JPS5846503B2 - Novel hepta- and octapeptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel hepta- and octapeptides.

The hebuta- and octapeptides of the present invention are represented by the general formula (wherein R represents a hydrogen atom, an L-aspartyl group, or a sarcosyl group).

The compound of formula I is a novel compound and is useful as an angiotensin antagonistic drug.

In order to produce the compound of formula (■), ordinary peptide synthesis methods can be applied, such as N , N'-dicyclohexylcarbodiimide method, carbonyldiimidazole method, acid chloride method, azide method, mixed acid anhydride method, active ester method,
A liquid phase method such as an isocyanate method or a phosphazo method, a solid phase method, etc. can be used.

Compounds of formula I can be prepared, for example, by protecting L with a carboxyl group.
-α-Methyl-(3,4-dibenzyloxy)-phenylalanine is reacted with L-proline with a protected amine group to form a peptide bond, then the protecting group of the amine group is removed, and then L-proline with a protected amine group is reacted. - Can be produced by peptide bonding histidine, L-isoleucine, L-tyrosine, L-valine, and L-arginine, or by peptide bonding each of these amino acids to form a dipeptide, tripeptide, etc., and further bonding these. .

In these peptide synthesis methods, it is generally advantageous to protect the carboxyl groups of amino acids or peptides that do not participate in the reaction by esterification.

Preferred examples of these esters include lower alkyl esters such as methyl ester, ethyl ester, flobyl ester, and t-7' methyl ester, and aralkyl esters such as benzyl ester, p-nitrobenzyl ester, and p-methoxybenzyl ester.

Furthermore, amino groups that do not participate in the reaction can be protected with a common protecting group in peptide synthesis.

Examples of heat-retaining groups for amino groups include aralkyloxycarbonyl groups, benzyloxycarbonyl groups,
Alkyloxycarbonyl groups such as p-nitrobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-phenylazobenzyloxycarbonyl group, etc.
A butoxycarbonyl group, a t-amyloxycarbonyl group, etc., a trityl group, a formyl group, a trifluoroacetyl group, an o-nitrophenylsulfenyl group, etc. are used.

It is also preferable to protect the hydroxyl group, imidazole residue, guanidino group, etc. of the amino acid or peptide with an appropriate protecting group.

The protecting group can be removed from hepta- and octapeptides having a protecting group by a commonly used method such as acid treatment, catalytic reduction, liquid ammonia-sodium reduction, hydrolysis, and the like.

Examples of solvents in the liquid phase method include chloroform,
Methylene chloride, tetrahydrofuran, ether, ethyl acetate, acetonitrile, dioxane, dimethylformamide, dimethyl sulfoxide, hexamethylphosphoryltriamide, methanol, ethanol, butanol, acetic acid, and the like are appropriately used.

The reaction time varies depending on the peptide synthesis method used and each step, but is generally 30 minutes or more.

The desired hepta- and octapeptide (I) can be produced by appropriately selecting the reaction conditions for the introduction of the protecting group, condensation reaction, and removal of the protecting group.

A preferred method for producing the present compound is, for example, as follows.

First, L-α-methyl-(3,4-dibenzyloxy)-
Nt- to phenylalanine benzyl ester (II)
Butoxycarbonyl-L-florin (2) is reacted in a solvent with cooling and stirring to produce N-t-butoxycarbonyl-L-prolyl-L-α-methyl-(3,4-dibenzyloxy)-phenylalanine. Benzyl ester (
1) Manufacture.

Next, compound (1) was hydrolyzed to remove the benzyl ester group, and then mixed and stirred with chloromethyl resin in a solvent, and further heated and stirred to form N-1-phthoxycarbonyl-L-prolyl-L-α-methyl- (3,4-dibenzyloxy)-phenylalanyl resin ester (7) is obtained.

The t-butoxycarbonyl group, which is the protective group of the resulting resin ester (■), is removed by treatment with trifluoroacetic acid in a solvent.

Next, an amino acid with a protected amine group is applied to this to form a peptide bond, and then the amine protecting group is removed by the same treatment to sequentially prepare L-histidine, L-isoleucine, L-tyrosine, L-valine and L-valine. - When the protecting group is removed by the action of arginine, the heptapeptide resin ester (2) is obtained.

By treating the obtained heptapeptide resin ester (b) with hydrogen fluoride, the desired heptapeptide (
IKR=H) is obtained.

Furthermore, when L-aspartic acid or sarcosine is peptide-bonded to the heptapeptide resin ester (b) in the same manner as above, and then released from the resin, the target octapeptide (I) (R-aspartyl group or sarcosyl group) is obtained. can get.

When the amino acid used in the reaction has a hydroxyl group, a guanidino group, an imidazole residue, etc., it is preferable that these groups are also protected with an appropriate protecting group.

The above reaction can be expressed as follows.

In the formula, R1 and R2 are amino group-protecting groups, R3 is a hydroxyl group-protecting group, and P is a resin polymerization moiety.

Isolation and purification of the obtained hepta- and octapeptides are carried out by methods commonly used in peptide chemistry, preferably gel filtration or ion exchange column chromatography.

The desired peptide (2) can be obtained as a free acid or an acid addition salt.

L-α-methyl-(3,4-
(dibenzyloxy)-phenylalanine benzyl ester is a new compound and can be produced from L-α-methylphenylalanine (see Japanese Patent Application No. 51-60588).

Example I L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-pro9JL/-I,
-α-methyl-3,4-dihydroxyphenylalanine.

N-t-butoxycarbonyl-L-proline 2.471
i' and 1.601 rIl of triethylamine are mixed and dissolved in ethyl acetate (55), and 1.49 (1.49) of inbutyl chloroformate is added while stirring and cooling at 0 to -5°C to form a jelly.

To this, L-α-methyl-(3,4-dibenzyloxy)
-Phenylalanine benzyl ester paratoluene sulfonate5. of and triethylamine 1.07rr
Add a solution prepared by mixing and dissolving 1 ml of ethyl acetate and stir.

The mixture was further stirred for 1 hour under water cooling, then stirred for 1 day and night at room temperature, and then 201 rll of water was added and stirred for 1 hour.

The reaction solution was washed successively with IN hydrochloric acid, 4φ sodium bicarbonate solution, and saturated brine, and then dried over anhydrous sodium sulfate.

After distilling off the solvent under reduced pressure, elution was carried out with chloroform using a silica gel column, and the solvent was distilled off from the eluate.
Oily N-t-butoxycarbonyl-L-prolyl-L
-α-Methyl-(3,4-dibenzyloxy)-phenylalanine benzyl ester 4.96 y (yield 95
．． 6%) is obtained.

Elemental analysis value: CHN as C41H46N207 Calculated value (to) 72.546.834.13 Experimental value φ 7
2.486.954.10 Obtained N-1-butoxycarbonyl-L-prolyl-
L-d-methyl-(3,4-dibenzyloxy)-phenylalanine benzyl ester z842 in methanol 7
0m1! Dissolved in IN aqueous sodium hydroxide solution 36.
Add 8 ml and stir at room temperature for 6 hours.

After concentrating the reaction solution and washing with ether, the aqueous layer was adjusted to pH 2-3 with IN hydrochloric acid, and extracted with 300 ml of ethyl acetate.

The ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off to give N as a colorless powder.
-butoxycarbonyl-L-prolyl-L-α-methyl-(3,4-dibenzyloxy)-phenylalanine monohydrate 14.7S' (yield: 92.9 yen) is obtained.

Elemental analysis value: C34H4, C as N207/H2O
HN Calculated value (to) 67.306.814.62 Experimental value (to)
67.276.874.74 Product obtained 7.58
r and triethylamine 1.751 rll, chloromethyl resin 5.0? , 15r111 of ethanol and 7.5ml of chloroform, and stirred gently at room temperature for 1 hour.

After further heating at 65°C for 24 hours, the reaction mixture was washed successively with ethanol, acetic acid, water and methanol, dried under reduced pressure at room temperature, and Nt-butoxycarbonyl-L-
Prolyl-L-α-methyl(3,4-dibenzyloxy)-phenylalanyl resin ester 6.45S' is obtained.

L-prolyl-L-α-methyl- in this resin ester
The (3,4-dibenzyloxy)-phenylalanine content is 0.39 mmolfy.

The obtained resin ester 4.805' was washed three times with 501 rll of methylene chloride, pre-washed with 40% trifluoroacetic acid-methylene chloride solution, and further washed with 40 φ trifluoroacetic acid-methylene chloride solution 50
rnl for 30 minutes to remove the t-butoxycarbonyl group.

Then methylene chloride 50r111. After sequentially washing three times with 50 ml of t-butyl alcohol and 50 d of methylene chloride, the mixture was stirred twice for 2 minutes each time with a 50-ml pot of 5% triethylamine-methylene chloride solution to neutralize.

Methylene chloride 50- and t-butyl alcohol 50
- When washed in the same manner as above, L-prolyl-L
-α-methyl-(3,4-dibenzyloxy)-phenylalanyl resin ester is obtained.

To this resin ester, N-t-butoxycarbonyl-(
A solution of Nim-tosyl)-L-histidine 2,301 in methylene chloride 40r111 was added and stirred to give 15
After a minute, a solution of 1.24 S' of N,N'-dicyclohexylcarbodiimide in 10 rrll of methylene chloride is added and stirred for 2 hours at room temperature.

After completion of the reaction, 30 mt of methylene chloride, 50 mt of butyl alcohol and 50 mt of methylene chloride were added in sequence.
After washing several times, N-t-butoxycarbonyl-(N
im), syl)-L-histidyl-L-prolyl-
L-α-methyl-(3,4-dibenzyloxy)-phenylalanyl resin ester is obtained.

Similarly, N-t-butoxycarbonyl-L-isoleucine, N-t-butoxycarbonyl-(0-2-
Brombenzyloxycarbonyl)-L-tyrosine, N
-1-Butoxycarbonyl-L-valine and Nt-amyloxycarbonyl-(NG-1sil)-L-arginine were used to sequentially remove the protecting group, wash, neutralize, wash, and condensate reaction. Afterwards, when dried under reduced pressure at room temperature, N-
-amyloxycarbonyl-(NG-1sil)-L-
Arginyl-L-valyl-(O-2-brombenzyloxycarbonyl)-L-tyrosyl-2-ynleucyl-
(Nim-tosyl)-L-histidyl-L-prolyl-
L-α-methyl-(3,4-dibenzyloxy)-phenylalanyl resin ester 7,10P is obtained.

The completion of the condensation reaction was examined by a ninhydrin color reaction test.

Obtained heptapeptide resin ester with protecting group 1
．． Add anisole 5- to 78@ and leave at room temperature for 1 hour.

Next, 50 ml of hydrogen fluoride was introduced, and after stirring for 1 hour while cooling with water, the hydrogen fluoride was removed at 0° C. under reduced pressure.

The residue was extracted with a monovalent acetic acid aqueous solution and passed through a DOWEX 1×2 acetate (manufactured by Dow Chemical Company, strong basic ion exchange resin) column to completely remove hydrogen fluoride.
The desired hebutapeptide acetate is obtained.

This was purified by gel filtration using Biogel P-2 (Bio-Rad Labora, manufactured by IJ- Inc.) using 2N acetic acid as an elution solvent, the fraction containing the target substance was lyophilized, and this was further added to a pyridine-acetate buffer. When the liquid (PH5,40) is purified using a column of carboxymethyl cellulose (manufactured by Seikagaku Corporation) as an elution solvent, L is obtained as a colorless powder.
-Arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-7'lolyl-L-α-methyl-3,4-dihydroxyphenylalanine diacetate 215 mlII is obtained.

The physical constants of this heptapeptide are as follows.

Specific optical rotation: [α)2f3=-40,4° (c=0.76
, IN acetic acid) Silica gel thin layer chromatography: Rf = 0.16 (n-butanol/acetic acid/water = 4:1: 5) Rf = 0.38 (n-butanol/acetic acid/pyridine/water = 4:1: 1:2) Example 2 N-1-amyloxycarbonyl- obtained in Example 1
(NG-)sil)-L-arginyl-L-halyl(0
-2-Brombenzyloxycarbonyl)-L-tyrosyl-L-isoleucyl-(Nim-tosyl)-L-histidyl-L-prolyl-L-α-methyl-(3,4-dibenzyloxy)-phenylalanyl Resin ester 3.
55F was treated in the same manner as in Example 1, removing the protective group, washing,
After neutralization and washing, a solution of 0.91 Nt-butoxycarbonyl-β-benzyl-L-aspartate in 25 methylene chloride is added and stirred.

Then N,N'-dicyclohexylcarbodiimide 0162? After the completion of the reaction, the mixture was washed three times with 30ml of methylene chloride, 20ml of methylene chloride, and 20rnl of methylene chloride, and then stirred at room temperature for 2 hours to condense. When dried under reduced pressure, N-1-butoxycarbonyl-β-benzyl-L-aspartyl-(NG-)yl)-ri-valyl(0-2-brombenzyloxycarbonyl)
-L-tyrosyl-L-isoleucyl-(Nim)-L-histidyl-L-prolyl-L-α-methyl-(3,4-dibenzyloxy)-phenylalanyl resin ester 3.89? is obtained.

After treatment with hydrogen fluoride and purification in the same manner as in Example 1, L-aspartyl-L-
Alkynyl L-valyl L40yl-L-isoleucyl-L-histidyl-L-prolyl-L-α-methyl-
3,4-dihydroxyphenylalanine acetate 485■
is obtained.

The physical constants of this octapeptide are as follows.

Specific optical rotation: [α) 20 = -67, 4° (c = 0.
5, IN acetic acid) Silica gel thin layer chromatography: Rf = 0.13 (n-butanol/acetic acid/water = 4:1: 5) Rf = 0.27 (n-butanol/acetic acid/pyridine/water = 4:1 :1:2) Example 3 N-1-amyloxycarbonyl- obtained in Example 1
(NG-tosyl)-L-arginyl-L-valyl-(0
-2-Brombenzyloxycarbonyl)-L-tyrosyl-L-isoleucyl-(Nim-tosyl)-L-histidyl-L-prolyl-L-α-methyl-(3,4-dibenzyloxy)-phenylalanyl Resin ester 1.
78S' was condensed with N-benzyloxycarbonyl-sarcosine in the same manner as in Example 2, and the following Example 1
After treatment with hydrogen fluoride and purification in the same manner as above, sarcosyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L--j'lolyl-L-α-methyl is obtained as a colorless powder. 252 ml of -3,4-dihydroxyphenylalanine acetate is obtained.

The physical constants of this octapeptide are as follows.

Specific optical rotation: [α) 20 = -70,3° (c = 1.06
, IN acetic acid) Silica gel thin layer chromatography: Rf value = 0.12 (n-butanol/acetic acid/water = 4: 1:5) Rf value = 0.30 (n-butanol/acetic acid/pyridine 7/water = 4 :1:1:2)

Claims (1)

[Scope of Claims] 1 Hepta- and octapeptides represented by the general formula (wherein R represents a hydrogen atom, an L-aspartyl group, or a sarcosyl group).