JPS61197597A - Synthetic peptide - Google Patents

Abstract

NEW MATERIAL:The compound of formula I or its salt. USE:A remedy and diagnostic for hypertension. PREPARATION:For example, a chloromethylated resin is made to react with a C-terminated amino acid of formula BoC-Cys(MBzl)OH (Boc is t- butoxycarbonyl; MBzl is p-methoxybenzyl) having protected amino group and sulfhydryl group in the presence of tetramethyl-ammonium hydroxide, and the amino-protecting group is removed to obtain the compound of formula II (X1 is supporting resin; X1 is protecting group of sulfhydryl). The product is made to react successively with amino acids having protected amino group and side- chain functional group according to the amino acid sequence of the peptide in the presence of a condensation agent, and the amino-protecting group is eliminated. The procedure is repeated to obtain a peptidyl resin. The produced peptide is separated from the resin and all the protecting groups are eliminated to obtain the objective compound of formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Objective] The present invention relates to a novel synthetic peptide and its salts having diuretic, natriuretic, vasodilatory, and hypotensive effects.

Atrial natriuretic hormone (hereinafter abbreviated as ANF) (hereinafter abbreviated as ANF), which was isolated from rat atrium and has the following structure, is known as a peptide having diuretic effects.

N-terminus (Formula A, the side chain of Cys forms an intramolecular disulfide bond) (Inagami et al.

Biochem, Biophys, Res, Com
mun, , 119, 524-529゜(I984
) and idem, 1bid,, 123.338~3
44.

(I984)].

The present inventors conducted intensive research on the synthesis of derivatives with ANF-like action and their pharmacological activities, and found that ANF
The present invention was completed by discovering that a peptide having a unique structure that is much simpler than (2) and in which the carboxylic acid moiety is unprotected has an excellent ANF-like effect.

〔composition〕

The novel synthetic peptides and salts thereof of the present invention have the amino acid sequence of formula (I).

N-terminal C-terminal In the above formula (I) and the formula showing ANF■, Cys
Hasistine residue, Phe is phenylalanine residue,
Gly is a glycine residue, Arg is an arginine residue, Il
e is an inleucine residue, ABp is an aspartic acid residue,
Ala is an alanine residue, [)in is a glutamine residue, 8
er is a serine residue, L13u is a leucine residue, Asn is an asparagine residue, and Tyr is a tyrosine residue,
In accordance with the general notation, the N-terminal amino group is written on the left and the C-terminal carboxyl group on the right.

If the amino acid residue has isomers, 2 for each
Although species isomers exist, the compound (I) of the present invention can include all or mixtures of these isomeric combinations, preferably L- unless otherwise specified. It is a compound consisting only of type isomer amino acids.

However, the compound having the general formula (I) of the present invention can be converted into a pharmacologically acceptable salt.

Such salts include, for example, mineral salts such as hydrochlorides, sulfates, phosphates, organic salts such as oxalates, maleates, succinates, citrates, mimethanesulfonates, Acid addition salts such as benzenesulfone di salt, p-1 toluenesulfonate salt, etc.); alkali metal salts or alkaline earth metal salts such as IJium salts, potassium salts, calcium salts, and magnesium salts; Organic base salts such as dicyclohexylamine salts may be mentioned.

The ANFm derivative (I) of the present invention can be produced by the solid phase method described below.

-+ X -CH-0-Cys(X2)-Gly-x3
-) X, -CH2-0-Cys(X2)-Gly
-H (where Xl is a solid resin support and X2 is C
It is a protective group for the sulfhydryl group of ys, for example, an aralkyl group such as a benzyl group (hereinafter abbreviated as Bzl), a p-methoxypenzyl group (hereinafter abbreviated as MBzl), a trityl group, or an acetamidomethyl group. , preferably MBzl, and X3 is a protecting group for the α-amino group, such as benzyloxycarbonyl group (hereinafter abbreviated as 2), p-chlorobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, p -Nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, aralkyloxycarbonyl group, t-butyloxycarbonyl group (hereinafter abbreviated as Boc), t-amyloxycarbonyl group (hereinafter A
(abbreviated as OC), inbornyloxycarbonyl group,
an adamantyloxycarbonyl group or a 9-fluorenylmethyloxycarbonyl group, preferably Boc
shows. ] The α-amino group of Cys was protected with X3 and the sulfhydryl group, which is a side chain functional group, was protected with X2.
ro,et,al,,J,Am,Chem,Soc,,
95.

1310 (I973), tetramethylammonium hydroxide, cesium bicarbonate as a base in for example amides such as dimethylformamide, unahalogenated hydrocarbons such as methylene chloride or chloroform, preferably dimethylformamide. or using potassium bicarbonate, preferably tramethylammonium hydroxide, at room temperature to 90°C for 10 to 48°C.
Synthesis is initiated from the C-end of the peptide by coupling to a halogenated methyl resin such as chloromethyl resin over time.

First, the protecting group of the α-amine group of Cys coupled to the resin support is removed using an acid, for example, from 20 to 20% in methylene chloride.
55 trifluoroacetic acid (hereinafter abbreviated as TFA)
, using TFA alone or hydrochloric acid in dioxane, 06
~ It is released by reacting in the chamber for 10 minutes to 1 hour.

To this, Cy
Approximately a 3-fold excess of coupling is applied to the s residue. Examples of the coupling agent include N,N-dicyclohexylcarbodiimide (hereinafter abbreviated as DCC).

) or a carbodiimide such as N,N-diisopropylcarbodiimide, preferably using DCC, for example in a mixed solvent of methylene chloride-dimethylformamide or in methylene chloride at 0.6 to room temperature for 1 hour to
It is carried out by reacting for 3 days. The coupling reaction was completed by Kaiaer, st. al., Anal.

If the coupling is incomplete, as confirmed by the ninhydrin reaction described in Biochem, 34, 595 (I970), the same step is repeated before deprotection of the α-amine group of the amino acid. . The coupling reaction was performed on a Beckman 990B peptide synthesizer by Yamaghiro, at, al.
J.

Am, Chem, Soc, 95, 1310 (I9
This can be done automatically using the program described in 73).

In this way, the steps of deprotection of the α-amino group of the terminal amino acid attached to the resin support and subsequent coupling with the preferred protected amino acid are performed sequentially on the protected Leua GIY 5Ser, Gin,
Ala, Gly, Ile, Arg, As
pjIIs, Arga Gly, Gly
m Phe and Cys in repeated steps.

As the amino acid having a functional group on the removed side chain, in addition to the α-amine group, a compound in which this functional group is protected is used. For example, the side chain functional groups of Arg are X4 group, and Asp has X group and 8
er is protected with an X6 group.

Here, X4 may be a protecting group for a guanidino group, such as Evanitro M, p-)luenesulfonyl group (hereinafter TO8
), 2 groups, an adamantyloxycarbonyl group or an inbornyloxycarbonyl group, preferably a p-toluenesulfonyl group. x5 may be any ester-forming protecting group for a side chain carboxyl group, such as a benzyl group or methyl, ethyl or t-
It is an alkyl group such as butyl, preferably a benzyl group.

X6 may be any protecting group for a side chain hydroxyl group, such as an acetyl group, a benzoyl group, a t-butic group, a tetrahydrobilanyl group, or a benzyl group, preferably a benzyl group.

Alternatively, several amino acids can be coupled in advance by the solid-phase method or liquid-phase method to synthesize short peptide fragments, which can be used as raw materials for the solid-phase method. In this case, coupling at the C-terminus of Gly allows synthesis without amino acid isomerization, so such a peptide fragment is preferably X3-Leu -Gly-OH X -Ala -Gin-8sr (x6) - Gly
-OHX -Arg(X)-11e-Asp(X
)-Arg(X4)-11e-()ly-ORX-C
A coupling reaction is carried out by a solid phase method using ys(X2)-Phs-GAY-OH.

By performing the above operations, an intermediate having formula (I) can be obtained.

X↓-Cys (X 2)-Ph e-Gly-Gly
-Ar g (X 4)-41e-Asp (X5)-
Arg(X)-11a-Gly-Ala-Gin-
8er(X6)-Gly-Leu-Gly-Cys (
x2)-X, ([) (where xl, x2
．． x4. X5o and x6 have the same meanings as above, and x
3 is defined as the above definition of x3 plus a hydrogen atom. ) A compound ([) in which x3 represents a hydrogen atom can be produced by removing the protecting group x3 as described above.

Compound (II) is not only useful as an important intermediate for compound (I) of the present application, but also as a synthetic intermediate for other long-chain peptides (eg, ANFff, etc.).

The compound ([) can be prepared by treating it with a strong acid such as liquid hydrogen fluoride for 30 minutes to 4 hours at room temperature.
Removal of all protecting groups and cleavage from the resin support is performed. In the case of 1F, 1=, it is preferable to contain anisole as a carbonium cation scavenger in the reaction vessel.

In the intermediate having the formula ([) produced in this way, the synthetic peptide having neither a protecting group nor a resin support has two sulfhydryl groups in the molecule, so the compound of the present invention ( In order to synthesize I), it must be immediately oxidized by a high dilution method to form a disulfide compound. As the solubilizing agent, for example, air, potassium ferricyanide or hydrogen peroxide solution is used, and potassium ferricyanide is preferably used.

al,, J, Med, Chsm, 19.1010 (I
Intramolecular disulfide bonds are formed by reacting at O@~room temperature for 12 hours~2 days according to the method described in 976).

〔effect〕

The object compound (I) of the present invention has natriuretic, diuretic, vasodilatory, and hypotensive effects, and is useful as a therapeutic agent and diagnostic agent for hypertension.

The administration form includes oral administration or injection in the form of tablets, capsules, granules, powders, syrups, etc.
Examples include parenteral administration using suppositories and the like. The amount used varies depending on the purpose of use, symptoms, age, etc., but for example, one mouthful is approximately
It is the body weight and can be administered once or in divided doses.

Next, an example of synthesizing a peptide having ANF activity of the present invention using a solid phase method will be given and explained in more detail.

Examples Peptide Boc-Cys (MBzl)OH2,74f (8
mmol) f, bathed in methanol 8 mA', 7.3
The mixture was concentrated under reduced pressure together with a methanol solution of 10% tetramethylammonium hydroxide (4d), a small amount of toluene was added thereto, and the mixture was azeotropically distilled several times to thoroughly dehydrate. The residue was dissolved in dimethylformamide 40M and 4.76 f.
Chloromethyl resin (purchased from Peptide Institute Co., Ltd.)
75-8 by adding chlorine content 0.6 mmol/t)
Stirred at 0°C for 10.5 hours. The resin was collected on a glass filter and washed with dimethylformamide, methanol, water, and methanol in this order.1. Vacuum dry in a desiccator containing phosphorus pentoxide, and remove resin 1? 0.49 mmol of cya per 5.83 f
I got it.

The stepwise synthesis was carried out on a Peckman 990B peptide synthesizer using the following program. Naori O(
! -Cya (MBzl) binding resin has 1.52 (however, the groups described above for each amino acid represent a protecting group for an α-amine group, and the group written in parentheses represents a protecting group for a side chain functional group. ) to carry out the coupling reaction step by step.

At the end of the above operation, the following formula %formula% An intermediate compound represented by 2.71f was obtained.

When this intermediate was hydrolyzed with 5.7N hydrochloric acid in a sealed glass tube at 110°C for 20 hours,
The amino acid analysis value is as follows: 1.51 of this intermediate and 2.31 nl of anisole! and about 25 d of hydrogen fluoride, 0
The reaction mixture was allowed to react for 1 hour at 0.degree. C., and the hydrogen fluoride was evaporated under high vacuum, washed with 100 g of ethyl acetate, extracted with 2007 IL of a degassed 5% acetic acid aqueous solution, and separated from the resin by filtration. Next, this extract was diluted to about 10% with degassed distilled water, concentrated aqueous ammonia was added, the pH was adjusted to 7.5, and the mixture was stirred at room temperature. Add a01M potassium ferricyanide aqueous solution to this aqueous solution until the yellow color no longer disappears,
The mixture was stirred at room temperature for 15 hours. After the reaction, acetic acid was added to adjust the pH to 5.0, and anion exchange resin AG3-X4A (200
-400 mesh, chloride type) 102 was added and stirred for 30 minutes. The resin was removed by filtration, the P solution was subjected to IM compression under reduced pressure, and the residue was freeze-dried. The obtained residue 0.47f was separated from Sephadex ()-25, fine column (2.5X95
cm), eluted with a 10% aqueous acetic acid solution, and the fraction containing the target compound was lyophilized to obtain 154 peptides having crude ANF activity.

This crude peptide 10211f'e was purified twice by reverse phase high performance liquid chromatography to obtain pure compound (I) of the present invention 341R9. The column used was Chemcopack Nucleosil 5C18 (2'φX 250
M), containing 0.1% TFA until 20 minutes to 02
0% acetonitrile aqueous solution was flowed at 5d per minute, 20 to 10
Until 0 minutes, only the content of acetonate was gradually changed from 20% to 40% using a concentration gradient method. However, at this time, TFA was adjusted so that it always contained 11%.

, +

Claims (1)

[Claims] A synthetic peptide having the amino acid sequence of formula (I) and its salt ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I)