanesulfonylphenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester , 2,4,6
Phenyl esters such as trichlorophenyl ester and pentachlorophenyl ester; dicarboxylic acid imide esters such as N-hydroxysuccinimide ester, N-hydroxyphthalic acid imide ester and HONB;
Examples thereof include hydroxylamine derivatives such as hydroquinoline ester, N-hydroxypiperidine ester and 2-hydroxypyridine ester. (2) Coupling methods include carbodiimide method using DCC, WSC, etc .; DCC-additive method; CD
Method I: A Woodward method, wherein N-ethyl-5-phenylisoxazolium-3′-sulfonate or N-ethyl-2′-hydroxybenzisoxazolium trifluoro is used as a reactant. Isoxazolium salts such as borate, EEDQ, BOP, HBT
A method using U and DPPA; a redox method by Mukaiyama et al .; a four-component synthesis (Ugi) method, and the like. (3) WSC includes EDC, N-cyclohexyl-N ′
-Morpholinoethylcarbodiimide, N-cyclohexyl-N '-(N, N-diethylamino) cyclohexylcarbodiimide and the like. WSC may be a salt, such as a hydrochloride salt. (4) DCC-HOSu includes the DCC-HOSu
Method, DCC-HOOBt method, DCC-HOOBt method, DC
C-HONB method, DCC-2-hydroxyimino-2-
Cyanoacetic acid ethyl ester method, WSC-HOSu method, W
The SC-HOBt method, the WSC-HOOBt method and the like are also included.

(Addition Salts and Complexes) The peptide derivatives, intermediates and starting materials according to the production method of the present invention, when they can be formed, are acid addition salts or complexes (hereinafter simply referred to as “salts”). And may be preferred. If those salts are pharmaceutically unacceptable salts, they can be changed to acceptable salts. Examples of the acid addition salt include salts of inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, and phosphoric acid, formic acid, acetic acid, TFA, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, and apple. Acids, tartaric acid, citric acid, benzoic acid, salicylic acid, salts of organic acids such as benzenesulfonic acid, toluenesulfonic acid and the like can be mentioned. As the complex, for example, calcium, magnesium, aluminum, cobalt, inorganic compounds derived from metals such as zinc, particularly phosphates of these metals,
Examples include salts of slightly soluble salts such as pyrophosphates and polyphosphates, complexes of hydroxides and alkali metal polyphosphates. The compounds in the following description are:
Unless otherwise specified, this shall include salts.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION

[Production Method of Compound (I)] The compound (I), which is the object of the present invention, is produced by forming a cyclic structure from the compound (II) as an intermediate by an intramolecular amide bond.

(Starting Compound) In the process for producing compound <I> of the present invention, compound <II> or a salt thereof is used.
Compound <II> is characterized by having a disulfide bond in the molecule. As a result, the step of synthesizing the intramolecular SS bond can be avoided, and the compound <I> can be produced with high yield. Compound <II> is a compound represented by the formula: where R ¹ is a protecting group for an indolyl group of D-Trp, a formyl group, a Z group, a 2,4-dichlorobenzyloxycarbonyl group, a trichloroethyloxycarbonyl group, Mt
It is preferably r or 2,4,6-trimethoxybenzenesulfonyl group, particularly preferably formyl group. This is because removal is easy. Further, when R ¹ is a hydrogen atom, it is preferable in that the target product can be obtained immediately after the formation of the cyclic structure, but R ¹ is preferably a protective group because by-products derived from Trp can be suppressed. Further, the protecting group R ² is preferably a Boc group or an Fmoc group.

(Manufacturing process) Hereinafter, compound <II> in which R ¹ is a hydrogen atom and R ² is a Boc group is referred to as compound <IIa>, wherein R ¹ is a protecting group for an indolyl group of D-Trp; The production process will be described with reference to the case where R ² is a Boc group as compound <IIb>.

(1) Preparation of Compound (I) Using Compound (IIa) Boc is removed from compound (IIa) and a new amide bond is formed by an intramolecular dehydration condensation reaction at low temperature to give compound (I). To manufacture. For the amide bond, a general peptide synthesis method can be used, but among the above-mentioned dehydration condensation methods, the WSC-HOBt method, DCC-HOBt method or WSC-HOOBt method is particularly preferable. For example, when the WSC-HOBt method is used, the compound <IIa> is used in combination with indole at a molar ratio of 10 to 100, dissolved in TFA at a temperature of -10 to 25 ° C, and stirred to obtain an α-carbon of D-Trp. The Boc group which is a protecting group for an amino group bonded to is removed. Then, TFA is distilled off, and 4N-HCl / DOX and isopropyl ether are added to form a hydrochloric acid addition salt. The Boc-dehydrate is precipitated and collected by filtration.
Next, this was dissolved in DMF, the pH was adjusted to about 8 with NMM, and WSC was added to compound <IIa> by 1.0 to 1.5.
HOBt is added 1.0 to 1.5 mole times, and the temperature is reduced to 0.
Stirring at ４０40 ° C. allows intramolecular cyclization. DMF is distilled off, and the obtained oil is crystallized by adding ethyl acetate, isopropyl alcohol, acetone and the like, whereby a cyclic structure precipitates to obtain the compound <I>.

(2) Preparation of Compound (I) Using Compound <IIb> Boc is removed from compound <IIb>, a new amide bond is formed by an intramolecular dehydration condensation reaction, and then the protecting group R ¹ is removed. By doing so, compound <I> is produced. A general peptide synthesis method can be used for the amide bond. Among the above dehydration condensation methods, WSC-
HOBt method, DCC-HOBt method or WSC-HOO
The Bt method is particularly preferred. For example, WSC-H
When the OBt method is used, indole is used in an amount of 10 to 100 times by mole the amount of compound <IIb>,
By dissolving in TFA at 0 ° C. and stirring, DT
Boc which is a protecting group for an amino group bonded to the α carbon of rp
Remove the group. TFA is distilled off and 4N HCl / DOX
And isopropyl ether, and the precipitate was collected by filtration.
The oc form is obtained as a precipitate. Then dissolved in DMF and N
The pH was adjusted to about 8 with MM, and for compound <IIb>,
WSC is 1.0 to 2.0 mole times, HOBt is 1.0 to 2.0
Add 1.5 mole times and stir at 0-35 ° C. DMF
Then, ethyl acetate, isopropyl alcohol, acetone and the like are added to the obtained oil to crystallize, whereby a cyclic structure is obtained as a precipitate. Then dissolved annular structure in ammonium acetate buffer, a small amount of adjusted to about pH9 with aqueous ammonia, and stirred 10 to 48 hours at a temperature 15 to 35 ° C., the protecting group R ¹ can be removed Compound <I> Is obtained.

[Method for producing compound (II)] (Starting compound)

[0024]

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Here, compound <V> is obtained by subjecting compound <IX> to dehydration condensation of compound <IX> shown below to compound <XI>
And then subjecting the compound <XII> to dehydration condensation to produce the compound (XII).

[0026]

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(1) Compound <IX> and compound <XII>, which are the starting materials for compound <V>, can be produced by utilizing a usual peptide synthesis method. For example, the compound <X> can be produced by condensing glycinamide with arginine having an amino group protected to give a dipeptide derivative, condensing this with proline having an imino group protected, and then removing the protecting group for the imino group. (2) Compound <V> can be produced by the following method. First, the compound <IX> and the compound <X> are subjected to a condensation reaction. For the condensation reaction, a general peptide synthesis method can be used. Among them, the DCC-HOBt method, DCC
-HOOBt method or WSC-HOBt method is preferred. For example, when the DCC-HOBt method is used, first, the protecting group Z of the compound <IX> is removed, and the compound <X> is used in an amount of 1.0 to 1.5 times, and H is added to the obtained compound.
OBt 1.0-1.5 mole times was added and the solution was brought to 0 ° C.
３５35 ° C. and then add 1.0-1.5 molar times of DCC. The white precipitate is separated by filtration, fractionated, and crystallized from ethyl acetate or the like to give compound <XI>. Next, the protecting groups Acm and Boc of the compound <XI> are removed. For the removal of the protecting group, general means for removing the protecting group can be used. For example, compound <XI> can be dissolved in TFA at a temperature of -10 to 30 ° C and removed by stirring. Then, TFA was distilled off under reduced pressure and 4N
After treating with -HCl / DOX and adding a solvent to cause crystallization, a compound is obtained in which the protecting group Boc has been removed from the compound <XI>. Next, the compound obtained by removing the protecting group Boc from the compound <XI> and the compound <XII> are subjected to dehydration condensation. The dehydration condensation is performed by DCC-HOB among the dehydration condensation methods described above.
The t method, the DCC-HOOBt method, or the WSC-HOBt method is preferable. For example, in the case of performing the DCC-HOBt method, a compound obtained by removing the protecting group Boc from the compound <XI> is dissolved in a solvent such as NMP, DMF, DMSO or the like, and the pH is adjusted to 7.0 to 9.0 with NMM, and the compound is dissolved. <X
Compound <XII> is 1.0 to 1.5 times the amount of compound obtained by removing protective group Boc from I>, HOBt 1.0 to 1.0
1.5 mole times and 1.0 to 1.5 mole times of DCC were added, and 10
Stir for ~ 48 hours. The reaction temperature is preferably -20 to 30C, particularly preferably -20 to 0C. The insolubles produced after the reaction are filtered off and the filtrate is concentrated to give compound <V>.

(Production process) Compound <I, which is an intermediate
The production method according to the present invention of I> includes the following (1) and (2)
Characterized by passing through a manufacturing process. (1) First, the compound <V> is oxidized to give the following compound <V>
I> is synthesized. For example, the compound <V> is dissolved in pure water, an aqueous solution of hydrogen peroxide, sodium perborate or the like is added as an oxidizing agent, and the mixture is stirred at a temperature of 10 to 40 ° C. When hydrogen peroxide is used as the oxidizing agent, manganese dioxide is added to decompose excess hydrogen peroxide, and then lyophilized to obtain compound <VI>.

[0029]

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(2) Next, a compound <IV> is added to the compound <VI> in the presence of a sulfide compound, and the compound <VII> is synthesized by intramolecular disulfide bond. Here, as the sulfide compound used in the present invention, for example,
Examples thereof include dimethyl sulfide, methyl ethyl sulfide, diethyl sulfide, dipropyl sulfide, dibutyl sulfide and the like. Of these, dimethyl sulfide is simply used. For example, a solvent such as TFA containing dimethyl sulfide is kept at 10 ° C., and the compound <VI> is added to the solution at a temperature of 1.0 to 1.0 with respect to the compound <IV>.
Add 1.5 mole times and then add compound <IV>. The reaction solution is maintained at a temperature of 5 to 30 ° C., stirred for 0.5 to 3.0 hours, and the solvent is distilled off under reduced pressure. A solvent such as isopropyl ether, ethyl acetate, diethyl ether or the like is added to the obtained oil, and the mixture is precipitated and filtered to obtain the following compound <VII>. As a protecting group for the thiol group of penicillamine (the skeleton may be abbreviated as Pen in the chemical formula) of compound <V>, Acm is suitable.
V> does not proceed sufficiently. (3) Compound <VII> was added to compound <VII>
Compound <II> is synthesized by subjecting I> to a condensation reaction. For example, the compound <VII> is dissolved in a solvent such as DMF, the pH is adjusted to 7.0 to 9.0 with triethylamine, and the compound <VI> is dissolved.
Compound <VIII> is added 1.0 to 1.5 times by mole to I>, and the solution is stirred at a temperature of 0 to 40 ° C. The resulting oil is crystallized from ethyl acetate or the like to give compound <I>
I> is obtained.

[0031]

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[Method for Producing Compound <III>] Compound <III> of the present invention is an intermediate for producing compound <I>. A in the formula is an R ² -D-Trp (-R ¹ )-group,
Compounds in which R ¹ is a protecting group for an indolyl group of D-Trp or a hydrogen atom and R ² is a protecting group for an α-amino group of D-Trp are compound <II>. (1) In order to produce the compound <III>, besides the method for producing the compound <II>, HD-Trp (-R ¹ ) -OH or R ² -is added to the amino group of Ile of the compound <VII>.
D-Trp (-R ¹⁾ and -OH can be obtained by dehydration condensation. As a result, A becomes HD-Trp (-R ¹ )
- group, or ^{R 2 -D-Trp (-R 1} ) - compound when a group is <III> is obtained. R ¹ represents a hydrogen atom or a protecting group for the amino group of the indolyl group of Trp. (2) Similarly, A is ^{H-D-Trp (-R 1} ) - group, or ^{R 2 -D-Trp (-R 1} ) - compound when a group <III> is penicillamine compound <VII> and compounds in which the side chain Acm-Gln-Asn- attached to an amino group, H-D-Trp (-R 1) -Ile-OH or R ²
-D-Trp (-R ¹⁾ and -Ile-OH can be obtained by dehydration condensation. (3) In addition, also A is ^{H-D-Trp (-R 1} ) - group, or ^{R 2 -D-Trp (-R 1} ) - compound when a group <III> is penicillamine compound <VII> A compound in which a side chain R ⁵ -Asn- is bonded to the amino group of
^{D-Trp (-R 1) -Ile} -Gln-OH or R ²
-D-Trp (-R ¹⁾ and -Ile-Gln-OH can be obtained by dehydration condensation. The protecting group R ⁵ here, Boc, can be exemplified is Fmoc and the like, preferably a Boc Of these. (4) In addition, A is ^{H-D-Trp (-R 1} ) - group, or ^{R 2 -D-Trp (-R 1} ) - compound when a group <III> is penicillamine compound <VII> And a compound in which a protecting group R ⁶ is bonded to the amino group of HD-Trp
^{(-R 1) -Ile-Gln-} Asn-OH or R ² -
^{D-Trp (-R 1) -Ile} -Gln-Asn-OH
Can be obtained by dehydration condensation of Here, examples of the protecting group R ⁶ include Boc and Fmoc, and among them, Boc is preferred.

[Method for Purifying Compound <I>] Compound <I>
Can be purified using various chromatography methods and according to a conventional method. As a purification method by a chromatography method, an ion exchange chromatography method,
Gel filtration chromatography, reversed-phase high-performance liquid chromatography, affinity chromatography,
Examples thereof include reversed-phase silica gel chromatography and silica gel chromatography. Among these, more preferred methods are ion exchange chromatography,
They are a gel filtration chromatography method and a reversed phase high performance liquid chromatography method, and these may be appropriately combined.
The compound <I> purified by the above method can be further purified, if necessary, by a method such as a recrystallization method and a reprecipitation method.

[0034]

EXAMPLES The present invention will now be described specifically with reference to examples, but the present invention is not limited to these examples. In addition, when the compounds (1) to (11) form an addition salt with an acid, it is indicated by “• acid” after the structural formula.

Example 1 Synthesis of Compound (1) (Hydrochloride of Compound <I>) 46.1 g of Compound (2) was dissolved in 196 ml of TFA under ice cooling and stirred at room temperature for 1 hour. TFA was distilled off under reduced pressure, and 4N-HCl / DOX 32.8 ml was added to the remaining oil.
And 250 ml of isopropyl ether, and the precipitate was collected by filtration. The crystals obtained as a precipitate were thoroughly washed with isopropyl ether and dried under reduced pressure to obtain a de-Boc form of compound (2). The amount collected was 45.0 g. De-Boc form 13.
0 g was dissolved in 1700 ml of DMF, the pH was adjusted to about 8 with NMM, 3.75 g of WSC.HCl, 3.75 g of HOBt.
91 g was added and the mixture was stirred at room temperature for 72 hours. DMF at reduced pressure
After distilling off, 100 ml of ethyl acetate was added to the remaining oil. The resulting precipitate is washed with about 100 m of isopropyl alcohol.
The precipitate was collected by filtration and dried to obtain an intramolecular cyclized product. The yield was 13.8 g. The total amount of the obtained cyclized product was added to a 30 mM ammonium acetate buffer (pH 9.5).
Dissolve in 1300 ml, and add a small amount of aqueous ammonia to pH
The mixture was adjusted to 9.5 and stirred at room temperature for 19 hours to remove formyl groups. The reaction solution is adjusted to pH 5.5 with acetic acid, and this aqueous solution is adsorbed on “SP-Toyopearl 650s” (Tosoh, sulfonic acid type cation exchange resin), eluted with an ammonium acetate buffer, and the fraction containing the target substance is eluted. Was collected, desalted, and freeze-dried to obtain 3.8 g of compound (1).

[0036]

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Example 2: Synthesis of compound (1) (hydrochloride of compound <I>) 600 mg of compound (3) and 0.3 g of indole were dissolved in 3 ml of TFA under ice-cooling, followed by stirring at room temperature for 1 hour. TFA was distilled off under reduced pressure, and 4N-HCl /
0.5 ml of DOX and 10 ml of isopropyl ether were added. The resulting precipitate was collected by filtration, and the obtained crystals were thoroughly washed with isopropyl ether and further dried under reduced pressure to obtain a Boc-deprotected crystal. The amount collected was 890 mg. Boc removal
The whole body was dissolved in 210 ml of DMF, the pH was adjusted to about 8 with NMM, 178 mg of WSC.HCl, HOBt6
2 mg was added and the mixture was stirred at room temperature for 24 hours. DMF at reduced pressure
After distilling off, 10 ml of ethyl acetate was added to the remaining oil. The resulting precipitate was dispersed in about 10 ml of isopropyl alcohol, then collected by filtration and dried to obtain an intramolecular cyclized product.
The yield was 440 mg. The obtained intramolecular cyclized product was 3
30 ml of 0 mM ammonium acetate buffer (pH 5.5)
And dissolve this aqueous solution in "SP-Toyopearl 650s"
And eluted with an ammonium acetate buffer. The fractions containing the target compound were collected, desalted, and lyophilized to obtain 240 mg of compound (1).

[0038]

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Example 3: Compound (2) (Compound <II>)
Synthesis of TFA salt of A) A. Synthesis of compound (4) (Z-Pro-Arg-Gly-NH ₂ .HCl: hydrochloride of compound <IX>) Z-Arg-OH (Kokusan Chemical Co., Ltd.) was added to 200 ml of DMF.
36.8 g) and 13.1 g of H-Gly-NH ₂ .HCl (manufactured by Kokusan Chemical Co., Ltd.) were suspended in HOBt.H ₂
O (manufactured by Peptide Research Laboratories) 18.3 g and DCC
24.5 g (manufactured by Kokusan Chemical Co., Ltd.) was added, and the mixture was stirred at room temperature for 24 hours. The resulting precipitate was filtered off and the filtrate was concentrated to dryness to give an oil. Ethyl acetate was added to the obtained oil to crystallize it. The crystals were repeatedly washed with ethyl acetate and thoroughly dried. The resulting Z-Arg-Gly-
The whole amount of NH ₂ .HCl was dissolved in 190 ml of methanol, 4.8 g of 10% by weight Pd / C was added, and the mixture was stirred in a stream of hydrogen to remove the protecting group. When the raw materials disappeared, the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The obtained oil was dissolved in 125 ml of DMF, 30.1 g of Z-Pro-OH (manufactured by Kokusan Chemical Co., Ltd.), and HOBt.H ₂ O 18.5.
g, 25.5 g of DCC, and the mixture was stirred at room temperature for 24 hours. The precipitate formed was removed by filtration, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the obtained oil to crystallize it,
Further, the crystals were repeatedly washed with ethyl acetate and dried under reduced pressure to obtain a compound (4). The yield was 43.3 g (73
%)Met.

B. Compound (5) (Boc-Ile-Gln-Asn-OH:
Synthesis of Compound (XII) in which R ³ is a Boc Group 41.1 g of Boc-Ile-OH · 1 / 2H ₂ O (manufactured by Peptide Research Laboratories) was dissolved in 191 ml of THF.
OSu (manufactured by Wako Pure Chemical Industries, Ltd.) 20.6 g and DCC3
7.0 g was added, and the mixture was stirred at room temperature for 20 hours. The resulting precipitate was removed by filtration, and the filtrate was concentrated to dryness. The residue was recrystallized from a mixed solution of isopropyl ether and isopropyl alcohol to obtain 52.7 g of Boc-Ile-OSu.
On the other hand, glutamine (manufactured by Peptide Research Laboratories) 24.6
g of triethylamine was suspended in 500 ml of pure water.
3 g was added and dissolved. The Boc obtained earlier was added to the solution.
A solution of 52.6 g of -Ile-OSu dissolved in 184 ml of THF was added, and the mixture was stirred at room temperature for 48 hours. T under reduced pressure
After HF was distilled off and the remaining aqueous solution was washed with ethyl acetate, the aqueous layer was adjusted to pH 2.8 with a 2N-HCl aqueous solution. The aqueous layer was extracted with ethyl acetate, and the ethyl acetate layer was washed with water and dried over anhydrous sodium sulfate. After the anhydrous sodium sulfate was filtered off, the mixture was concentrated to dryness under reduced pressure, and the obtained gelatinous substance was crystallized with a mixture of ethyl acetate and hexane. The yield was 53.0 g (86%). next,
53.0 g of the obtained Boc-Ile-Gln-OH was dissolved in 271 ml of DMF, 17.0 g of HOSu and 30.4 g of DCC were added, and the mixture was stirred at 10 ° C. or lower for 4 hours. The precipitate formed was removed by filtration, and the filtrate and the washing solution were combined to form D.
The MF solution (DMF amount: 430 ml) was used for the next reaction. Next, asparagine (manufactured by Peptide Research Institute) 2
3.2 g was suspended in 590 ml of pure water, 18.6 g of triethylamine was added and dissolved, and the solution was cooled to 5 ° C. or lower. The DMF solution obtained above was added dropwise thereto. After stirring at the same temperature for 12 hours, the mixture was concentrated to dryness under reduced pressure. The residue was dissolved by adding 214 ml of a 5% by weight aqueous sodium hydrogen carbonate solution, and the aqueous solution was washed with ethyl acetate. The pH of the aqueous layer was adjusted to 2.8 with 2N-HCl aqueous solution.
Left for hours. The precipitated crystals were collected by filtration, dried, and recrystallized from aqueous ethanol to obtain Compound (5). Yield 38.
0 g, melting point 225-227 ° C (decomposition), [α] _D -4
5.7% (1% by weight in 0.1 N NaOH).

C. Compound (6) 4- [4- (4-methoxyphenylmethyl) thio] thianylacetic acid (compound <I
Synthesis of V>) First, ethyl-4-thianilidene acetate was synthesized. Ethyl diethyl phosphonoacetate (manufactured by Wako Pure Chemical Industries, Ltd.), 88.0 g, was added dropwise to a suspension of 15.0 g of sodium hydride (60% by weight oil) in 100 ml of toluene while maintaining the temperature at 20 ° C. or lower. Further, the mixture was stirred at room temperature for 1 hour to obtain a pale yellow transparent solution. This solution was added dropwise to a suspension of 43.5 g of tetrahydrothiopyran-4-one (manufactured by Wako Pure Chemical Industries, Ltd.) in 200 ml of toluene at 10 ° C. or lower. After completion of the dropwise addition, the mixture was further stirred for 1 hour, and then 200 ml of water was added to the reaction solution to carry out liquid separation. Add water layer to toluene 1
The mixture was extracted with 00 ml, the toluene layers were combined, washed with saturated saline, and dried over anhydrous magnesium sulfate. After the desiccant was removed by filtration, toluene was distilled off under reduced pressure, and the obtained oil was distilled under reduced pressure (94 ° C, 4 mmHg) to obtain ethyl-4-thianilidene acetate. The yield was 65.6 g (94
%)Met. Then, ethyl-4- [4-
(4-Methoxyphenylmethyl) thio] thianyl acetate was synthesized. 60 g of ethyl-4-thianilidene acetate and 4-methoxybenzylmercaptan (purity 90
%, Manufactured by Wako Pure Chemical Industries, Ltd.), and cooled with ice.
Sodium methoxide (28% by weight methanol solution)
2 ml was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 4.5 hours. 0.4 ml of acetic acid was added to the reaction mixture, and then 200 ml of diisopropyl ether and the same amount of saturated saline were added. The diisopropyl ether layer was separated, washed with a small amount of saturated saline, and dried over anhydrous magnesium sulfate.
After removing the drying agent by filtration, the solvent was distilled off under reduced pressure to obtain an oily substance.
The yield was 103 g (94%). Finally, 4- [4- (4-methoxyphenylmethyl) thio] thianylacetic acid was synthesized using the compound obtained in the above. 103 g of ethyl-4- [4- (4-methoxyphenylmethyl) thio] thianyl acetate in methanol 4
00 ml suspension, potassium carbonate 250 ml in water 200 ml
g in a nitrogen atmosphere and a bath temperature of 80-9.
The mixture was heated to reflux at 0 ° C. for 8 hours with vigorous stirring. After completion of the reaction, the reaction solution was allowed to cool, 250 ml of water was added, and methanol was distilled off under reduced pressure. To the remaining aqueous solution, 500 ml of water was further added, and the mixture was washed twice with 150 ml of diisopropyl ether. About 300 ml of concentrated hydrochloric acid was added to the aqueous layer to adjust the pH to 1-2. After extracting the precipitated crystals with diethyl ether, the solvent was distilled off, and the obtained crystals were recrystallized from a mixed solution of ethyl acetate and hexane to obtain a compound (6). Yield 73.2 g
(77%), mp 110 ° C.

D. Compound (7) (Boc-Pen (-Acm) -Pro-Ar
Synthesis of g-Gly-NH ₂ .HCl: hydrochloride of compound (XI)) 41.5 g of compound (4) was dissolved in 170 ml of methanol, and 2.1 g of 10% by weight Pd / C was added. Was stirred until the disappearance of the protecting group Z. Pd
After the / C was filtered off, the filtrate was concentrated under reduced pressure to obtain an oil.
The obtained oil was dissolved in DMF (170 ml) and the compound (8) (same as compound (X), Watanabe Chemical Industry Co., Ltd.) was added to the solution.
32.0 g of HOBt monohydrate (HOBt.H
₂ O) was added 13.2g. Then, the solution was cooled to 10 ° C. or lower, 17.7 g of DCC was added, and the mixture was stirred at room temperature for 24 hours. The resulting white precipitate was filtered off, and the filtrate was concentrated under reduced pressure to obtain an oil. Ethyl acetate was added to the obtained oil to crystallize, and the mixture was washed well with ethyl acetate to obtain compound (7). The yield was 46.5 g (77%). The amino acid composition (acid degradation) of compound (7) was Gly: 1.04
(1), Arg: 1.02 (1), Pro: 0.94
(1), and the result of FAB-MS was m / z: 679.
(M + 1).

E. Compound (9) Boc-Ile-Gln-Asn-Pen (-A
cm) -Pro-Arg-Gly-NH ₂ .HCl: Synthesis of the hydrochloride of compound (V) in which the protecting group R ³ is a Boc group (45.7 g of compound (7) at a temperature of 5 ° C. or lower at TFA 150
and stirred for 1 hour. TFA was distilled off under reduced pressure to obtain an oil. A solution obtained by dissolving hydrogen chloride gas in DOX equivalent to 4N in the obtained oily substance (4N-HCl
/ DOX, manufactured by Wako Pure Chemical Industries, Ltd.), and crystallized by further adding 183 ml of isopropyl ether.
The precipitate was collected by filtration, washed well with isopropyl ether, dried under reduced pressure, and dissolved in 440 ml of NMP. This solution was adjusted to pH 8 to 9 with NMM, and then compound (5) 3
0.3 g and then HOOBt (Kokusan Chemical Co., Ltd.)
13.6 g). The mixture was cooled to −10 ° C. or lower, 12.3 g of a hydrochloride of WSC (WSC · HCl, manufactured by Peptide Research Laboratories) was added, and the mixture was stirred at the same temperature for 24 hours. The precipitated insoluble matter was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. After the residue was precipitated by adding ethyl acetate, the precipitate was collected by filtration and washed well with ethyl acetate.
The precipitate was subjected to a reprecipitation operation using isopropyl alcohol and isopropyl ether, and the precipitate was collected by filtration to obtain a compound (9). The yield was 65.9 g (96%). The amino acid composition (acid degradation) of compound (9) is determined by As
p: 0.85 (1), Glu: 0.92 (1), Gl
y: 1.11 (1), Ile: 0.99 (1), Ar
g: 1.13 (1), Pro: 1.00 (1),
The result of FAB-MS was m / z: 1035 (M + 1).

F. Compound (10) (Boc-Ile-Gln-Asn-Pe
Synthesis of n (-Acm) (O) -Pro-Arg-Gly-NH ₂ .HCl: HCl of compound <VI> in which R ³ is Boc 44.0 g of compound (9) was dissolved in 440 ml of pure water. And 45.3 g of an aqueous hydrogen peroxide solution (30% by weight), and the mixture was stirred at room temperature for 20 hours. 1.74 g of manganese dioxide
Was added to decompose the remaining hydrogen peroxide, and the obtained aqueous solution was freeze-dried to obtain a compound (10). The yield is 43.
It was 5 g.

G. Synthesis of compound (11) (TFA salt of compound corresponding to compound <VII>) A mixture of 380 ml of TFA and 38 ml of dimethyl sulfide was kept at 10 ° C., and 11.7 g of compound (6) was added to the solution. 43.0 g) was added.
After stirring the reaction solution at about 15 ° C. for 1 hour, the solvent was distilled off under reduced pressure. Isopropyl ether 250 was added to the obtained oil.
The mixture was precipitated by adding ml, and the precipitate was collected by filtration. The collected material was washed with isopropyl ether and dried, dispersed in ethyl acetate containing 10 v / v% methanol, and the precipitate was collected by filtration and dried to obtain Compound (11). The yield was 48.9 g. The amino acid composition (acid degradation) of compound (11)
Asp: 0.88 (1), Glu: 0.91 (1), G
ly: 1.09 (1), Ile: 1.03 (1), Ar
g: 1.09 (1) and Pro: 1.03 (1).

H. Compound (2) (TFA salt of compound <II> in which R ¹ is a formyl group and R ² is a Boc group)
The compound (11) (45.0 g) was dissolved in DMF (180 ml) and adjusted to pH 8 with triethylamine.
16.0 g of -D-Trp (CHO) -OSu was added, and the mixture was stirred at room temperature for 5 hours. DMF was distilled off under reduced pressure, and 360 ml of ethyl acetate was added to the remaining oil. The resulting precipitate was collected by filtration, and dispersed and washed with 30 v / v% isopropyl alcohol-containing ethyl acetate. The obtained precipitate was collected by filtration and dried under reduced pressure to obtain 49.7 g of compound (2). The amino acid composition (acid degradation) of compound (2) was Asp: 0.91 (1), Gl
u: 0.89 (1), Gly: 1.12 (1), Il
e: 1.05 (1), Arg: 1.05 (1), Pr
o: 1.01 (1).

I. Compound (3) (TFA salt of compound <II> in which protective group R ¹ is a hydrogen atom and R ² is a Boc group)
Compound (11) 739 mg was dissolved in DMF 3 ml,
The pH was adjusted to 8 with triethylamine, and Boc-D was added there.
313 mg of -Trp-OSu was added, and the mixture was stirred at room temperature for 2 hours. DMF was distilled off under reduced pressure, and the remaining oil was precipitated by adding 15 ml of ethyl acetate. The resulting precipitate was collected by filtration, dispersed and washed with isopropyl alcohol-isopropyl ether (2: 1), and the obtained precipitate was collected by filtration and dried under reduced pressure to obtain 635 mg of compound (3). The amino acid composition (acid degradation) of compound (3) was Asp: 0.88
(1), Glu: 0.95 (1), Gly: 1.09
(1), Ile: 1.11 (1), Arg: 1.03
(1), Pro: 0.99 (1).

[0048]

Embedded image

[0049]

According to the method of the present invention, a peptide derivative having a pharmacological action such as prevention of premature miscarriage (compound <I>)
Can be industrially and efficiently produced with high purity and high yield. Since the present invention uses a compound having a disulfide bond in the molecule to produce the compound <I>, the step of synthesizing an intramolecular SS bond can be avoided, and a strong alkali is used to remove the protecting group. Without this, compound <I> can be produced in good yield. The present invention employs a skeletal synthesis method by fragment condensation in synthesizing the compound <I>, so that amino acid deficiency can be prevented and a highly pure compound <I> can be produced.

Claims (4)

[Claims] 1. A compound represented by the formula <I> wherein the protecting group R ² is removed from the compound <II> or a salt thereof to form a cyclic structure by an amide bond between an amino group and a carboxyl group in the molecule. A method for producing a peptide derivative or a salt thereof. Embedded image 2. The method for producing a peptide derivative or a salt thereof according to claim ¹ , wherein the protecting group R ¹ is removed after forming a cyclic structure by an intramolecular amide bond. 3. A compound <III> or a salt thereof. Embedded image 4. A compound <V> or a salt thereof is oxidized with a peroxide to a compound <VI> or a salt thereof, and then the compound <IV> is reacted in the presence of a sulfide compound to form a disulfide bond, and A method for producing compound <II> or a salt thereof, comprising removing compound (VII) or a salt thereof by removing protecting group R ³ of VI>, and then reacting compound <VIII>. Embedded image