JP2525798B2 - Peptide derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a peptide derivative represented by the following formula.

R ¹ -Asp (OR ⁴ ) -Tyr-Pro-R ^{2 In the} formula, R ¹ is an α-amino protecting group or hydrogen atom used in peptide synthesis, and R ² is OR ⁶ , NHNH ₂ or NHNHR ¹ (R ⁶ means a hydrogen atom, a carboxyl group-protecting group used in peptide synthesis or an active ester residue used in peptide synthesis), R ⁴ represents a side-chain carboxyl-group protecting group or hydrogen used in peptide synthesis Means an atom.

These compounds are novel compounds not described in the literature, and are useful as intermediates for peptide synthesis having physiological activity, especially fibrinolytic activity.

<Prior Art> There are plasmin as a fibrinolytic enzyme in blood, and several antifibrinolytic factors called plasmin inhibitors that antagonize the fibrinolytic action of plasmin, and α ₂ -plasmin inhibitor (α ₂ PI ) Or α ₂ -antipramine has an immediate and strong inhibitory effect on plasmin, and is known to be the physiologically most important plasmin inhibitor.

Japanese Patent Application Laid-Open No. 59-163355 discloses a hexacosapeptide (H-Gly-Asp-Lys-Leu-Phe-Gly-Pro-A in α ₂ PI.
sp-Leu-Lys-Leu-Val-Pro-Pro-Met-Glu-Glu-A
sp-Tyr-Pro-Gln-Phe-Gly-Ser-Pro-Lys-OH) has a strong affinity for plasmin, indicating that this hexacosapeptide can be a fibrinolysis inhibitor. .

In JP-A-59-163355, the above-mentioned hexacosapeptide was isolated from human α ₂ PI, and human α ₂ PI was obtained from human plasma. It is far from practical use.

<Problems Solved by the Present Invention> The present inventors have found that the chemical synthesis is cheaper than the isolation of the above-mentioned hexacosapeptide from human plasma.
Moreover, considering that it can be obtained in a large amount, as a result of diligent studies, a method for synthesizing the above hexacosapeptide using the compounds of formulas (I) to (VII) as a part of the fragment was found.

^{R 1 -Ser-Pro-Lys (} R 3) -R 2 (I) R 1 -Gln-Phe-Gly-R 2 (II) R 1 -Asp (OR 4) -Tyr-Pro-R 2 (III) ^{R 1 -Met (O) -Glu (} OR 4) -R 2 (IV) R 1 -Leu-Val-Pro-Pro-R 2 (V) R 1 -Leu-Phe-Gly-Pro-R 2 (VI ^{) R 5 -Gly-Asp (OR} 4) -Lys- ( in R ³⁾ -OH (VII) ^{formula, R 1, R 2, R} 4, are as defined above. R ³ means a side chain amino protecting group or hydrogen atom used for peptide synthesis, and R ⁵ means an α-amino protecting group used for peptide synthesis.

Formula (I) is the 24-26 position of the above hexacosapeptide, Formula (II) is the 21-23 position, Formula (III) is the 18-20 position, Formula (IV) is 1
5th to 16th, formula (V) is 11 to 14th, formula (VI) is 4 to 7th,
Formula (VII) is a useful intermediate corresponding to positions 1-3.

<Structure of Invention> The meanings of the abbreviations and abbreviations used in the present specification are as follows.

1. Regarding amino acid residues Asp: Aspartic acid Gln: Glutamine Glu: Glutamic acid Gly: Glycine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Tyr: Tyrosine Val: Valine All amino acids are L-form .

2. Protecting groups Boc: tertiary butyloxycarbonyl For: formyl Z: benzyloxycarbonyl Z (OMe): 4-methoxybenzyloxycarbonyl Me: methyl Bzl: benzyl Np: paranitrophenyl Tcp: 2,4,6 -Trichlorophenyl Pcp: Pentachlorophenyl O: oxide 3. Examples of protecting groups α-Amino protecting group: Boc, Z, Z (OMe) Carboxyl protecting groups: methyl, ethyl, benzyl,
Tertiary butyl Active ester residue: para-nitrophenyl, 2,4,6-trichlorophenyl, pentachlorophenyl, succinimidoyl Side chain amino protecting group: Boc, Z, For, CF ₃ CO 4. Reagents and solvents DCC : N, N'-dicyclohexylcarbodiimide DMF: N, N-dimethylformamide DMSO: Dimethylsulfoxide HOBT: Hydroxybenzotriazole IPE: Isopropyl ether NMM: N-Methylmorpholine TEA: Triethylamine TFA: Trifluoroacetic acid TEMSA: Trifluoromethanesulfonic acid THE : Tetrahydrofuran The compounds of formulas (I) to (VII) can be produced by the following reaction formula.

Process for producing compounds of formula (I) The compound of formula (VIII) and the compound of formula (IX) are reacted in DMF.

The raw material compound (VIII) can be obtained by reacting R ¹ -Ser-NHNH ₂ with isoa (X) nitrite.

Raw material compound (IX) And are obtained by removing Boc group after the reaction with DCC in DMF.

Process for producing compound of formula (II) The compound of formula (XIII) and the compound of formula (XIV) are reacted in DMF.

The raw material compound (XIV) is And are obtained by removing the Boc group after the reaction with DCC-HOBT in THF-chloroform.

Process for producing compound of formula (III) The compound of formula (XVII) and the compound of formula (XVIII) are reacted in DMF.

Raw material compound (XVIII) And are reacted in DMF-DMSO and then the Boc group is removed.

Process for producing compound of formula (IV) The compound of formula (XXI) is oxidized with sodium periodate in alcohol.

Process for producing compound of formula (V) The compound of formula (XXII) and the compound of formula (XXIII) are reacted in DMF.

The raw material compound (XXII) is Was reacted with DCC-HOBT in DMF-chloroform and then with hydrazine hydrate. And is obtained by reacting this with isoamyl nitrite.

Raw material compound (XXIII) After reacting with DCF-HOBT in DMF-chloroform, Boc
Obtained by removing the group.

Process for producing compound of formula (VI) It can be obtained by reacting a compound of formula (XXIX) with a compound of formula (XXX) in DMF.

Raw material compound (XXIX) Was reacted with DCC-HOBT in dioxane-THF-chloroform to form R ¹ -Leu-Phe-NHNH ₂ with hydrazine hydrate,
It is obtained by reacting this with isoamyl nitrite.

Raw material compound (XXX) It is obtained by reacting and with DCC in THF-chloroform and then removing the Boc group.

Process for producing compound of formula (VII) The compound of formula (XXXV) and the compound of formula (XXXVI) are reacted in THF.

Raw material compound (XXXVI) When It is obtained by reacting and in DMF and then removing the Boc group.

<Effects of the Invention> Using the compound of the present invention, the above-mentioned hexacosapeptide can be obtained synthetically by the following fragment condensation method and deprotection group. An example is shown.

Deprotection group → H-Gly-Asp-Lys-Leu-Phe-Gly-Pro-Asp-Leu-L
ys-Leu-Val-Pro-Pro-Met-Glu-Glu-Asp-Tyr-P
ro-Gln-Phe-Gly-Ser-Pro-Lys-OH Example 1 Synthesis of Z (OMe) -Ser-Pro-Lys (Z) -OBzl 8.45 g of Boc-Pro-Lys (Z) -OBzl in 15 ml of TFA and ice. The mixture was added under cooling, stirred at the same temperature for 45 minutes, the excess TFA was distilled off under reduced pressure, the residue was washed with hexane, dissolved in 20 ml of DMF and neutralized with TEA. This was designated as solution A.

Z (OMe) -Ser-NHNH ₂ 4.64 g was added to 3.87 N HCl-DMF 12.71
Add to ml at −20 ° C. and stir at the same temperature while isoamyl nitrite 3.
31 ml was added dropwise, and after stirring at -20 to -5 ° C, TEA 8.89 ml
Was added. This was designated as solution B.

The solution B was added to the solution A stirred with ice cooling and stirred at 4 ° C. for 48 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water and dried over sodium sulfate. The solvent was distilled off under reduced pressure and IPE was added to the residue.
After adding and solidifying, it was recrystallized from methanol-ether to obtain 5.78 g of crystals having a melting point of 78 to 80 ° C.

Elemental analysis Calculated value as C ₃₈ H ₄₆ N ₄ O ₁₀ 1 / 2H ₂ O (%) C62.71, H6.51, N7.70 Measured value (%) C62.81, H6.36, N7.94 ▲ [Α] ²⁰ _D ▼ -49.4 ° (C = 1.1, methanol) Example 2 Synthesis of Boc-Gln-Phe-Gly-OMe 6.12 g of Boc-Phe-Gly-OMe was added to 15 ml of ice-cooled TFA, and at the same temperature 45 After stirring for a minute, excess TFA was distilled off under reduced pressure, the residue was washed with hexane, dissolved in 30 ml of DMF and neutralized with TEA. To this solution was added Boc-Gln-ONp6.69g and TEA2.52ml, stirred at room temperature for 18 hours, the solvent was distilled off under reduced pressure, ether and 5% citric acid were added to the residue to solidify, and then 5% citric acid, Five%
Wash sequentially with sodium hydrogen carbonate and water, methanol-THF
Recrystallization from IPE yielded 6.83 g of crystals with a melting point of 164-165 ° C.

Elemental analysis Calculated as C ₂₂ H ₃₂ N ₄ O ₇ (%) C56.88, H6.94, N12.06 Measured value (%) C56.66, H6.77, N12.14 ▲ [α] ²⁴ _D ▼ −30.4 ° (C = 1.0, methanol) Example 3 Synthesis of Boc-Gln-Phe-Gly-NHNH ₂ 6.83 g of Boc-Gln-Phe-Gly-OMe was dissolved in methanol-dioxane (15 ml-15 ml) and 80% hydrazine was added. Hydrate
8.93 ml was added and the mixture was allowed to stand overnight and the precipitate was recrystallized from methanol-dioxane-ether to give crystals with a melting point of 174-176 ° C
89 g were obtained.

Elemental analysis Calculated as C ₂₁ H ₃₂ N ₆ O ₆ 1 / 2H ₂ O (%) C53.26, H7.02, N17.75 Measured value (%) C53.31, H6.83, N17.59 ▲ [Α] ²⁵ _D ▼ -39.0 ° (C = 1.0, DMF) Example Synthesis of Boc-Asp (OBzl) -Tyr-Pro-OH 6.50 g of Boc-Tyr-Pro-OH was added to 20 ml of ice-cooled TFA,
The mixture was stirred at the same temperature for 1 hour, excess TFA was distilled off under reduced pressure, IPE-hexane was added to the residue, the precipitate was collected by filtration, dried, dissolved in DMF (50 ml) and neutralized with TEA. Boc-Asp (OBzl) -ONp7.
64 g and NMM 1.89 ml are added and stirred at room temperature overnight, the solvent is distilled off under reduced pressure, the residue is dissolved in 5% sodium carbonate, washed with ethyl acetate, the aqueous layer is acidified with citric acid and extracted with ethyl acetate. The ethyl acetate solution was washed successively with 5% citric acid and water, dried over sodium sulfate, evaporated to remove the residue.
IPE was added to solidify and recrystallized from methanol-ethyl acetate-IPE to obtain 8.56 g of a crystal having a melting point of 93 to 96 ° C.

Elemental analysis Calculated as C ₃₀ H ₃₇ N ₃ O ₉ (%) C61.74, H6.39, N7.20 Measured value (%) C61.48, H6.43, N6.98 ▲ [α] ²⁴ _D ▼ -43.2 ° (C = 1.0, methanol) Example 4 Synthesis of Boc-Met (O) -Glu (OBzl) -OH 5.69 g of H-Glu (OBzl) -OH and 3.33 ml of TEA were added to DMF-DMS.
O was added to the mixture (20 ml to 20 ml). Boc-Met was added to this solution.
-Add 8.58 g of OTcp and 2.20 ml of NMM, stir overnight, evaporate the solvent, and partition the residue with ether-5% sodium hydrogen carbonate. Acidify the aqueous layer with citric acid and extract with ethyl acetate,
The extract is washed successively with 5% citric acid and water, dried, the solvent is distilled off, the residue is dissolved in 75 ml of methanol, an aqueous solution of 4.49 g of sodium periodate (90 ml of water) is added, and the mixture is stirred for 2 hours.
The precipitate was filtered off, the solvent in the filtrate was evaporated, the residue was extracted with ethyl acetate, the extract was washed with water, dried and evaporated to remove the solvent. The solidified residue was recrystallized from ethyl acetate-methanol-IPE to give the melting point. 8.15 g of crystals at 93-95 ° C were obtained.

Elemental analysis Calculated as C ₂₂ H ₃₂ N ₂ O ₈ S (%) C54.53, H6.66, N5.78 Measured value (%) C54.56, H6.58, N5.73 ▲ [α] ²⁴ _D ▼ -5.2 ° (C = 1.0, methanol) Example 5 Synthesis of Boc-Leu-Val-Pro-Pro-NHNH ₂ Add 3.78 g of Boc-Pro-Pro-OBzl to 15 ml of ice-cooled TFA and stir at the same temperature for 1 hour. After that, excess TFA is distilled off under reduced pressure.
The residue was washed with ether-hexane and dissolved in DMF (10 ml).
The solution was neutralized with A to obtain a solution A.

Boc-Leu-Val-NHNH ₂ 3.07 g 3.87 N HCl-DMF 6.10 ml
Dissolve in water and add 1.58 ml of isoamyl nitrite at -20 to -5 ℃
After stirring at 4.33 ml of TEA was added and diluted with 10 ml of DMF to prepare a solution B.

Solution B is added to solution A under ice cooling and stirred at 4 ° C for 48 hours.
The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed successively with 5% citric acid, 5% sodium carbonate and water and dried over sodium sulfate, then the solvent was distilled off and the residue was diluted with 15 ml of methanol.
Dissolve in, add 4.57 ml of 80% hydrazine hydrate, stir overnight at room temperature, and collect the precipitate by filtration. Reprecipitation from methanol-IPE yielded 3.36 g of powder with a melting point of 118-121 ° C.

Elemental analysis Calculated as C ₂₆ H ₄₆ N ₆ O ₆ H ₂ O (%) C56.09, H8.69, N15.10 Measured value (%) C56.31, H8.56, N15.02 ▲ [α] ²⁴ _D ▼ -155.8 ° (C = 1.0, methanol) Example 6 Synthesis of Boc-Leu-Phe-Gly-Pro-OBzl 2.62 g of Boc-Gly-Pro-OBzl was added to 5 ml of ice-cooled TFA, and the mixture was kept at the same temperature for 45 minutes. After stirring, the excess TFA was distilled off under reduced pressure,
The residue was washed with IPE and hexane, dissolved in DMF (10 ml) and neutralized with TEA to obtain solution A.

The _{Boc-Leu-Phe-NHNH 2} 2.35g at -15 ℃ 3.87N HCl-DM
Dissolve in F and add 0.97 ml of isoamyl nitrite at the same temperature.
The mixture was stirred at 0 to -5 ° C, 2.83 ml of TEA was added, and this was designated as solution B.

After diluting solution B with DMF (10 ml), combine with solution A, stir at 4 ° C for 48 hours, evaporate the solvent under reduced pressure, dissolve the residue in ethyl acetate, and wash with 5% citric acid, 5% sodium carbonate and water successively. After drying over sodium sulfate, the solvent was distilled off, and IPE and hexane were added to the residue to solidify it, which was dissolved in ether and recrystallized by adding IPE-hexane to obtain 3.40 g of a powder having a melting point of 78-80 ° C.

Elemental analysis Calculated value as C ₃₄ H ₄₆ N ₄ O ₇ 1 / 2H ₂ O (%) C64.64, H7.50, N8.87 Measured value (%) C64.82, H7.30, N8.85 ▲ [ α] ²⁵ _D ▼ -84.4 ° (C = 1.0, methanol) Example 7 Synthesis of Boc-Leu-Phe-Gly-Pro-NHNH ₂ 3.94 g of Boc-Leu-Phe-Gly-Pro-OBzl in methanol 4
Dissolve in 0 ml, add 3.84 ml of 80% hydrazine hydrate under ice cooling, and let stand overnight. The solvent was distilled off, the residue was extracted with ethyl acetate, the extract was washed with water and dried, the solvent was distilled off, the residue was reprecipitated with methanol IPE, and a powder having a melting point of 113 to 115 ° C was 3.04 g.
I got

Elemental analysis Calculated as C ₂₇ H ₄₂ N ₆ O ₆ 1 / 2H ₂ O (%) C58.36, H7.80, N15.13 Measured value (%) C58.36, H7.80, N15.19 ▲ [ α] ²⁵ _D ▼ -80.5 ° (C = 1.1, methanol) Example 8 Synthesis of Boc-Gly-Asp (OBzl) -Lys (Z) -OH Boc-Asp (OBzl) -Lys (Z) OH 5.65 g of ice Chilled TFA
Dissolve in 15 ml and stir at the same temperature for 45 minutes, then distill off excess TFA under reduced pressure. IPE was added to the residue, the insoluble material was collected by filtration, dried under reduced pressure, added to 20 ml of THF and neutralized with TEA. Boc-Gly-
Add ONp3.27g and TEA1.33ml, stir at room temperature for 48 hours,
The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed successively with 5% citric acid and water, dried over sodium sulfate, the solvent was distilled off under reduced pressure, ether was added to the residue, and the insoluble matter was removed with acetic acid. It was dissolved in ethyl-methanol. Ether-IPE was added and reprecipitated to obtain 3.32 g of powder having a melting point of 81 to 83 ° C.

Elemental analysis Calculated as C ₃₂ H ₄₂ N ₄ O ₁₀ (%) C59.80, H6.59, N8.72 Measured value (%) C59.95, H6.62, N9.01 ▲ [α] ²⁵ _D ▼ -5.6 ° (C = 1.0, methanol) Reference Example 1 Synthesis of Boc-Pro-Lys (Z) -OBzl 10.81 g of Boc-Pro-OH and H-Lys (Z) -OBzl (TsOH salt
27.13 g TEA 6.93 ml was added) was dissolved in DMF 90 ml, DCC 11.24 g was added, and the mixture was stirred overnight at room temperature.
After evaporating the solvent under reduced pressure, the residue was dissolved in ether, washed successively with 5% citric acid, 5% sodium hydrogencarbonate and water and dried over sodium sulfate. The solvent was evaporated to obtain 24.20 g of an oil.

Thin-layer chromatography (solvent chloroform-methanol-water 8: 3: 1) Rf value 0.87 Reference example 2 Synthesis of Boc-Phe-Gly-OMe Dissolve 7.96 g of Boc-Phe-OH in 50 ml of THF and prepare H-Gly-OMe.
Chloroform (made from 4.24 g of hydrochloride and 4.57 ml of TEA)
It was dissolved in 50 ml and both were combined. DCC 6.13g and HOBT
Hydrate (4.59 g) was added, the mixture was stirred overnight at room temperature, the solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 5% citric acid, 5% sodium hydrogen carbonate, water successively and dried over sodium sulfate. Was distilled off, hexane was added to the residue to solidify it, and it was recrystallized from ether-hexane.
Got 54g.

Elemental analysis Calculated value as C ₁₇ H ₂₄ N ₂ O ₅ (%) C60.70, H7.19, N8.33 Measured value (%) C61.09, H7.13, N8.67 ▲ [α] ²⁴ _D ▼ -4.2 ° (C = 1.0, Methanol) Reference Example 3 Synthesis of Boc-Tyr-Pro-OH 8.86 g of Boc-Tyr-NHNH ₂ was dissolved in 18.6 ml of 3.87N HCl-DMF, and 4.85 ml of isoamyl nitrite was added. After stirring at 20 to -5 ° C, 9.98 ml of TEA was added to prepare a solution A.

H-Pro-OH (4.14 g) was added to a mixed solution of water (5 ml) -DMSO (45 ml), and TE was added.
A solution (9.15 ml) was added to prepare solution B.

Solution B is added to solution A, stirred at 4 ° C. for 48 hours, the solvent is distilled off under reduced pressure, the residue is dissolved in 5% sodium hydrogen carbonate and washed with ethyl acetate. Citric acid was added to this aqueous layer to make it acidic, and the mixture was extracted with ethyl acetate. The ethyl acetate solution was washed with water and dried over sodium sulfate, then the solvent was distilled off to give a residue.
Add IPE to solidify, recrystallize from methanol IPE, melting point
7.63 g of crystals at 114-116 ° C were obtained.

Elemental analysis Calculated as C ₁₉ H ₂₆ N ₂ O ₄ 1 / 4H ₂ O (%) C59.95, H7.24, N7.32 Measured value (%) C59.70, H7.07, N7.07 ▲ [ α] ²⁴ _D ▼ -26.6 ° (C = 1.0, methanol) Reference Example 4 Synthesis of Boc-Pro-Pro-OBzl 6.49 g of Boc-Pro-OH was dissolved in 30 ml of DMF to obtain 6.13 g of DCC and
HOBT hydrate (1.0 g) was added, and the mixture was stirred at room temperature for 15 minutes. To this solution was added H-Pro-OBzl (hydrochloric acid salt 8.70 g and TEA 4.99 ml) dissolved in chloroform (25 ml), and the mixture was stirred at room temperature for 18 hours, then the insoluble material was filtered off, and the filtrate solvent was evaporated under reduced pressure. You The residue was dissolved in ethyl acetate, washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water, dried over sodium sulfate and the solvent was distilled off to obtain 12.0 g of an oily substance.

Thin layer chromatography (solvent: chloroform-methanol-water 8: 3: 1) Rf value 0.81 Reference example 5 Synthesis of Boc-Leu-Val-OMe 4.10 g of Boc-Leu-OH hydrate was dissolved in 10 ml of DMF to obtain DCC3.57.
g and 2.43 g HOBT hydrate. After stirring for 15 minutes at room temperature, H
A solution of -Val-OMe (hydrochloride 2.91 g and TEA 2.42 ml) in 10 ml chloroform is added. After stirring at room temperature for 18 hours, the insoluble material is filtered off, and the solvent of the filtrate is evaporated under reduced pressure. Dissolve the residue in ethyl acetate, wash with 5% citric acid, 5% sodium hydrogen carbonate, and water successively, and dry over sodium sulfate. After evaporating the solvent, the residue was recrystallized from ethyl acetate-hexane to give a melting point of 139-1.
4.19 g of powder at 41 ° C. was obtained.

Elemental analysis Calculated as C ₁₇ H ₃₂ N ₂ O ₅ (%) C59.28, H9.36, N8.13 Measured value (%) C59.09, H9.25, N8.16 ▲ [α] ²⁴ _D ▼ -14.1 ° (C = 1.1, Methanol) Reference Example 6 Synthesis of Boc-Leu-Val-NHNH ₂ 4.19 g of Boc-Leu-Val-OMe was dissolved in 15 ml of methanol, and 7.39 ml of 80% hydrazine hydrate was added, After stirring at room temperature for 1 hour, the solvent is distilled off under reduced pressure. Water was added to the residue, and the precipitate was collected by filtration and reprecipitated from methanol-water with a melting point of 143-145.
3.07 g of powder having a temperature of ℃ was obtained.

Elemental analysis Calculated value as C ₁₆ H ₃₂ N ₄ O ₄ (%) C55.79, H9.36, N16.27 Measured value (%) C55.74, H9.27, N16.01 ▲ [α] ²⁴ _D ▼ −52.4 ° (C = 1.0, methanol) Reference Example 7 Synthesis of Boc-Gly-Pro-OBzl 2.51 g of Boc-Gly-OH and H-Pro-OBzl (hydrochloride 3.80 g)
(Manufactured from 2.18 ml of TEA) is added to a mixed solution of 10 ml of THF and 15 ml of chloroform, 3.21 g of DCC is added, and the mixture is stirred at room temperature for 18 hours. The insoluble material was removed by filtration, the solvent of the filtrate was evaporated under reduced pressure, the residue was dissolved in ethyl acetate, washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water, dried over sodium sulfate and the solvent was removed. Evaporation under reduced pressure gave 4.98 g of an oily substance.

Thin layer chromatography (solvent chloroform-methanol-water 8: 3: 1) Rf value 0.67 Reference Example 8 Synthesis of Boc-Leu-Phe-OMe 5.17 g Boc-Leu-OH hydrate dioxane 10 ml-THF 20 ml
Dissolve it in the mixed solution and add 4.49 g of DCC and 3.06 g of HOBT hydrate,
Stir for 15 minutes at room temperature. H-Phe-OMe (hydrochloride 4.31
20 ml of chloroform and 10 ml of dioxane (produced from 2.79 ml of TEA) and stirred at room temperature for 18 hours. After the insoluble matter is filtered off, the solvent of the filtrate is distilled off under reduced pressure. The residue is dissolved in ethyl acetate, washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water, dried over sodium sulfate and evaporated under reduced pressure. The residue was solidified with IPE and hexane and recrystallized from ether-IPE-hexane to obtain 7.61 g of crystals with a melting point of 86-87 ° C.

Elemental analysis Calculated value as C ₂₁ H ₃₂ N ₂ O ₅ (%) C64.24, H8.22, N7.14 Measured value (%) C64.19, H8.25, N7.08 ▲ [α] ²⁵ _D ▼ −20.7 ° (C = 1.2, methanol) Reference Example 9 Synthesis of Boc-Leu-Phe-NHNH ₂ 7.55 g of Boc-Leu-Phe-OMe was dissolved in 50 ml of methanol, and 11.70 ml of 80% hydrazine hydrate was added. Stir overnight at room temperature. The precipitate was collected by filtration and recrystallized from methanol IPE,
6.95 g of crystals with a melting point of 165-166 ° C. were obtained.

Elemental analysis Calculated value as C ₂₀ H ₃₂ N ₄ O ₄ (%) C61.20, H8.22, N14.28 Measured value (%) C61.06, H8.08, N14.17 ▲ [α] ²⁵ _D ▼ −36.0 ° (C = 1.0, methanol) Reference Example 10 Synthesis of Boc-Asp (OBzl) -Lys (Z) -OH 6.92 g of H-Lys (Z) -OH was suspended in 70 ml of DMF, and TEA3.6 was used.
After adding 3 ml and 2.20 ml of NMM, Boc-Asp (OBzl) -ONp8.
Add 89 g and stir at room temperature for 48 hours. The solvent was distilled off under reduced pressure,
Dissolve the residue in 5% sodium carbonate and wash with ethyl acetate. Acidify the aqueous layer with citric acid and extract with ethyl acetate,
The ethyl acetate solution was washed successively with 5% citric acid and water, dried over sodium sulfate and the solvent was distilled off to obtain 10.23 g of an oily substance.

Thin layer chromatography (solvent chloroform-methanol-water 8: 3: 1) Rf value 0.54 Reference Example 11 Boc-Gln-Phe-Gly-Ser-Pro-Lys (Z)
Synthesis of -OBzl 5.78 g of Z (OMe) -Ser-Pro-Lys (Z) -OBzl and 2.0 ml of anisole are stirred with 20 ml of TFA at 0 ° C for 1 hour. TFA was distilled off under reduced pressure, IPE-hexane was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in DMF (15 ml), and neutralized with TEA to obtain solution A.

Boc-Gln-Phe-Gly- NHNH 2 4.48g at -15 ℃ 3.87NH
Dissolve in 7.48 ml of Cl-DMF, add 1.95 ml of isoamyl nitrite, stir at -20 to -5 ℃, add 5.12 ml of TEA, add DMF-DMSO.
It was diluted with (10 ml-10 ml) to prepare a solution B.

Solution A and solution B were combined and stirred at 4 ° C. for 48 hours, a few drops of acetic acid were added, the solvent was distilled off, water-ether was added to the residue, and the solidified portion was collected by filtration. This was washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water, and reprecipitated from methanol-THF-ether to obtain 7.05 g of a powder having a melting point of 149-151 ° C.

Elemental analysis Calculated as C ₅₀ H ₆₆ N ₈ O ₁₃ · H ₂ O (%) C59.74, H6.82, N11.15 Measured value (%) C59.87, H6.69, N11.59 ▲ [α ] ²⁵ _D ▼ −37.5 ° (C = 1.0, DMF) Reference Example 12 Boc-Asp (OBzl) -Tyr-Pro-Gln-Phe-Gl
Synthesis of y-Ser-Pro-Lys (Z) -OBzl 2.41 g of the compound of Reference Example 11 and 10 ml of TFA are stirred at 0 ° C. for 45 minutes. TFA was distilled off, ether was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in 10 ml of DMF, and TEA was added to neutralize it.
This was designated as solution A.

2.14 g of Boc-Asp (OBzl) -Tyr-Pyo-OH was dissolved in 10 ml of DMF, 0.90 ml of DCC and 0.62 g of HOBT hydrate were added at 0 ° C.
Stir for 1 minute, and use this as solution B.

Solution A was added to solution B, the pH was adjusted to near neutral with TEA, and the mixture was stirred overnight. The precipitate is filtered off, the solvent of the filtrate is evaporated, the residue is dissolved in ethyl acetate, washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water and dried. The solvent was distilled off, ether was added, the precipitate was collected by filtration, and reprecipitated with ethyl acetate-ether to obtain 3.24 g of a powder having a melting point of 117 to 119 ° C.

Elemental analysis Calculated value as C ₇₅ H ₉₃ N ₁₁ O ₁₉ (%) C60.15, H6.60, N10.29 Measured value (%) C60.01, H6.34, N10.45 ▲ [α] ²⁵ _D ▼ −52.8 ° (C = 1.1, DMF) Reference Example 13 Boc-Glu (OBzl) -Asp (OBzl) -Tyr-Pro
Synthesis of -Gln-Phe-Gly-Ser-Pro-Lys (Z) -OBzl 3.24 g of the compound of Reference Example 12 and 15 ml of TFA were stirred at 0 ° C for 45 minutes. TFA was distilled off, ether was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in 15 ml of DMF, and neutralized with TEA.
Add Boc-Glu (OBzl) -ONp (1.53 g) and NMM (0.25 ml) and stir overnight. The solvent was distilled off, the residue was dissolved in ethyl acetate, and
Wash sequentially with% citric acid, 5% sodium carbonate, and water. After drying, the solvent was distilled off, ether was added to the residue, the precipitate was collected by filtration, subjected to silica gel chromatography, and eluted with chloroform-methanol-acetic acid (85: 10: 5). The desired product was reprecipitated with ethyl acetate-ether and had a melting point of 115-1.
1.70 g of powder at 18 ° C. was obtained.

Elemental analysis _{C 87 H 106 N 12 O 22} · 2H 2 O Calculated (%) C61.18, H6.49, N9.84 Found (%) C61.14, H6.28, N9.77 ▲ [α ] ²⁵ _D ▼ -52.9 ° (C = 1.1, DMF) Reference Example 14 Boc-Met (O) -Glu (OBzl) -Glu (OBzl)
-Asp (OBzl) -Tyr-Pro-Gln-Phe-Gly-Ser-Pro-
Synthesis of Lys (Z) -OBzl 1.67 g of the compound of Reference Example 13 and 5 ml of TFA were stirred at 0 ° C for 45 minutes. TFA is distilled off, ether is added to the residue, and the precipitate is collected by filtration. After drying under reduced pressure, it was dissolved in DMF (7 ml) and neutralized with TEA to obtain solution A.

0.63 g of Boc-Met (O) -Glu (OBzl) -OH was dissolved in 5 ml of DMF, 0.32 g of DCC and 0.15 g of HOBT hydrate were added, and a solution B was obtained by stirring for 15 minutes.

Solution A is added to solution B, TEA is added to adjust the pH to near neutral, the mixture is stirred overnight, and the precipitate is filtered off. The solvent of the filtrate was distilled off, the residue was extracted with ethyl acetate, the extract was washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water and dried, then the solvent was distilled off and IPE was added to the residue. , The obtained powder was reprecipitated from methanol-ether and powder with a melting point of 105-108 ℃
1.97 g were obtained.

Elemental analysis Calculated as C ₁₀₄ H ₁₂₈ N ₁₄ O ₂₇ S-3H ₂ O (%) C59.70, H6.46, N9.37 Measured value (%) C59.92, H6.33, N9.20 ▲ [ α] ²⁵ _D ▼ -42.0 ° (C = 1.1, DMF) Reference Example 15 Boc-Leu-Val-Pro-Pro-Met (O) -Glu
(OBzl) -Glu (OBzl) -Asp (OBzl) -Tyr-Pro-Gln
Synthesis of -Phe-Gly-Ser-Pro-Lys (Z) -OBzl 1.86 g of the compound of Reference Example 14 and 6 ml of TFA are stirred at 0 ° C for 45 minutes. TFA was distilled off, ether was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in 3 ml of DMF, and neutralized with TEA to obtain solution A.

0.79 g of Boc-Leu-Val-Pro-Pro-NHNH ₂ at -15 ° C 3.
Dissolve in 0.99 ml of 87N HCl-DMF, add 0.26 ml of isoamyl nitrite at the same temperature and stir at -20 to -5 ° C, then 0.66 ml of TEA
After adding, the solution was diluted with DMF 2.0 ml-TEA 0.13 ml to prepare a solution B.

Add solution A to solution B and stir overnight. After distilling off the solvent,
Extract the residue with ethyl acetate and extract with 5% citric acid, 5
% Sodium hydrogencarbonate, water, and then dried, the solvent is distilled off, ether is added to the residue, and the obtained powder is reprecipitated with methanol-ethyl acetate-ether to give a melting point of 123-1.
2.07 g of powder at 26 ° C. was obtained.

Elemental analysis Calculated as C ₁₂₅ H ₁₆₂ N ₁₈ O ₃₁ S ・ H ₂ O (%) C59.65, H6.81, N10.02 Measured value (%) C59.70, H6.74, N10.05 ▲ [ α] ²⁵ _D ▼ -63.1 ° (C = 1.1, DMF) Reference Example 16 Boc-Asp (OBzl) -Leu-Lys (Z) -Leu-V
al-Pro-Pro-Met (0) -Glu (OBzl) -Glu (OBzl)
-Asp (OBzl) -Tyr-Pro-Gln-Phe-Gly-Ser-Pro-
Synthesis of Lys (Z) -OBzl 2.07 g of the compound of Reference Example 15 and 10 ml of TFA are stirred at 0 ° C. for 45 minutes. TFA was distilled off, ether was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in DMF (10 ml) and neutralized with TEA to prepare solution A.

0.83 g of Boc-Asp (OBzl) -Leu-Lys (Z) -OH was added to DMF5.
Dissolve in 0 ml and add DCC 0.26g at 0 ° C, then HOBT hydrate 0.13g and stir for 15 minutes. Add solution A to this, adjust the pH to near neutral with TEA, stir overnight and filter off insolubles. . The solvent of the filtrate was evaporated, ether-5% citric acid was added to the residue, and the solidified portion was collected by filtration, washed successively with 5% citric acid, 5% sodium hydrogen carbonate and water, and reprecipitated from methanol-ethyl acetate. Let
2.35 g of a powder with a melting point of 139-141 ° C. were obtained.

Elemental analysis Calculated as C ₁₅₆ H ₂₀₂ N ₂₂ O ₃₈ S / 3H ₂ O (%) C60.84, H6.81, N10.01 Measured value (%) C60.78, H6.72, N10.02 ▲ [ α] ²⁵ _D ▼ -58.9 ° (C = 1.1, DMF) Reference Example 17 Boc-Leu-Phe-Gly-Pro-Asp (OBzl) -Le
u-Lys (Z) -Leu-Val-Pro-Pro-Met (O) -Glu
(OBzl) -Glu (OBzl) -Asp- (OBzl) -Tyr-Pro-Gl
Synthesis of n-Phe-Gly-Ser-Pro-Lys (Z) -OBzl 1.70 g of the compound of Reference Example 16 was stirred in 10 ml of TFA at 0 ° C for 45 minutes. TFA was distilled off under reduced pressure, ether was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in 5.0 ml of DMF, and neutralized with TEA.
This was designated as solution A.

Boc-Leu-Phe-Gly-Pro-NHNH ₂ 0.46 g at -15 ° C 3.8
Dissolve it in 0.65 ml of 7N HCl-DMF, add 0.17 ml of isoamyl nitrite at the same temperature, and stir at -20 ° C to -5 ° C. TEA0.43 ml
Was added and diluted with 5 ml of DMF, which was designated as solution B.

Solution A and solution B were combined, stirred for two nights, the solvent was distilled off under reduced pressure, water-ether was added to the residue, and the solidified portion was collected by filtration, 5% citric acid, 5% sodium hydrogen carbonate, water. Sequentially wash with, reprecipitate from methanol-ether, melting point 133-1
1.86 g of powder at 36 ° C. was obtained.

Elemental analysis Calculated as C ₁₇₈ H ₂₃₂ N ₂₆ O ₄₂ S-4H ₂ O (%) C60.87, H6.89, N10.37 Measured value (%) C60.64, H6.69, N10.44 ▲ [ α] ²⁵ _D ▼ -62.7 ° (C = 1.1, DMF) Reference Example 18 Boc-Gly-Asp (OBzl) -Lye (Z) -Len
-Phe-Gly-Pro-Asp (OBzl) -Len-Lys (Z) -Leu
-Val-Pro-Pro-Met (O) -Glu (OBzl) -Glu (OBz
l) -Asp (OBzl) -Tyr-Pro-Gln-Phe-Gly-Ser-Pr
Synthesis of o-Lys (Z) -OBzl 1.40 g of the compound of Reference Example 17 was added to 10 ml of ice-cooled TFA, and the mixture was stirred at the same temperature for 45 minutes. TFA was distilled off under reduced pressure, ether was added to the residue, the precipitate was collected by filtration, dried under reduced pressure, dissolved in DMF 7.0 ml, and neutralized with TEA to obtain solution A.

0.39 g of Boc-Gly-Asp (OBzl) -Lys (Z) -OH was added to DMF3.
Dissolve in 0 ml, add 0.15 g of DCC and 0.10 g of HOBT hydrate at 0 ° C., stir for 15 minutes, add solution A, further add TEA to adjust pH to near neutral, and stir overnight. The insoluble material was filtered off, the solvent in the filtrate was evaporated under reduced pressure, water-ether was added to the residue, and the solidified portion was collected by filtration, washed successively with 5% citric acid, 5% sodium hydrogencarbonate and water, and methanol was added. Reprecipitation from -THF-ether gave 1.41 g of powder with a melting point of 198-202 ° C.

Elemental analysis Calculated as C ₂₀₅ H ₂₆₄ N ₃₀ O ₄₉ S-4H ₂ O (%) C60.99, H6.79, N10.41 Measured value (%) C60.77, H6.63, N10.40 ▲ [ α] ²⁵ _D ▼ -58.6 ° (C = 1.0, DMF) Reference Example 19 H-Gly-Asp-Lys-Leu-Phe-Gly-Pro-A
sp-Leu-Lys-Leu-Val-Pro-Pro-Met-Glu-Glu-A
Synthesis of sp-Tyr-Pro-Gln-Phe-Gly-Ser-Pro-Lys-OH To 200 mg of the compound of Reference Example 18, 0.53 ml of metacresol, 0.59 ml of thioanisole, 3.97 ml of TFA, 0.44 ml of TFMSA were sequentially added, and
The mixture was stirred at 0 ° C for 1 hour. The solvent was evaporated under reduced pressure, ether was added to the residue, the precipitate was collected by filtration, washed with ether, and dried under reduced pressure. Dissolve this in 4 ml of water and use Amberlite IRA
Add 410 (acetic acid type) at 0 ° C., stir for 30 minutes, and then filter out the amberlite. The filtrate is maintained at 0 ° C, 5% aqueous ammonia is added to adjust the pH to 8, and the mixture is stirred for 3 minutes. Acetic acid was added to adjust the pH to 4.0, water was added, and the mixture was freeze-dried. The obtained lyophilized product and 200 mg of dithiothreitol were added to 5 ml of water, and the mixture was stirred at 37 ° C for 19 hours under a nitrogen stream. Elution with 1N acetic acid was applied to the Sephadex G-25 column, and the desired fraction was lyophilized. The lyophilizate obtained was dissolved in 3 ml of water and applied to a DEAE-Cellulose column, and 0.01 M ammonium hydrogen carbonate buffer (pH 8.
1) and 0.20 M ammonium hydrogen carbonate buffer were eluted with a linear concentration gradient, the desired phthalation was freeze-dried, acetic acid was added, and freeze-dried again to obtain 52 mg of powder with a melting point of 201-208 ° C (decomposition). .

Elemental analysis _{C 134 H 202 N 30 O 40} S · 4CH 3 COOH15.5H 2 O Calculated (%) C49.80, H7.33, N12.27 Found (%) C49.97, H7.52, N12 .53 ▲ [α] ²⁵ _D ▼ -76.0 ° (C = 0.2,0.1N acetic acid) Amino acid analysis after hydrolysis with 6N hydrochloric acid (calculated values in parentheses) Aspartic acid 3.01 (3) Serine 0.85 (1) Glutamic acid 3.01 ( 3) Proline 5.21 (5) Glycine 3.01 (3) Valine 1.02 (1) Methionine 0.79 (1) Leucine 3.11 (3) Tyrosine 0.92 (1) Phenylalanine 2.00 (2) Lysine 2.85 (3)

Claims (1)

(57) [Claims] 1. A peptide derivative represented by the formula R ¹ -Asp (OR ⁴ ) -Tyr-Pro-R ² . In the formula, R ¹ is an α-amino protecting group or hydrogen atom used for peptide synthesis, R ² is OR ⁶ , NHNH ₂ or NHNHR ¹ (R ⁶ is a hydrogen atom, protection of a carboxyl group used for peptide synthesis) Group or an active ester residue used in peptide synthesis), R ⁴ represents a protecting group of a side chain carboxyl group used in peptide synthesis or a hydrogen atom.