JPH0597891A - Vasohypotonic peptides - Google Patents

Abstract

PURPOSE:To provide the subject new compound useful for treating and preventing hypertension, cardiac infarction, angina pectoris and cerebral infarction as a vasohypotonic agent exhibiting a strong vasohypotonic action because of selectively activating the ETB receptor of endothelin. CONSTITUTION:A peptide of formula I [X is H, acyl, etc.; X<2> is Glu, D-Glu; X<3> is Val, Ala; X<4> is His, Ala; X<5> is Lle, Ala] or its salt, e.g. a compound of formula II. The compound is obtained by covalently bonding an a-amino group-protected C-terminal amino acid to an insoluble carrier such as a chloromethyl resin by a conventional solid-phase process removing the alpha-amino- protecting group, successively polycondensing amino acids with the C-terminal amino acid in the order of the sequence and subsequently separating the objective peptide derivative from the carrier resin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to physiologically active peptides having a strong vasorelaxant activity and their uses.

Of the endothelin receptors, the compound of the present invention is on vascular smooth muscle, does not act on the receptor subtype (ET _A ) involved in vasoconstriction, is on vascular endothelium, and is effective for vascular relaxation. It has potent vasorelaxant activity by acting on the involved receptor subtypes (ET _B ), and in the field of medicine, especially hypertension, myocardial infarction, angina, acute renal failure,
It can be used as a therapeutic agent for cerebral infarction and cerebral vasospasm.

[0003]

BACKGROUND OF THE INVENTION Endothelin is a polypeptide consisting of 21 amino acids. It is produced by human and porcine endothelial cells and has a strong vasoconstrictor action. , Has a continuous and strong pressor action. It is known that endothelin has three types of endothelins (endothelin-1, endothelin-2, endothelin-3) as family peptides having similar structures in animals including humans [Nature ( Nature), 332 , 411-415 (198).
8), Febs Letters (FEBS Letter)
s), 231 , 440-444 (1988), Biochemical and Biophysical Research Communications (Biochem. Biophys.
Res. Commun. ), 154 , 868-875
(1988), ibid., 164 , 74-80 (1989) and the Proceeding National Academy of.
Science (Proc. Natl. Acad. S
ci. USA), 86 , 2863-2867 (198).
9)]].

Furthermore, endothelin-1 has an endothelium-dependent vasorelaxant activity in addition to a vasoconstrictor activity, and it is known that vasoconstrictor and relaxant actions are caused by activating different endothelin receptor subtypes. ing. Endothelin-induced vasoconstriction is mainly caused by activation of endothelin receptor (ET _A ) on vascular smooth muscle,
The activity of ET-1 is higher than that of ET-3.
On the other hand, vascular relaxation is due to endothelin receptor (E
The activation of T _B ) produces a vasorelaxant (EDRF), which acts on vascular smooth muscle to cause vasorelaxation, and this activity is equivalent in endothelin-1 and endothelin-3 [European Journal of・ Pharmacology (Eur. J. Pharmacol.), 159 , 3
25-326 (1989), Japanese Journal of Pharmacology Supplement First (Jap. J. Pharmacol.), 55 (sup).
pl. I), 185 (1991)]. Endothelin receptor subtypes with different selectivity among these endothelin receptors have different tissue distributions, with many ET _A receptors in the cardiovascular system excluding vascular endothelium and ET _B receptors in vascular endothelium and other tissues. It has been shown that there are many bodies and the respective receptor proteins are also different [Japanese Journal of Pharmacology Supplement First (Japan Pharmaco.), 52 .
(Suppl.I), 203 (1990), Nature, 348 , 730-735 (199).
0)]. In addition, since renal proximal tubule-derived LLC-PK ₁ cells have ET _B receptors and promote cyclic GMP production by endothelin, activation of ET _B receptors also has a diuretic effect. Is suggested [Japanese Journal of Pharmacology Supplement First (J.P.Pharmac)
ol), 55 (suppl.I), 70 (199)
1)].

Therefore, a substance which selectively activates only the ET _B receptor exhibits vasorelaxant activity and renal diuretic action, and is considered to be useful as a pharmaceutical.

[0006]

[Problems to be Solved by the Invention] ET _{B of} endothelin
It is to create a novel peptide having a vasorelaxant effect by selectively activating a receptor.

[0007]

SUMMARY OF THE INVENTION The present inventors have synthesized a fragment of endothelin by the general formula X ¹ -X ² -Ala-X ³ -Tyr-Phe-Ala-X ⁴
-Leu-Asp-X ⁵ -Ile-Trp [I] [In the formula, X ¹ represents a hydrogen atom, an acyl group, or any amino acid residue in which the amino group may have an acyl group, or any 2
Represents a peptide residue which may have an acyl group at the N-terminal having an arbitrary sequence consisting of ~ 4 amino acids, X
² represents Glu or D-Glu, X ³ represents Val or Al
a, X ⁴ represents His or Ala, X ⁵ represents Ile
Or Ala]] or a pharmaceutically acceptable salt thereof has a vasorelaxant action,
The present invention has been completed.

[0008] The endothelin receptor affinity of the compound according to the present invention is described in the following items of the inhibition test for endothelin-1 binding in porcine arterial smooth muscle and cerebellar membrane fraction and the inhibition test for norepinephrine contraction in porcine isolated pulmonary artery preparation. It was measured by the method described in.

The compound according to the present invention has a strong vasorelaxant action as shown in the following vasoconstriction inhibition test. Therefore, the present invention is applicable to hypertension, acute renal failure, myocardial infarction, angina, cerebral infarction, brain. It is expected as a therapeutic and / or preventive agent for vasospasm.

In the present specification, any amino acid or any amino acid residue means alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, Serine, threonine, tryptophan, tyrosine,
Means valine or its residue, and the acyl group is an acetyl group, a propanoyl group, an isopropanoyl group, a butanoyl group, an isobutanoyl group, a tert-butanoyl group, a pentanoyl group or an isopentanoyl group having 2 to 5 carbon atoms. And an aromatic acyl group such as a benzoyl group and a naphthoyl group, and the meanings of various abbreviations described in the present specification are as follows. Meanings of the abbreviations Abbreviations Ala L-alanine Asp L-aspartic acid Glu L-glutamic acid His L-histidine Ile L-isoleucine Leu L-leucine Lys L-Lysine Met L-Methionine Phe L-Phenylalanine Trp L-Tryptophan Tyr L-tyrosine Val L-valine Boc tert-butoxycarbonyl ^t Bu tert-butyl Fmoc 9-fluorenylmethoxycarbonyl Trt trityl Tos p-toluenesulfonyl TFA trifluoroacetic acid DCC N, N′-dicyclohexylcarbodiimide DIPC N, N′-diisopropylcarbodiimide HOBT. H ₂ O 1-hydroxy -1H- benzotriazole-monohydrate and Z benzyloxycarbonyl MOPS 3- morpholino propane Acid HEPES 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid Tris tris (hydroxymethyl) aminomethane PMSF phenylmethanesulfonyl = fluoride Next, a method for producing the novel peptide of the present invention will be described. To do.

The compound of the present invention can be produced by carrying out a condensation reaction of amino acids by a usual solid phase method or liquid phase method in the field of peptide chemistry. (A) Solid Phase Method The target peptide derivative is a commercially available peptide synthesizer, for example, Model 431A manufactured by Applied Biosystems, and chloromethyl resin [Biochemistry, 3 , 1385].
(1964)], oxymethyl resin [Chemistry and Industry (Chem. Ind.
on)), 1966 , 1597], p-alkoxybenzyl alcohol resin [Journal of the American Chemical Society (J. Am. Chem. So.
c. ), 95 , 1328 (1973)], functionalized polyamide resin [Bioorganic Chemistry, 8 ,
351-370 (1979)] and the like, and the condensation reaction is successively carried out on an insoluble carrier. First, the α-amino group of the selected amino acid is protected as the C-terminal amino acid in the peptide derivative, and if the side chain has a reactive functional group, the side chain functional group is also protected, and then according to a known method. Covalently bound to an insoluble carrier in the form of a carboxylic ester. Then, after removing the α-amino protecting group, the amino-protected derivative having the following sequence order (which also protects the side chain functional group if necessary) is condensed with a condensing agent such as DCC or DIPC and HOBT · H ₂ if necessary. It is added at the same time as an additive such as O and condensed. This amino protected derivative may be used in the form of a carboxy activated amino acid such as pentafluorophenyl ester, acid azide and the like. Such deprotection and condensation are repeated to obtain the desired peptide derivative. Amino protecting groups are usually well known in the art,
For example, it is selected from urethane type protecting groups such as Z group, Boc group, Fmoc group, p-methoxybenzyloxycarbonyl group and p-nitrobenzyloxycarbonyl group. Regarding protection of α-amino group, Fmoc group or Bo
The use of group c is preferred. The Fmoc group can be easily removed after the condensation reaction by the action of a relatively mild base, for example, a piperidine / N-methylpyrrolidone solution, a piperidine / DMF solution, or the like, while the Boc group is a relatively mild acid. It can be easily removed by action, eg TFA.

When the Fmoc group is used for protecting the α-amino group, the carboxy group of aspartic acid is tert.
-Butyl ester or trityl ester, serine, isoserine and tyrosine hydroxyl groups as tert-butyl ether, and histidine imidazolyl group as Tr
It may be protected with a t group. Since these protecting groups are stable under the condition of removing Fmoc group, all the protecting groups of the peptide derivative cleaved from the resin after completion of the peptide chain extension are treated with TFA.
It can be removed at the same time by treating with a mild acid such as. On the other hand, when the Boc group is used to protect the α-amino group, the hydroxyl groups of serine and tyrosine are used as benzyl ether, and the imidazolyl group of histidine is used as Tos.
The indolyl group of tryptophan may be protected with a formyl group. Since these protecting groups are stable under the conditions for removing the Boc group, after completion of the peptide chain extension, all the protecting groups of the peptide derivative cleaved from the resin are subjected to catalytic hydrogenolysis treatment, hydrogen fluoride treatment, trimethylsilyl trifluoromethanesulfonate / Thioanisole / TFA treatment [Chemical and Pharmaceutical Bulletin (Ch
em. Pharm. Bull. ), 35 , 3447-5
2 (1987)] and the like.

Removal of the peptide derivative from the resin after completion of the peptide chain extension can be carried out by various methods well known to those skilled in the art. For example, when P-alkoxybenzyl alcohol resin is used as the resin, TFA
A peptide derivative having a carboxy group at the C-terminal can be obtained by the action of a mild acid such as, and preferably a TFA / m-cresol / ethanedithiol solution.

The carrier resin can be removed from the reaction mixture by suspending the mixture in a solvent capable of dissolving the peptide component and then filtering the resin, or by separating the resin and the peptide component from the reagents by once precipitating the peptide. Only the components may be dissolved in acetic acid or the like and the resin may be filtered off. In order to isolate and purify the compound of the present invention after removing the resin, after concentrating the solution, the residue is subjected to a method known per se (for example, recrystallization, reprecipitation, partitioning operation, normal phase or reverse phase chromatography or Ion exchange chromatography). (B) Liquid Phase Method The peptide of the present invention can also be produced by a liquid phase method of general peptide derivatives. That is, by condensing amino acids one by one, condensing condensates composed of a plurality of amino acids, or a combination thereof, further protecting the C-terminal and / or side chain of the peptide after the condensation reaction, if necessary. It can be produced by removing the group by alkali hydrolysis or catalytic hydrogenolysis. The condensation reaction is D
Known methods such as the CC method, the azide method, the active ester method, and the mixed acid anhydride method [for example, M. Bodanski (M.
anky) and M. Ondetti (MA).
Ondetti, Peptide Synthesis, Interscience, New York (Peptide Synt)
hesis, Interscience, NewYor
k) 1966; FM Finn (FM Fi)
nn) and K. Hofmann.
The Proteins, Volume 2, Edited by H. Nenrath and RL Hill, Academic Press Inc., New York (Academic Press Inc.). New
York) 1976; Nobuo Izumiya et al., Peptide synthesis,
Maruzen Co., Ltd., 1975, etc.].

The α-amino protecting group is usually well known to those skilled in the art, and examples thereof include urethane type protecting groups such as Z group, Boc group, p-methoxybenzyloxycarbonyl group and p-nitrobenzyloxycarbonyl group. To be selected. On the other hand, the α-carboxy protecting group is selected from methyl, ethyl, benzyl, tert-butyl ester and the like, and is selected according to the N-terminal protecting group so that each protecting group after condensation can be selectively removed. Should. For example, when the N-terminal protecting group is a Boc group, the C-terminal side is methyl,
It is preferably protected as an ethyl or benzyl ester. The Boc group is easily removed by the action of relatively mild acids such as TFA, but under these conditions these carboxy protecting groups are stable. On the other hand, methyl and ethyl esters are easily removed by alkaline hydrolysis, and benzyl esters are easily removed by catalytic hydrogenolysis.

It is possible to obtain an N-terminal acylated peptide derivative by reacting the peptide derivative produced as described above with an acid anhydride or an acid halide in the presence of a base, if necessary. it can. Further optionally, salts of alkali or alkaline earth metals such as sodium, potassium, calcium, etc., salts with non-toxic organic amines such as dimethylamine, triethylamine, benzylamine, dicyclohexylamine, basic such as lysine, arginine, etc. Addition salts with amino acids, for example addition salts with amide derivatives of amino acids such as phenylalanine amide, leucine amide, etc., acid addition salts with mineral acids such as hydrochloric acid, sulfuric acid, etc. Acid addition with acidic amino acids such as aspartic acid, glutamic acid, etc. It can be led to salts, for example acid addition salts with organic acids such as maleic acid, fumaric acid, tartaric acid, malic acid, citric acid and the like.

The pharmacological test examples are shown below to explain the vasorelaxant action of the compounds of the present invention.

Pharmacological test (1) Endothelin binding inhibition test Porcine aortic smooth muscle and cerebellum were tested at 10 mM MOP at 4 ° C.
Homogenized with Polytron in S pH 7.4 buffer. Add 20% sucrose to the homogenate, centrifuge at 1000 xg for 15 minutes, and add 1 to the supernatant.
It was centrifuged at 0000 xg for 15 minutes. This supernatant is further
Centrifuge at 90,000 xg for 40 minutes, and precipitate the resulting precipitate to 5
The membrane fraction was prepared by suspending it in the mM HEPES / Tris pH 7.4 buffer solution to 25 mg / ml.

4 μl of this membrane fraction was added to 50 mM Tris /
HCl pH 7.4 buffer A (10 μM calcium chloride, 10 μM magnesium chloride, 0.1 mM PMS
F, 1 μM pepstatin A, 2 μM leupeptin,
352 μl containing 1 mM 1,10-phenanthroline, 0.1% bovine serum albumin). In this suspension, (A) unlabeled endothelin-1 (for nonspecific binding) having a final concentration of 0.2 μM, (B) buffer A
(For total binding), or (C) 4 μl of the test compound at various concentrations was added, and further 40 μl of ¹²⁵ I-endothelin-1 (12000 to 18000 cpm) was added to each. These mixtures were incubated at 25 ° C. for 4 hours, filtered with a glass filter GF / C, and
After washing with mM HEPES / Tris pH 7.4 (containing 0.3% bovine serum albumin), the ¹²⁵ I-endothelin-1 binding inhibition rate D (%) of the test compound was determined from the following formula by measuring the radioactivity on a glass filter. Sought by.

[0020]

[Equation 1] The 50% binding inhibition concentration was determined from the ¹²⁵ I-endothelin-1 binding inhibition rate by various concentrations of the compound of the present invention.

As shown in Table 1, it was found that the compound of the present invention has an extremely strong endothelin binding inhibitory activity selective to the cerebellum.

[0022]

[Table 1] (2) Effect on Norepinephrine Contraction in Swine Extracted Pulmonary Artery Specimens A pulmonary artery having an outer diameter of about 1 mm was excised from a pig lung to prepare a spiral specimen (endothelium-preserving). The specimen was suspended in an organ bath filled with Krebs Henseleit nutrient solution and the tension was recorded isometrically. After contraction was induced by norepinephrine (1 μM) and the contraction was stabilized, the test compound was administered and the relaxation reaction was observed. The degree of relaxation reaction is 1 norepinephrine contraction
It was expressed as% when it was set to 00%. From the results, the concentration required for each test compound to cause 40% relaxation (ED ₄₀ ) was calculated and shown in Table 2.

[0023]

[Table 2] As shown in Table 2, each test compound was found to have a vasorelaxant effect. (3) Effect on intracellular calcium concentration in cells derived from renal proximal tubules Cells derived from porcine renal proximal tubules LLC-PK ₁ (American Type Culture Collection CRL 1
392), the effect of the compound of each Example on the intracellular calcium concentration was measured by the following method.

LLC-PK ₁ cultured on cover glass
8 μM fura2-acetoxymethyl ester (Dojindo Laboratories), 2.5 mM probenecid, 0.0
Krebs Henseleit Hepes (Kr) with 5% Cremophor EL (manufactured by Sigma)
ebs-Henseleit-HEPES) buffer (p
Incubated in H7.4) for 1 hour at room temperature.
This was excited at 340 nm and 380 nm, fluorescence at 500 nm was measured to monitor the 340/380 ratio, and the intracellular calcium concentration was calculated from this. Table 3 shows the action of the compound of the present invention to increase intracellular calcium concentration (difference between maximum values before and after addition of compound: Δ [Ca ²⁺ ]).

[0025]

[Table 3] As shown in Table 3, the compound of the present invention was found to have an action of increasing intracellular calcium concentration in cells derived from renal proximal tubules. The intracellular calcium concentration-increasing action in the renal proximal tubule cells causes activation of guanylate cycle, resulting in a diuretic action.

As described above, the compound of the present invention has an excellent vasorelaxant action and is useful as a vasodilator in the field of pharmaceuticals, and it has high blood pressure, acute renal failure, myocardial infarction, angina, cerebral infarction, and cerebral blood vessel. It can be a therapeutic drug for spasms. When used as a therapeutic agent for such diseases, the compound of the present invention can be used alone or in combination with other therapeutic agents.

The compound of the present invention can be used in the form of a pharmaceutical formulation suitable for parenteral administration, oral administration or external administration by mixing with a solid or liquid excipient carrier known in the art. Examples of the pharmaceutical preparation include liquid preparations such as injections, inhalants, syrups and emulsions, solid preparations such as tablets, capsules and granules, and external preparations such as ointments and suppositories. In addition, these preparations may optionally contain additives such as auxiliary agents, stabilizers, wetting agents, emulsifiers, absorption promoters or surfactants which are usually used.
As additives, distilled water for injection, physiological saline, Ringer's solution, glucose, sucrose syrup, gelatin, edible oil,
Examples thereof include cocoa butter, ethylene glycol, hydroxypropyl cellulose, lactose, sucrose, corn starch, magnesium stearate and talc.

The dose of the compound of the present invention as a vasorelaxant varies depending on the administration method, the age and weight of the patient, the condition of the patient to be treated, etc., but typical administration methods for adults are oral administration and non-administration. Oral administration, 0.05-10 mg / kg / day for oral administration to adult patients
Body weight, 0.1 to 5m per day for parenteral administration
g / kg body weight.

[0029]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Asp-Lys-Glu-Ala-Val-Tyr-P
he-Ala-His-Leu-Asp-Ile-Il
Synthesis of e-Trp (Compound of SEQ ID NO: 4) Applied Biosystems (Applied Bisystems)
p-alkoxybenzyl alcohol resin (273 mg: 0.25 mm) by the standard Fmoc method using a peptide synthesizer model 431A manufactured by O. Systems).
ol equivalent amount), Fmoc-Trp, Fmoc-Il
e, Fmoc-Ile, Fmoc-Asp (O ^t B
u), Fmoc-Leu, Fmoc-His (Tr
t), Fmoc-Ala, Fmoc-Phe, Fmoc
-Tyr ( ^t Bu), Fmoc-Val, Fmoc-A
la were sequentially coupled (454 mg: intermediate-
1). Using 304mg of them, Fmoc-G
^{lu (O t Bu), Fmoc} -Lys (Boc), Fm
oc-Asp (O ^t Bu) was sequentially coupled (3
29 mg: Intermediate-2). This coupling reaction is
-DCC as a condensing agent at room temperature in methylpyrrolidone
-HOBt was used, and Fmoc was removed by using a piperidine / N-methylpyrrolidone solution. This intermediate-2
A mixed liquid of 9.5 ml of TFA, 0.5 ml of m-cresol and 0.25 ml of ethanedithiol was added to 113 mg, and the mixture was stirred at room temperature for 90 minutes. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was cooled with diethyl ether.
It was washed with tell and dried to obtain 22.5 mg.

13.2 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid-water and subjected to reversed-layer high performance liquid chromatography [Capsule Pack (CAPCELL PAK) C ₁₈ : 2].
0 mm × 250 mm, solvent: 0.1% TFA aqueous solution containing 35% acetonitrile, flow rate: 10 ml / min], and freeze-dried to obtain 4.1 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1720 Example 2 Leu-Met-Asp-Lys-Glu-Ala-V
al-Tyr-Phe-Ala-His-Leu-As
Synthesis of p-Ile-Ile-Trp (Compound of SEQ ID NO: 1
The product) 150 mg of Intermediate-2 obtained in Example 1 was used, and under the same conditions as in Example 1, Fmoc-Met, Fmoc-Le.
u were sequentially coupled (164 mg). To the resulting coupled resin 109 mg, TFA 9.5 m
1, m-cresol 0.5 ml, ethanedithiol 0.
25 ml of the mixed solution was added and stirred at room temperature for 90 minutes. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate is washed with cold diethyl ether and dried to 32.1 m.
g was obtained.

12.3 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid / water and subjected to reverse phase high performance liquid chromatography [CAPCELL PAK C ₁₈ : 2].
0 mm × 250 mm, solvent: 0.1% TFA aqueous solution containing 35% acetonitrile, flow rate: 10 ml / min], and freeze-dried to obtain 3.8 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1964 Example 3 Leu-Met-Asp-Ala-Glu-Ala-V
al-Tyr-Phe-Ala-His-Leu-As
Synthesis of p-Ile-Ile-Trp (Compound of SEQ ID NO: 3
Thing) Applied Biosystems (Applied Bi)
o.systems) peptide synthesizer model 431A, and p-alkoxybenzyl alcohol resin (256 mg: 0.25 mm) according to the standard Fmoc method.
ol equivalent amount) to Fmoc-Trp, Fmoc-Ile,
Fmoc-Ile, Fmoc-Asp (O ^t Bu), F
moc-Leu, Fmoc-His (Trt) and Fm
oc-Ala was sequentially coupled (298 mg: intermediate-3).

100 mg of Intermediate-3 was used and Fm
oc-Phe, Fmoc-Tyr ( ^t Bu), Fmoc
-Val, Fmoc-Ala, Fmoc- Glu (O t
Bu), Fmoc-Ala, Fmoc-Asp (O ^t B
u), Fmoc-Met and Fmoc-Leu were sequentially coupled under the same conditions as in Example 1 (108 m
g). 108 mg of the obtained coupled resin was taken, 9.5 ml of TFA, 0.5 ml of m-cresol,
A mixed solution of 0.25 ml of ethanedithiol was added, and the mixture was stirred at room temperature for 90 minutes. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was washed with cold diethyl ether and dried to obtain 22.1 mg.

9.7 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid / water and subjected to reversed phase high performance liquid chromatography [CAPCELL PAK C ₁₈ : 20].
mm × 250 mm, solvent: 0.1% TFA aqueous solution containing 35% acetonitrile, flow rate: 10 ml / min], and freeze-dried to obtain 1.1 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1907 Example 4 Leu-Met-Ala-Lys-Glu-Ala-V
al-Tyr-Phe-Ala-His-Leu-As
Synthesis of p-Ile-Ile-Trp (Compound of SEQ ID NO: 2
Using 99mg of things) obtained in Example 3 Intermediate -3, Fm
oc-Phe, Fmoc-Tyr ( ^t Bu), Fmoc
-Val, Fmoc-Ala, Fmoc- Glu (O t
Bu), Fmoc-Lys (Boc), Fmoc-Al
a, Fmoc-Met and Fmoc-Leu were sequentially coupled under the same conditions as in Example 1 (105 mg).
105 mg of the obtained coupled resin was added to TFA.
A mixed solution of 9.5 ml, m-cresol 0.5 ml, and ethanedithiol 0.25 ml was added, and the mixture was stirred at room temperature for 90 minutes. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was washed with cold diethyl ether and dried (23.4 mg).

8.5 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid / water and subjected to reversed-phase high performance liquid chromatography [CAPCELL PAK C ₁₈ : 20 m].
m × 250 mm, solvent: containing 35% acetonitrile.
The mixture was purified with a 1% TFA aqueous solution, flow rate: 10 ml / min] and freeze-dried to obtain 2.6 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1920 Example 5 N-acetyl-Glu-Ala-Val-Tyr-Ph
e-Ala-His-Leu-Asp-Ile-Ile
-Trp Synthetic Applied Biosystems (Applied BiSystems)
o-systems) peptide synthesizer model 431A, and p-alkoxybenzyl alcohol resin (257 mg: 0.25 mm) according to the standard Fmoc method.
ol equivalent amount), Fmoc-Trp, Fmoc-Il
e, Fmoc-Ile, Fmoc-Asp (O ^t B
u), Fmoc-Leu, Fmoc-His (Tr
t), Fmoc-Ala, Fmoc-Phe, Fmoc
-Tyr ( ^t Bu), Fmoc-Val, Fmoc-A
la, were successively coupling ^{Fmoc-Glu (O t Bu)} (485mg). This coupling reaction is N-
The reaction was carried out in methylpyrrolidone at room temperature, Fmoc was eliminated using a piperidine / N-methylpyrrolidone solution, and DCC-HOBt was used as a condensing agent. Take 120 mg of this coupled resin, and add 5 ml of methylene chloride,
Acetic anhydride (0.2 ml) and pyridine (0.2 ml) were added, and the mixture was reacted at room temperature for 1 hour to acetylate the N-terminal amino group. After completion of the reaction, the residue was washed with methylene chloride, then TFA (9.5 ml), m-cresol (0.5 ml),
A mixture of 0.25 ml of ethanedithiol was added, and the mixture was stirred at room temperature for 90 minutes. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was washed with cold diethyl ether and then dried to obtain 25.3 mg.

9.8 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid / water and subjected to reverse phase high performance liquid chromatography [CAPCELL PAK C ₁₈ : 20].
mm × 250 mm, solvent: 0.1% TFA aqueous solution containing 35% acetonitrile, flow rate: 10 ml / min], and freeze-dried to obtain 2.3 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1519 Example 6 N-acetyl-Leu-Met-Asp-Lys-Gl
u-Ala-Val-Tyr-Phe-Ala-His
-Leu-Asp-Ile-Ile-Trp synthesis Applied Biosystems (Applied Bisystems)
o-systems) peptide synthesizer-model 431A and p-alkoxybenzyl alcohol resin (311 mg: 0.3 mmo) by the standard Fmoc method.
1 equivalent amount), Fmoc-Trp, Fmoc-Ile,
Fmoc-Ile, Fmoc-Asp (O ^t Bu), F
moc-Leu, Fmoc-His (Trt), Fmo
c-Ala, Fmoc-Phe, Fmoc-Tyr ( ^t
Bu), Fmoc-Val, Fmoc-Ala, Fmo.
^{c-Glu (O t Bu)} , Fmoc-Lys (Bo
c), Fmoc-Asp (O ^t Bu), Fmoc-Me
t and Fmoc-Leu were sequentially coupled (102
2 mg). This coupling reaction is carried out in N-methylpyrrolidone at room temperature, and Fmoc is eliminated by piperidine /
An N-methylpyrrolidone solution was used, and DCC-HOBt was used as a condensing agent. 182 mg of this coupled resin was taken and N-methylpyrrolidone 4.5 m
1, and 0.5 ml of acetic anhydride were added and reacted at room temperature for 1 hour to acetylate the N-terminal amino group. After completion of the reaction, the residue was washed with N-methylpyrrolidone and methylene chloride, then TFA (9.5 ml) and m-cresol (0.5 m) were used.
1, a mixed solution of 0.25 ml of ethanedithiol was added,
Stir at room temperature for 90 minutes. The reaction solution was poured into a glass filter, the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was washed with cold diethyl ether and then dried to obtain 74.5 mg.

The precipitate (16.3 mg) was mixed with water (4 ml) and acetic acid (3.
Dissolve in a mixed solvent of 5 ml and 3 ml of acetonitrile, and perform reverse phase high performance liquid chromatography [Capsule Pack (C
APCELL PAK) C ₁₈ : 20 mm x 250 m
m, solvent: 0.1% TFA aqueous solution containing 40% acetonitrile, flow rate: 10 ml / min], and freeze-dried to obtain 5.9 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 2006 Example 7 N-acetyl-D-Glu-Ala-Val-Tyr-
Phe-Ala-His-Leu-Asp-Ile-I
le-Trp Synthesis Applied Biosystems (Applied Bisystems)
o.systems) peptide synthesizer-model 431A and p-alkoxybenzyl alcohol resin (316 mg: 0.3 mmol) according to the standard Fmoc method.
Fmoc-Trp, Fmoc-Ile, F
moc-Ile, Fmoc-Asp (OtBu), Fm
oc-Leu, Fmoc-His (Trt), Fmoc
-Ala, Fmoc-Phe, Fmoc-Tyr ( ^t B
u) were sequentially coupled (804 mg: intermediate-
4). This coupling reaction is performed in N-methylpyrrolidone at room temperature, and Fmoc is eliminated by piperidine / N-
Methylpyrrolidone solution is used, and the condensing agent is DC
C-HOBt was used.

399 mg of Intermediate-4 was used, and Fm
oc-Val, Fmoc-Ala, Fmoc-D-Gl
u (O ^t Bu) was sequentially coupled under similar conditions (415 mg). 205 mg of this coupled resin
Then, 4.5 ml of N-methylpyrrolidone and 0.5 ml of acetic anhydride were added, and the mixture was reacted at room temperature for 1 hour to acetylate the N-terminal amino group. After completion of the reaction, the residue was washed successively with N-methylpyrrolidone and methylene chloride, and then a mixed solution of 9.5 ml of TFA, 0.5 ml of m-cresol and 0.25 ml of ethanedithiol was added, and the mixture was stirred at room temperature for 90 minutes. did. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was washed with cold diethyl ether and dried to obtain 86.3 mg.

20 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid / water, and reversed phase high performance liquid chromatography- [CAPCELL PAK] C ₁₈ : 20 m was used.
m × 250 mm, solvent: containing 40% acetonitrile.
The mixture was purified with a 1% TFA aqueous solution, flow rate: 10 ml / min] and freeze-dried to obtain 7.2 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1519 Example 8 N-acetyl-Glu-Ala-Ala-Tyr-Ph
e-Ala-His-Leu-Asp-Ile-Ile
-Trp Synthetic Applied Biosystems (Applied BiSystems)
ossystems Peptide Synthesizer Model 4
31A using standard Fmoc method and p-alkoxybenzyl alcohol resin (258 mg: 0.25 mmol).
Fmoc-Trp, Fmoc-Ile, F
moc-Ile, Fmoc-Asp (O ^t Bu), Fm
oc-Leu, Fmoc-His (Trt), Fmoc
-Ala, Fmoc-Phe, Fmoc-Tyr ( ^t B
u), Fmoc-Ala, Fmoc-Ala, Fmoc
-Glu (O ^t Bu) was sequentially coupled (665
mg). Take 192 mg of this coupled resin,
4.5 ml of N-methylpyrrolidone, 0.5 ml of acetic anhydride
Was added and reacted at room temperature for 1 hour to acetylate the N-terminal amino group. After completion of the reaction, the residue was washed successively with N-methylpyrrolidone and methylene chloride and then with TFA.
A mixed solution of 9.5 ml, m-cresol 0.5 ml, and ethanedithiol 0.25 ml was added, and the mixture was stirred at room temperature for 90 minutes. The reaction solution is poured into a glass filter and the resin is TFA.
After washing with, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The precipitate obtained is washed with cold diethyl ether and dried,
5 mg was obtained. Dissolve 11.0 mg of the precipitate in a mixed solvent of 50% acetic acid / water, and perform reverse phase high performance liquid chromatography.
[Capsule Pack (CAPCELL PAK)
C ₁₈ : 20 mm × 250 mm, solvent: 0.1% TFA aqueous solution containing 36% acetonitrile, flow rate: 10 ml / mi
n] and freeze-dried to give 4.6 mg of the title compound as a white powder.

FAB-MS [M + H] ⁺ : 1491 Example 9 N-acetyl-Glu-Ala-Val-Tyr-Ph
e-Ala-Ala-Leu-Asp-Ile-Ile
-Trp Synthetic Applied Biosystems (Applied BiSystems)
ossystems Peptide Synthesizer Model 4
31A using p-alkoxybenzyl alcohol resin (309 mg: 0.3 mmol equivalent amount) according to the standard Fmoc method, and Fmoc-Trp, Fmoc-Ile, Fm.
oc-Ile was sequentially coupled (472 mg: Intermediate-5). 158 mg of Intermediate-5 was used and Fm
^{oc-Asp (O t Bu)} , Fmoc-Leu, Fmo
c-Ala, Fmoc-Ala, Fmoc-Phe, F
moc-Tyr ( ^t Bu), Fmoc-Val, Fmo
c-Ala, Fmoc-Glu ( O t Bu) was sequentially coupled under similar conditions (220 mg). 109 mg of this coupled resin was taken, 4.5 ml of N-methylpyrrolidone and 0.5 ml of acetic anhydride were added, and the mixture was reacted at room temperature for 1 hour to acetylate the N-terminal amino group. After completion of the reaction, the residue was washed successively with N-methylpyrrolidone and methylene chloride, then a mixed solution of 9.5 ml of TFA, 0.5 ml of m-cresol and 0.25 ml of ethanedithiol was added, and the mixture was stirred at room temperature for 90 minutes. did. After the reaction solution was poured into a glass filter and the resin was washed with TFA, the filtrate and the washing solution were combined and concentrated under reduced pressure, and cold diethyl ether was added to the residue to precipitate the peptide. The obtained precipitate was washed with cold diethyl ether and dried to obtain 32 mg.

14 mg of the precipitate was dissolved in a mixed solvent of 50% acetic acid / water and subjected to reversed phase high performance liquid chromatography- [CAPCELL PAK] C ₁₈ : 20 m.
m × 250 mm, solvent: containing 43% acetonitrile.
It was purified with a 1% TFA aqueous solution, flow rate: 10 ml / min] and freeze-dried to obtain 1.1 mg of the title compound as a white powder.

FAB-MS [M + H]⁺: 1453 Example 10N-acetyl-Glu-Ala-Val-Tyr-Ph
e-Ala-His-Leu-Asp-Ala-Ile
-Trp synthesis Applied Biosystems (Applied Bi)
ossystems) peptide synthesizer model
P-alkoxy according to the standard Fmoc method using 431A.
Benzyl alcohol resin (258 mg: 0.25 mmo
1 equivalent amount), Fmoc-Trp, Fmoc-Ile,
Fmoc-Ala, Fmoc-Asp (O^tBu), F
moc-Leu, Fmoc-His (Trt), Fmo
c-Ala, Fmoc-Phe, Fmoc-Tyr (^t
Bu), Fmoc-Val, Fmoc-Ala, Fmo.
c-Glu (O^tBu) was sequentially coupled (66
3 mg). 182 mg of this coupled resin
, N-methylpyrrolidone 4.5 ml, acetic anhydride 0.5
ml, and react at room temperature for 1 hour.
The acetylation of the amino group was performed. After completion of the reaction, N-methylpi
After washing the residue with rolidone and methylene chloride sequentially, TF
A 9.5 ml, m-cresol 0.5 ml, ethanedith
Add a mixture of 0.25 ml of oil and keep it at room temperature for 90 minutes.
It was stirred. Pour the reaction solution into a glass filter and put the resin in TF.
After washing with A, the filtrate and washing solution are combined and concentrated under reduced pressure to leave a residue.
Cold diethyl ether was added to the residue to precipitate the peptide.
The obtained precipitate is washed with cold diethyl ether and dried,
77 mg was obtained. 14.5 mg of precipitate was added to 50% acetic acid /
Dissolve in a mixed solvent of water and reverse phase high performance liquid chromatography
-[Capsule Pack (CAPCELL PAK)
C ₁₈: 20 mm x 250 mm, solvent: 36% acetonite
Rill-containing 0.1% TFA aqueous solution, flow rate: 10 ml / mi
n] and then freeze-dried to obtain a white powder.
6.6 mg of the title compound was obtained.

FAB-MS [M + H] ⁺ : 1477

[0050]

INDUSTRIAL APPLICABILITY Since the compound of the present invention has a strong vasorelaxant action, it is useful as a therapeutic drug for human hypertension, acute renal failure, myocardial infarction, angina, cerebral infarction and cerebral vasospasm. ..

[0051]

[Sequence list]

SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 4 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kyoko Nakamichi 2-9-3 Shimomeguro, Meguro-ku, Tokyo Manyu Pharmaceutical Co., Ltd. Central Research Laboratory (72) Inventor Mitsuo Yano 2-chome, Shimomeguro, Meguro-ku, Tokyo 9th-3 Manyu Pharmaceutical Co., Ltd. Central Research Laboratory

Claims (5)

[Claims] 1. A general formula X ¹ -X ² -Ala-X ³ -Tyr-Phe-Ala-X ⁴
-Leu-Asp-X ⁵ -Ile-Trp [I] [In the formula, X ¹ represents a hydrogen atom, an acyl group, or any amino acid residue in which the amino group may have an acyl group, or any 2
Represents a peptide residue which may have an acyl group at the N-terminal having an arbitrary sequence consisting of ~ 4 amino acids, X
² represents Glu or D-Glu, X ³ represents Val or Al
a, X ⁴ represents His or Ala, X ⁵ represents Ile
Or Ala]] or a pharmaceutically acceptable salt thereof. 2. X ¹ is a hydrogen atom, an acyl group, or Ala,
Gly, Ile, Leu, Met, Val, Phe, A
Amino acid residue in which an amino group selected from the group consisting of sp, Glu and Lys may have an acyl group, or A
la, Gly, Ile, Leu, Met, Val, Ph
N having an arbitrary sequence consisting of 2 to 4 amino acid residues selected from the group consisting of e, Asp, Glu and Lys
-The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, which is a peptide residue having an acyl group at the terminal. 3. X ¹ is a hydrogen atom or Leu-Met-As.
p-Lys, Leu-Met-Asp-Ala, Leu
-Met-Ala-Lys, Asp-Lys or A
The peptide according to claim 1, which is la-Lys, or a pharmaceutically acceptable salt thereof. 4. A compound represented by the following chemical formula or a pharmaceutically acceptable salt thereof. Asp-Lys-Glu-Ala-Val-Tyr-P
he-Ala-His-Leu-Asp-Ile-Il
e-Trp, Leu-Met-Asp-Lys-Glu
-Ala-Val-Tyr-Phe-Ala-His-
Leu-Asp-Ile-Ile-Trp, Leu-M
et-Asp-Ala-Glu-Ala-Val-Ty
r-Phe-Ala-His-Leu-Asp-Ile
-Ile-Trp, Leu-Met-Ala-Lys-
Glu-Ala-Val-Tyr-Phe-Ala-H
is-Leu-Asp-Ile-Ile-Trp, N-
Acetyl-Glu-Ala-Val-Tyr-Phe-
Ala-His-Leu-Asp-Ile-Ile-T
rp, N-acetyl-Leu-Met-Asp-Lys
-Glu-Ala-Val-Tyr-Phe-Ala-
His-Leu-Asp-Ile-Ile-Trp, N
-Acetyl-D-Glu-Ala-Val-Tyr-P
he-Ala-His-Leu-Asp-Ile-Il
e-Trp, N-acetyl-Glu-Ala-Ala-
Tyr-Phe-Ala-His-Leu-Asp-I
le-Ile-Trp, N-acetyl-Glu-Ala
-Val-Tyr-Phe-Ala-Ala-Leu-
Asp-Ile-Ile-Trp, N-acetyl-Gl
u-Ala-Val-Tyr-Phe-Ala-His
-Leu-Asp-Ala-Ile-Trp 5. Hypertension, acute renal failure, myocardial infarction, angina, cerebral infarction, which comprises the compound represented by the formula [I] or the pharmaceutically acceptable salt thereof according to claim 1. ,
A therapeutic and / or preventive agent for cerebral vasospasm.