JPS5934176B2 - Method for producing bioactive peptides - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for synthesizing physiologically active peptides.

It is known that a peptide hormone called somatostatin exists as a factor that suppresses the growth function of the pituitary gland in humans and animals. Since somatostatin also has the effect of lowering blood sugar, it has recently been viewed as a promising drug for the treatment of diabetes.

However, problems have been raised that the action time is short, and there are side effects such as blood disorders such as bleeding tendency, and once this amino acid arrangement was clarified [P. Br
azeauetal,. Science, Vol. 79, p. 77 (1973)], synthetic studies of various similar compounds have begun to be carried out with the aim of solving the above-mentioned problems. ★1
The present inventors have demonstrated that the N-terminal alanine 1 of somatostatin,
Focusing on the fact that glycine 2 is not essential for the expression of activity, and that the disulfide bond between the two cysteines in the molecule is not only not essential for the expression of activity, but also that it is extremely chemically unstable, we carried out a structural transformation of somatostatin. As a result of continuing independent research, we found that the cysteine at the C-terminus was changed to L=
It is represented by the following formula in which α-aminosuberic acid is replaced with ω-position carboxyl group and lysine 4 is condensed to form a cyclic structure.

The peptide (hereinafter referred to as L-Asu-type somatostatin) has a biological activity equivalent to or higher than that of natural somatostatin in inhibiting growth hormone secretion in experimental animals, is highly chemically stable, and has a long action in vivo. I learned that having time is something to be expected.
The present invention was completed based on these findings. On the other hand, DL-Asu type somatostatin was recently announced [J. Am. Chem. SOc. 982367(
1976), published on April 14, 1976], according to the same report, the growth hormone secretion suppressing ability of DL-Asu somatostatin is only half that of natural somatostatin.

In other words, in this report, since optically inactive DL-α-aminosuberic acid is used as a raw material, a target substance with low biological activity containing DL-α-aminosuberic acid as a constituent amino acid can be obtained. It's just a matter of getting caught. According to the same report, as a method for synthesizing DL-Asu type somatostatin, a peptide having DL-α-aminosuberic acid at its N-terminus is subjected to a cyclization reaction by the azide method, but according to the method of the present invention, Since the peptide with L-α=aminosuberic acid at the C-terminus is subjected to a cyclization reaction, cyclization can be performed by the technically easy active ester method, and the desired high biological activity can be achieved. L-As
U-type somatostatin can be produced with high purity and high yield.

Next, the method of the present invention will be explained in detail.

In the method of the present invention, first, a tetrapeptide hydrazide represented by the formula (in the formula, xl and X2 represent an amino-protecting group, and y represents a hydroxyl group-protecting group) and a and Z represents a protected carboxyl group together with a carbonyl group), and then the amino protecting group of the N-terminal ★-terminal amino acid is removed to form the formula (where X2, y and Z have the same meanings as above), and condense this heptapeptide with a hydrazide of a tetrapeptide represented by the formula (wherein xl and X2 have the same meanings as above), This condensate is subjected to active esterification of the ω-position carboxyl group of the C-terminal L-α-aminosuberic acid and elimination of the amino protecting group of the N-terminal L-lysine, and then subjected to a cyclization reaction, and then is removed to produce a physiologically active peptide represented by the formula, ie, L-Asu type somatostatin or a salt thereof.

In the method of the present invention, the above-mentioned tri- and tetrapeptides serving as structural units are obtained by sequentially condensing the constituent amino acids according to conventional methods such as the active ester method, the carbodiimide method, and the acid anhydride method.

In the method of the present invention, with regard to the protection of amino groups, carboxyl groups, or hydroxyl groups, protective groups can be appropriately selected and removed by well-known and commonly used means in the field of peptide chemistry.

Examples of amino protecting groups include acyl groups such as formyl group, trifluoroacetyl group, and putalol group, aralkyl groups such as benzyl group, diphenylmethyl group, and triphenylmethyl group, menzyloxycarbonyl group, and o-bromobenzyloxycarbonyl group. group, a substituted benzyloxycarbonyl group such as o-chlorobenzyloxycarbonyl group, aliphatic oxycarbonyl group such as t-butoxycarbonyl group, diisopropylmethoxycarbonyl group, and the like.

However, when selecting the amino protecting group, it goes without saying that the protecting groups for the side chain amino group and the α-amino group of lysine must be mutually selected so that they can be removed by different means. Next, the carboxyl group is mainly protected by esterification, but also by amide formation or the like.

For example, alkanols such as methanol, ethanol, t-butanol, alkanols such as benzyl alcohol, p-nitrobenzyl alcohol, benzhydryl alcohol, phenols such as 2,4,6-trichlorophenol, p-nitrophenol, or thiophenol. ,
It can be protected by esterification with thiophenols such as p-nitrothiophenol. The hydroxyl group can also be protected, for example, by esterification or etherification. Groups suitable for this esterification include:
Examples include alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethyloxycarbonyl groups. Examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. However, these hydroxyl groups do not necessarily need to be protected. In the method of the present invention, the condensation of a hydrazide of a tetrapeptide with a side chain amino group protected as a structural unit and another peptide having a free α-amino group is carried out by a known azide coupling method.

That is, for example, by reacting the hydrazide of a tetrapeptide with an organic nitrite ester such as t-butyl nitrite or isoamyl ester to give the corresponding azide, which is then condensed with the free amino groups of other peptides at low temperature with stirring. It will be done. In the method of the present invention, cyclization by condensation of the ω-position carboxyl group of L-α-aminosuberic acid and the α-amino group of N-terminal L-lysine is carried out by an active ester method. In this case, active esterification of the ω-position carboxyl group is carried out by a conventional method, for example, by converting this into cyanomethyl ester,
Thiophenyl ester, p-nitrothiophenyl ester, p-methanesulfonylphenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,4,5:trichlorophenyl ester, etc. This is done by Next, in the method of the present invention, L-α
The cyclization reaction by condensation of the active ester of the ω-carboxyl group of -aminosuberic acid and the free amino group of the N-terminal L-lysine is easily carried out in the presence of an organic base at room temperature or by heating according to a conventional method.

The protecting groups for the amino and hydroxyl groups can be easily removed from the cyclized product thus obtained by known means such as hydrolysis, acidolysis or reduction depending on the type of protecting group used. The cyclized product L-A thus produced
For su-type somatostatin, the organic solvent is removed from the reaction mixture and the residue is chromatographed using an ion exchange resin according to the conventional method for separating and purifying peptides.
They can be separated and purified by molecular sieve chromatography and rechromatography.

Is L-Asu type somatostatin according to the present invention? - Obtained as a base or its form depending on the separation and purification conditions.

Next, the growth hormone secretion suppressing effect of L-Asu type somatostatin obtained by the method of the present invention on experimental animals will be described.

〔experimental method〕

Using male Wistar rats (weighing approximately 2007 cm), the jugular vein was exposed under urethane anesthesia, and synthetic T was injected from one side of the jugular vein.
RH (ThyrOthrOpinReleasingH
OrmOne) 200n7/100y body weight was injected.

In some rats, synthetic somatostatin was injected subcutaneously at 20 μy/1007 body weight 5 minutes before TRH administration.
All drugs were dissolved in saline, and the control group received saline alone. Blood was collected from the jugular vein on the other side before, 5 and 10 minutes after TRH administration, and after separating the plasma, the plasma rat GH level was measured by radioimmunoassay (EndOcri).
nOlOgy95l6O8~16131974). As a standard substance, NIAMDD-RatGHRP-1
was used. (Minimum detectable amount 1.0n7/ml).

[Results] As shown in Table 1, TRH2OOn7/100
Intravenous injection of 7 body weight clearly increased plasma GH levels in all rats.

On the other hand, no significant increase in plasma GH was observed in the group in which TRH was intravenously injected after administration of synthetic somatostatin 20μ7/1007 body weight and in the group in which TRH was intravenously injected after administration of L-Asu somatostatin 20μ7/1007 body weight.
Furthermore, the maximum increases in plasma GH after TRH administration in the synthetic somatostatin administration group and L-Asu somatostatin administration group were 15.6±8.1n7/ml and 97±3n7/ml, respectively.
．． 1ny/ml'('significantly(p<0.01) lower than the TRH alone administration group.

In addition, the abbreviations in this specification have the following meanings.

For amino acid analysis, the sample was hydrolyzed with 6N hydrochloric acid (adding several drops of anisole) at 108°C for 24 hours, dried under reduced pressure, and subjected to amino acid analysis.

Next, the method of the present invention will be specifically explained with reference to Examples.
The present invention is not limited thereby. Example (1) Production of BOC-Asu-OH
t-butyloxycarbonyl-S-4.
React with 6-dimethylpyrimidi-2-ylthiocarbonate at room temperature for 24 hours.

After dioxane is distilled off under reduced pressure, excess reagent is extracted and removed with diethyl ether. After adjusting the aqueous layer to pH 2-3 with 2N hydrochloric acid, the resulting oil is extracted with ethyl acetate. After thoroughly washing the organic layer with water, it was dehydrated with sodium sulfate, concentrated, crystallized with n-hexane, and recrystallized with the same solvent system to obtain the target compound 46.27 (yield: 80%). (2)
Production of BOC-Asu-0Bz1-DCHABOC-
Asu-0H26.

07 and 12.6ml of triethylamine in 20ml of DMF
Add 17.17 liters of benzyl bromide and react at room temperature for 12 hours.

Excess water is added to the reaction solution and the resulting oil is extracted with ethyl acetate. Wash the organic layer thoroughly with water and 5% sodium bicarbonate solution. The target product is transferred to the aqueous layer by extracting the organic layer three times with 5% sodium carbonate. The aqueous layer is acidified with 6N hydrochloric acid, and the resulting oil is extracted again with ethyl acetate, washed with water, and then dried over sodium sulfate. The ethyl acetate solution is replaced with ether, and DCHA is added dropwise under cooling to neutralize. Then, collect a large amount of the crystals formed and wash thoroughly with diethyl ether. Mix this with methanol
The target compound 10.0y (yield 20%) was obtained by recrystallization from diethyl ether. 3) BOC
-Ser(Bzl)-Asu-0Bz1・CHA production BOC-Asu-0Bz1-DCHA9. DCHA is removed by suspending O7 in ethyl acetate and separating with 1N sulfuric acid.

After washing the organic layer with water, it is dehydrated with sodium sulfate and concentrated to obtain an oily substance. This material is dissolved in 20 ml of TFA and treated at room temperature for 45 minutes. After distilling off excess TFA under reduced pressure, the resulting oil is dissolved in 20 ml of DMF and neutralized with triethylamine. BOC while cooling to 0℃
Add -Ser(Bzl)-0SU7.57 and HOBTl7.

The reaction is left stirring at 0° C. for 4 hours and at room temperature for 20 hours.

After adding 1 ml of dimethylpropanediamine to the reaction solution to inactivate the active ester, 200 ml of ethyl acetate was added.
and then wash the organic layer thoroughly in the following order: 1N hydrochloric acid, water, 5% aqueous sodium bicarbonate, and water.

The organic layer is dried over sodium sulfate, concentrated, and then dissolved in diethyl ether. Add CHA dropwise to the ether layer to neutralize. Collect a large amount of the formed crystals and wash with ether. By recrystallizing this product in a solvent system of ethyl acetate-n-hexane, the above target compound 6.8y (yield: 6
5%). CHA3.67 in ethyl acetate 100ml
CHA is removed by suspending the solution in water and separating the solution with 1N hydrochloric acid.

After washing the organic layer with water, it is dehydrated with sodium sulfate and concentrated to obtain an oily substance. This material is dissolved in 30 ml of TFA and treated at room temperature for 45 minutes. After distilling off excess TFA under reduced pressure, the resulting oil is dissolved in 20 ml of CMF and neutralized with triethylamine. BOCT while cooling to O℃
hr(Bzl)-0SU3,5y and HOBTO. Add 57.

The reaction solution is left stirring at 0° C. for 2 hours and at room temperature for 20 hours. After adding 1 ml of dimethylpropanediamine to the reaction solution to inactivate the excess active ester, 100 ml of ethyl acetate was added and the organic layer was washed in this order with 1N hydrochloric acid, water, 5% aqueous sodium bicarbonate, and water. The organic layer is dried over sodium sulfate, concentrated, and then dissolved in diethyl ether. C in the ether layer
Add HA dropwise to neutralize. Collect a lot of the formed crystals and wash with ether. This product was recrystallized in a solvent system of ethyl acetate-n-hexane to obtain the target compound 3.67 (yield: 87%). Comb to 200m1,
Leave at 0 to -5° C. for 2 hours and at room temperature for 18 hours with stirring.

After replacing methylene chloride in the reaction solution with ethyl acetate, 1
Wash sequentially with N-hydrochloric acid, 5% sodium bicarbonate, and water, and dehydrate with magnesium sulfate. By concentrating the organic layer and crystallizing it with n-hexane, the target compound 39.0y (yield 81%) was obtained.
) was obtained. Dissolve in TFA and treat at room temperature for 45 minutes.

After removing excess TFA under reduced pressure, n-hexane is added to precipitate the residue. The precipitate was collected and dried under reduced pressure in a desiccator over NaOH. This was dissolved in 30ml of DMF, neutralized with 4.2ml of triethylamine, and then heated to 0°C.
BOC-LysCCbz(0-Cl)]
-0NP247 is added and reacted at 0° C. for 1 hour and at room temperature for 72 hours. After inactivating the excess activated ester by adding 1 ml of dimethylpropanediamine, 2 ml of ethyl acetate was added.
00ml was added and washed sequentially with 1N hydrochloric acid, water, 5% sodium bicarbonate solution, and water, and dehydrated with magnesium sulfate. The organic layer is concentrated under reduced pressure and then crystallized from n-hexane. This product was recrystallized from a methanol-diethyl ether solvent system to obtain the target compound 17.07 (yield 73%). Ph
Dissolve e-0Et in 50 ml of TFA and treat at room temperature for 45 minutes. After distilling off excess TFA under reduced pressure, the residue was precipitated by adding ether. The precipitate was collected and dried under reduced pressure in a desiccator over NaOH. Dissolve this in 50ml of DMF and add 4.2ml of triethylamine.
After neutralization with m1, BOCTrp-
0NP16.67 and HOBTl7 are added and reacted.

The reaction was left stirring at 0° C. for 2 hours and at room temperature for 48 hours.
Add 200 ml of chloroform to the reaction solution, wash thoroughly in the order of saturated sodium carbonate, water, 1N hydrochloric acid, and water, and dehydrate with magnesium sulfate. After distilling off the organic solvent, the above target compound 287 (yield 96%) was obtained by crystallization using a solvent system of chloroform diethyl ether. Me0H(-
2:1) mixture to 20 ml, and reacted with 5 ml of hydrazine hydrate (80%) at room temperature for 5 hours.

The resulting precipitate is suspended in ethyl acetate, collected, and washed repeatedly with the same solvent. This product was recrystallized from a DMF-ethyl acetate solvent system to obtain the target compound 4.37 (yield: 86%). 0Bz1-CHA4.47 in ethyl acetate 100m
C by suspending it in e and separating it with 50ml of 1N hydrochloric acid.
Remove HA.

After washing the organic layer thoroughly with water and dehydrating it with sodium sulfate,
Concentrate under reduced pressure. Dissolve in 20ml of TFA and treat at room temperature for 45 minutes. Excess TFA was distilled off under reduced pressure, and the resulting oil was dissolved in 5 ml of DMF, neutralized with triethylamine, and cooled to -10°C to obtain HThr(Bzl)-Se.
A DMF solution of r(Bzl)-Asu-0Bz1 is obtained.

Dissolve in 5 ml of DMF, and add 6.2 ml of 4N hydrochloric acid/dioxane at -15°C or lower.

To this was added 0.84 ml of isoamyl nitrite with vigorous stirring at -15°C, and the mixture was allowed to react for 10 minutes. After adding the DMF solution obtained above and neutralizing with triethylamine, a condensation reaction is carried out at -10°C for 48 hours and at 5°C for 48 hours.゛Add a large amount of water to the reaction solution and collect the resulting precipitate. This precipitate is suspended in ethyl acetate and boiled to remove insoluble materials. This was mixed with chloroform-n-hexane,
Next, reprecipitation was performed in a solvent system of ethanol lys diethyl ether to obtain the target compound 5.1f (yield: 57.9
%) was obtained. Dissolve 22.9y of 0Et-HCl in 150ml of chloroform and add WSCl while cooling to -10°C.
Add 84ml.

The reaction is left stirring at -10° C. for 2 hours and then at room temperature for 15 hours. Next, it was concentrated under reduced pressure, the residue was dissolved in 200 ml of ethyl acetate, washed with 1N hydrochloric acid, water, 5% aqueous sodium bicarbonate, and water in that order, dried over sodium sulfate, and concentrated again under reduced pressure. A shaped precipitate was collected and reprecipitated with the same solvent system to obtain the target compound 377 (yield: 84%). After dissolving to 35 ml and treating for 35 minutes at room temperature, excess TFA is distilled off under reduced pressure.

Ether was added to the residue, the resulting precipitate was collected, and NaOH
Dry under reduced pressure in a soil desiccator. This thing is DMF2
After diluting to 5 ml and neutralizing with 2.8 ml of triethylamine, BOC-Asn-0NP10 was added while cooling to 0°C.
．． 67 and 1-hydroxybenzotriazole 1y are added.

The reaction was stirred at 0° C. for 2 hours and at room temperature for 20 hours. A large amount of water was added to the reaction mixture, the resulting precipitate was collected, washed with water and diethyl ether, and then crystallized from methanol-diethyl ether and then recrystallized from methanol alone to obtain the above-mentioned target compound (8.0y). A yield of 73% was obtained. TF
After dissolving in 25 ml of A and treating at room temperature for 35 minutes, excess TFA was distilled off under reduced pressure.

Add diethyl ether to the residue, collect the resulting precipitate,
Dry under reduced pressure in a desiccator over NaOH. DM this
After dissolving in 20ml of F and neutralizing with 1.4ml of triethylamine, BOCLysCCbz (0-
C1)]-0NP77 and 0.57 of 1-hydroxybenzotriazole are added. After reacting the reaction solution at 0°C for 2 hours and then at room temperature for 20 hours, a large amount of water was added to the reaction solution, the resulting precipitate was collected, washed well with water and diethyl ether, and crystallized with methanol to obtain the above. Target compound 7.0
7 (yield 86%) was obtained. Phe-Phe-0Et5.
67 was dissolved in a mixture of 20 ml of DMF and 30 ml of methanol, 6.6 ml of hydrazine hydrate (80%) was added, and the mixture was allowed to react at room temperature for 48 hours.

After the reaction, concentrate under reduced pressure, add a large amount of water to the residue, and collect a lot of the resulting precipitate. This product was reprecipitated from DMF and water to obtain the target compound 4.17 (yield 70%). Ser(B
zl)-Asu-0Bz14.87 is dissolved in 25 ml of TFA containing 1 ml of ethanedithiol and reacted for 45 minutes at room temperature.

After distilling off excess TFA, diethyl ether is added to the residue, and the resulting precipitate is collected, washed with diethyl ether, and then dried under reduced pressure in a desiccator over NaOH. D this
Dissolve in MFlOml and neutralize with 4.3ml of triethylamine. Add 0.8 ml of isoamyl nitrate with stirring.

This reaction solution was kept at -10°C for 10 minutes, then -30°C.
The mixture is cooled to , neutralized with triethylamine, added to the DMF solution obtained above, and subjected to a condensation reaction at 5° C. for 1 hour and at 5° C. for 48 hours. After the reaction, a large amount of water was added to this, the resulting precipitate was collected, washed with methanol, and reprecipitated from DMF methanol to obtain the target compound 5.57 (yield 79).
．． 3%). Dissolve in 50ml of dry pyridine, TF
A-0NP7.07 was added, and after stirring at 45°C for 3 hours, the pressure was reduced to distill off the pyridine, diethyl ether was added to the residue, and the resulting precipitate was collected.

After washing with 35 liters of diethyl ether, reprecipitation was performed from DMF-diethyl ether to obtain 5.7y of the above target compound (yield: 10
0%) was obtained. TFA3O containing 1ml ethanediol
ml and process for 25 minutes at room temperature.

Excess TFA was distilled off under reduced pressure, and the residue was dissolved in 30 ml of DMF, and this was added dropwise to pyridine 2,31 at 50° C. over 1 hour. After further stirring at the same temperature for 24 hours, the mixture was concentrated under reduced pressure, 0.5N hydrochloric acid was added to the residue, and the resulting precipitate was thoroughly washed with water and diethyl ether. D this thing
Reprecipitation was performed from MF-diethyl ether to obtain 4.0 t (yield 77.7%) of the above-mentioned target compound. Mound RrTT) Production of 0H The compound 3.57 obtained in 06) above, 6 ml of anisole and 0.7 V of methionine were dissolved in 60 ml of anhydrous hydrogen fluoride and treated at 0 to 2°C for 60 minutes to distill off excess hydrogen fluoride. The residue was washed with diethyl ether and dried in a desiccator over NaOH to give a pale yellow powder.

Dissolve this in 5% acetic acid, dowex 1 x 2 (3 x 1
5CTL1 (acetic acid type), and the eluate was lyophilized to obtain about 2.37 g of the acetate salt of the target compound. A 2M acetic acid solution of 2.0f7 of the powder obtained above was added to Cephadex LH-20.
The sample was injected onto a column (3.5 x 135C711) packed with 2M acetic acid and eluted with 2M acetic acid, and the active fraction of the eluate was lyophilized.

A 0.1M acetic acid solution of this powder was injected into a column (3.5 x 30 (:Tn)) packed with CM-cellulose, and
Linear concentration gradient elution from 1000 ml of M acetic acid aqueous solution to 1000 ml of 0.5 M ammonium acetate aqueous solution (PH 7.0) is performed, and the active fraction of the eluate is freeze-dried. A 2M acetic acid solution of this powder was injected into a column packed with Cephadex G25, eluted with 2M acetic acid, and the active fraction of the eluate was lyophilized to yield an active powder of the target compound 785 ~ (yield 34
%) was obtained.

This product can be used for electrophoresis (PH4.8, 1500V, 60 minutes), TLC [solvent system: n-butanol-acetic acid monowater (=
4:1:5, upper layer) Rf = 0.52, solvent system: n-
Butanol-acetic acid-water-pyridine (= 30:6:2
4:20) with a single spot at Rf = 0.82].

[α]2T5-41.6s (C = 0.37, 1%
Acetic acid) elemental analysis [C73H99Ol7Nl5・7H2
Amino acid analysis (as 0.3AC0H) (measured spectroscopically)

Claims (1)

[Claims] 1 Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, x^2, y and z have the same meanings as above)
A heptapeptide represented by is produced, and this heptapeptide has the formula ▲ mathematical formula, chemical formula, table, etc. ▼ (in the formula, x^1 and x^2 represent an amino protecting group,
indicates a protecting group for the hydroxyl group) and the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, y indicates the same meaning as above, and Z is After condensing the tripeptide represented by (representing a carboxyl group), the amino protecting group of the N-terminal amino acid is removed to form the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (where x^1 and x^2 are (same meaning as above)
By condensing the hydrazide of the tetrapeptide represented by
After undergoing active esterification of the ω-position carboxyl group of the C-terminal L-α-aminosuberic acid of this condensate and removal of the amino protecting group of the N-terminal L-lysine, the condensate is subjected to a cyclization reaction,
A method for producing a physiologically active peptide represented by the formula or a salt thereof, which comprises subsequently removing a protecting group.