JPH03123797A - Oligopeptide - Google Patents

Abstract

NEW MATERIAL:The sarafotoxin derivative (salt) having a structure obtained by substituting the 6-Met of oligopeptide of formula with homoserine. USE:A blood pressure regulation agent. PREPARATION:The objective oligopeptide of formula can be produced e.g. by successively bonding alpha-amino acids and the amino acids having protected side chain functional group to a resin carrier from the C-terminal according to the amino acid sequence of the peptide by solid-shape synthesis process using methylbenzyl group as the protecting group for the 1- and 15-Cys groups and acetamidemethyl group as the protecting group for the 3- and 11-Cys groups, separating the peptide from the resin using HF after the completion of the synthesis of the peptide chain and, at the same time, removing the methylbenzyl group, oxidizing the product with air to form an SS crosslink between 1- and 15-sites, purifying by high performance liquid chromatography, removing the acetamidomethyl group by oxidizing with iodine and forming an SS crosslink between 3- and 11 sites.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a physiologically active compound of the following formula (1): % formula % (
) or sarafotoxin s6b, which is an oligopeptide represented by the following formula (II)
The present invention relates to providing a sarafotoxin derivative derived from s6c, and furthermore, the present invention also provides a blood pressure regulating drug characterized by containing a saraphotoxin derivative or a pharmaceutically acceptable salt thereof as an active ingredient. related.

[Prior art and its problems Kosalaphotoxin is Attractaspis engad
It is a snake venom isolated and extracted from the venom gland of S. densis, and the entire structure was elucidated by Takasaki, one of the inventors of the present invention, and it has the following formula (I
III: Cys-A-Cys-B-Asp-Met-C
-Asp-D-Glu-Cys-Leu-E- is an oligopeptide consisting of 21 amino acids represented by the formula (II
In II, AB (1: DE are Ser and Lys, respectively.

Thr/Ser, Lys, Asn saraphotoxin s6a+xSer, Lys, Thr, Glu
, saraphotoxin s6b, which is Phe, and saraphotoxin s6c, which is Thr, Asn, Thr, Glu, and Phe (Toxicon, No. 26).
(Vol., p. 543, 1988) Furthermore, a more detailed study of the structure of sarafotoxin revealed that Cys at positions 1 and 15 and simultaneously Cys at positions 3 and 11 form a loop by SS cross-linking, and the first loop was found to be hydrophilic, and the till part was found to be strongly hydrophobic.

The present invention shows that the above-mentioned sarafotoxin has a unique action of restoring abnormal blood pressure, that is, high blood pressure or low blood pressure, to normal values, and that its point of action is not through the central nervous system, but directly on vascular smooth muscle. This has been clarified through studies by researchers.

Due to its unique pharmacological action, saraphotoxin is expected to be applied as a completely new type of blood pressure regulating drug different from conventional ones, and the present inventors have completed a patent application (Japanese Patent Application No. 63-32801). (See application specification).

As clarified in the above-mentioned application specification, sarafotoxin has the excellent feature that its pharmacological activity is sufficiently exerted even in extremely small amounts.

However, on the other hand, it has become clear that the margin of safety between the lethal dose and the effective dose, that is, the effective dose/lethal dose, is relatively narrow.

Therefore, the present inventors synthesized various sarafotoxin derivatives and examined their structure-activity relationships, and found that the following formula (I): Cys-Leu-Tyr-Phe-C:ys-His-
Gin-Asp-Val-11e-Trp--+-(
1) Oligopeptide shown by (sarafotoxin s6b)
Sarafotoxin derivative in which Net at position 6 is substituted with homoserine (hereinafter sometimes referred to as 'Hse-s6b)
, a sarafotoxin derivative in which Tyr at position 3 in formula (I) is substituted with mono- or di-iodine (hereinafter also referred to as Iod-s6b); N-terminal Cys at position 4 in formula (I);
Sarafotoxin derivatives (hereinafter referred to as Ac-56) in which at least one or more of the amino groups of Lys at position 9 are acetylated
b), and the following formula (II): Cys-Tyr-Cys-Asn-Asp-Met-T
hr-Asp-Glu-Glu-Cys-Leu-As
n-Phe-Cys-Hi 5-Gin-Asp-Va
-1-Ile-Tr p ---(II) A saraphotoxin derivative in which Met at position 6 is substituted with homoserine in the oligopeptide (sarafotosin s6c) (hereinafter sometimes referred to as 'Hse-sec) ,
Sarafotoxin derivatives in which the amino group of the N-terminal Cys in formula (II) is acetylated (hereinafter sometimes referred to as Ac-56c), and the above five derivatives, have low toxicity when compared to natural products, and also have pharmacological effects. It was confirmed that these derivatives were comparable to natural products, and when these derivatives were used as medicines, they were found to be more effective than natural products, leading to the completion of the present invention.

[Means for Solving the Problems] The present invention provides a sarafotoxin derivative 'Hse-
s6b, formula (Sarafotoxin derivative Ac-56 in which the N-terminal Cys in Il, the amino groups at the 4th and 9th positions are acetylated)
b, or a saraphotoxin derivative 'Hse- in which Met at position 6 is substituted with homoserine in the oligopeptide represented by the following formula (II)
s6c, a sarafotoxin derivative Ac-56c in which the amino group of the N-terminal Cys in formula (IIl is acetylated), or a pharmaceutically acceptable salt of these compounds, as an active ingredient.

Each of the above-mentioned sarafotoxin derivatives can be synthesized by methods known in the field of peptide chemistry, and representative synthetic methods are shown in Examples 2 to 8 below.

Sarafotoxin derivatives obtained by the methods shown in Examples below can be made into pharmaceutical compositions as described below.

That is, the sarafotoxin derivative or its pharmaceutically acceptable salt can be used as a pharmaceutically widely used inorganic or organic solid or liquid carrier or diluent for preparation, such as starch, lactose, sucrose, crystalline cellulose, calcium hydrogen phosphate, etc. Excipients such as niacacia, hydroxypropyl cellulose, alginic acid, gelatin, binders such as polyvinylpyrrolidone; lubricants such as stearic acid, magnesium stearate, calcium stearate, talc, hydrogenated vegetable oil; modified starch, calcium carboxymethyl cellulose,
Disintegrants such as low substituted hydroxypropyl cellulose; nonionic surfactants, which can be anionized. Dosage forms suitable for oral administration include solid preparations such as tablets, coatings, capsules, troches, powders, fine granules, granules, and dry syrups, and liquid preparations such as syrups. Suitable dosage forms include, for example, injections, drops, suppositories, and the like. In addition, dosage forms suitable for topical administration include ointments, tinctures, gnomes, gels, aerosols, and the like. These formulations can be prepared in a manner well known per se in the pharmaceutical field. In addition, for administration forms other than injections, stabilizers such as proteolytic enzyme inhibitors may be added as appropriate.

In this way, a preparation containing the sarafotoxin derivative of the present invention as an active ingredient can be obtained. This pharmaceutical composition further provided by the present invention can be administered to all mammalian animals including humans. The dosage can be varied over a wide range depending on the age, weight, symptoms of the patient to be treated, the form of administration of the drug, the doctor's diagnosis, etc.
Doses in the mg range are standard and are usually administered once a day.
It can be administered in one or several divided doses.

The sarafotoxin derivatives provided by the present invention will be explained in further detail in Examples below.

The abbreviations used in the peptide field in this specification are as follows. Cys; cystine, Lys; lysine,
Asp: aspartic acid, Met: methionine, T
hr: threonine, Glu: glutamic acid, Leu
: Leucine, Tyr; Tyrosine, Phe; Phenylalanine, His; Histidine, Asn; Asparagine, Val; Valine, Ile; Isoleucine, Tr
p; tryptophan, Gin: glutamine, Hse;
homoserine.

Fruit '1 case'2: Pharmacological activity test 1.50% 'Hse-s6b, Iod-s6b, A, which is a derivative of saraphotoxin s6b obtained in Examples 2 to 8 written in Fuuma's postscript.
Attract c-56b, and 'Hse-s6c, a derivative of sarafotoxin s6c, Ac-56c.
A comparative study of toxicity was conducted with natural products s6b and s6c extracted from aspis engadden-sis.

Method: A group of three male mice (body weight 20 g) was given a physiological saline solution of a sarafotoxin derivative through the tail vein.
d et al.'s method (Reed L, J, and Muen
ch H, (1938) Am, J, Hy
g, 27. 493-497), the 50% lethal dose (LD, .) was determined.

<result> Table 1 2. Increase blood pressure in viv.

The pharmacological effects of sarafotoxin derivatives on blood pressure were investigated.

Method> Sarafotoxin derivatives were intravenously administered to spontaneously hypertensive rats at a dose of lnmol/kg, femoral artery blood pressure was measured, and the hypotensive and pressor effects of each derivative were compared with natural products.

Results〉 The results are shown in Table 2.

Table 2 N, D: Not measured As is clear from the results in Table 1, each sarafotoxin derivative has a higher LD value (lower toxicity) than the natural product.

2) As is clear from Table 2, which shows the range between the intracranial pressure and the systolic blood pressure, it can be seen that each derivative retains its pharmacological activity.

As understood from the pharmacological activity test in 1.2 above, the toxicity of sarafotoxin is reduced by making it into a derivative of the present invention, and the pharmacological activity is also sufficiently maintained, making it more clinically effective than natural products. It is expected that this drug will be an excellent drug.

Synthesis of 1 Gori Unisaraphotoxin S6b Cys-3er
-Cys-Lys-Asp-Met-Thr-Asp-
Lys-Glu-Cys-Leu-Tyr-Phe-C
ys-Hi 5-Gl n-Asp-Val-I 1
e-Trp---(s6b) Using a methylbenzyl group as the protecting group for Cys at the 1st and 15th positions and an acetamidomethyl group as the protecting group for the Cys at the 11th position, using a solid phase peptide synthesizer using a conventional method. Synthesis was carried out sequentially starting from the C-terminus.

After completion of the synthesis, the peptide was separated from the resin using a liver, the methylbenzyl group was removed at the same time, and SS crosslinks were formed at positions 1-15 by air oxidation.
Purification was carried out by. Then, the acetamidomethyl group was removed by iodine oxidation to form an SS crosslink between positions 3 and 11 to obtain sarafotoxin s6b.

! Synthesis of Gooku Unisaraphotoxin s6c Cys-Tyr
-Cys-Asn-Asp-Met-Thr-Asp-
Glu-Glu-Cys-Leu-Asn-Phe-C
ys-Hi 5-Gin-Asp-Val-I 1e
-Trp-1-(s6c) The same procedure as in Example 2 was carried out using a methylbenzyl group as the protecting group for Cys at the 1st and 15th positions and an amidomethyl group as the protecting group for the Cys at the 3rd and 11th positions, Sarafotoxin s6c was obtained.

7: Synthesis of 'H8e-86b Cys-3er-Cys-Lys-Asp-Met-T
hr-Asp-Lys-Glu-Cys-Leu-Ty
r-Phe-Cys-Hi 5-Gl n-Asp-V
al -I l e-Trp---(s6b) ↓ Cys-3er-(:ys-Lys-Asp-Hse-
Thr-Asp-Lys-Glu-Cys-Leu-T
yr-Phe-C: ys-His-Gln-Asp-V
al-I 1e-Trp-1 ('Hse-s6b) Sarafotoxin s6b obtained in Example 1 0.1m
g (40 nmol) in 0.2 ml of 70% formic acid, and a solution of 70% formic acid in which 1.3 mg of cyanogen bromide was dissolved.
Add 0 μm, tightly stopper, and leave in the dark at 30°C for 16 hours.

Next, 2 ml of distilled water was added, freeze-dried, and 1.0 ml of 1M formic acid was added.
Excess cyanogen bromide was removed by dissolving in 0 ml and freeze-drying again.

Next, purification was performed by HPLC to obtain 6Hse-s6b.

1st 1j154: Synthesis of Iod-s6b---(s
6b) ↓ ---1 (Iod-s6b) Sarafotoxin s6b obtained in Example 1 O,11
mg (40 nmol) in 0.2 M phosphate buffer (pH
7,5) Dissolved in 100 μl of NaI-Iz under ice cooling.
Solution (0.IM NaI 2ml, 0.4MHSO
Add 14 μm of 0.6% H2 (mixed with 2 ml of H2O and left under water cooling for 30 minutes), and left under water cooling for 30 minutes.

Then, it was desalted using a 5ephadex G-15 column,
It was further purified by HPLC to obtain a mono-iodinated product (rod-
s6bl was obtained.

One dish typeヱ: Synthesis of Ac-56b ↓ Ac-Cys-5er-Cys-Ac-Lys-Asp
-Hse-Thr-Asp-Ac-Lys-Glu-C
ys-Leu-Tyr-I-Phe-Cys-His-
Gln-Asp-Val-11e-Trp---(
Ac-s6b) Sarafotoxin s obtained in Example 1
Dissolve 300 μg (120 nmol) of 6b in 0.3 ml of 50% saturated sodium acetate solution, and add 2.5 LL1 of acetic anhydride at 15 minute intervals for a total of 4 times while stirring under water cooling (
During this time, keep the pH at 7-9 with 5M NaOH). After the reaction was completed, the temperature was returned to room temperature and purified by HPLC to obtain Ac-56b.

Synthesis of W Uni' Hse-s6c Cys-Tyr-Cys-Asn-Asp-Met-T
hr-Asp-Glu-GluCysFLeu-As
n-Phe-Cys-Hi5-Gl n-Asp-
Val -I 1 e-Tr p---(s6cl Cys-Tyr-Cys-Asn-Asp-1(se-
Thr-Asp-Glu-Glu---('Hse-
s6c) Sarafotoxin s6c O, 1m obtained in Example 2
'Hse-s' was processed in the same manner as in Example 1 using
I got 6c.

Mitate type top: Synthesis of Ac-56c --- (s6c) --- (Ac-s6c) Sarafotoxin s6c obtained in Example 2 300μ
Ac-56c was obtained by processing in the same manner as in Example 3 using
I got it.

'1(se-s6b powder) obtained according to 11li Uni Injection Preparation Example 4.
2 mg of mannitol is dissolved in 1 ml of an aqueous solution of 20 mg of mannitol. The solution is filtered under sterile conditions into 2 ml ampoules and lyophilized.

Support 1 Feng Bean: Injection Preparation 5 mg of Ac-56b powder obtained in Example 6 was prepared in the same manner as in Example 9 to obtain a freeze-dried product.

Support 1 Takudan: 'Hse-s6c powder obtained according to injection preparation Example 7 5 m
g is adjusted in the same manner as in Example 9 to obtain a lyophilized product.

11: Preparation for injection 5 mg of Ac-56c powder obtained in Example 8 is prepared in the same manner as in Example 9 to obtain a lyophilized product.

1 Directly and directly: Iod-s6b powder obtained according to Injectable Preparation Example 5 0.06
mg of mannitol is dissolved in 1.5 ml of an aqueous solution of 20 mg of mannitol. The solution is filtered under sterile conditions into 2 ml ampoules to obtain a lyophilized product.

1 plate: fine powder of Iod-s6b obtained from nasal administration composition Example 13 etc.
．． 06 mg are suspended in a mixture of 75 mg of benzyl alcohol and 1.395 g of a suspending agent, such as a mixture of commercially available semi-synthetic triglycerides of higher fatty acids. Place the turbid liquid into a 10 ml aerosol container with a metering valve;
Fill with aerosol propellant.

[Effects] Sarafotoxin is expected to become a new type of blood pressure regulator due to its unique pharmacological activity. One of the characteristics of sarafotoxin is that its pharmacological activity is sufficiently exerted even in minute amounts, but its safety margin as a drug has been relatively narrow.

The sarafotoxin derivatives provided by the present inventors solved this problem and succeeded in reducing toxicity while sufficiently retaining pharmacological activity. The sarafotoxin derivative provided this time is expected to become a blood pressure regulating drug that is easier to use clinically than natural products.

Claims (9)

[Claims] (1) Following formula (I): Cys-Ser-Cys-Lys-Asp-Met-T
hr-Asp-Lys-Glu-Cys-Leu-Ty
r-Phe-Cys-His-Gln-Asp-Val
-Ile-Trp---(I) A sarafotoxin derivative in which Met at position 6 is substituted with homoserine, or a pharmaceutically acceptable salt. (2) A sarafotoxin derivative of formula (I) according to claim 1, in which Tyr at position 13 is substituted with mono- or di-iodine, or a pharmaceutically acceptable salt thereof. (3) In formula (I) according to claim 1, N-terminal Cys, 4
A sarafotoxin derivative in which one or more of the amino groups at Lys and Lys at positions 9 and 9 is acetylated, or a pharmaceutically acceptable salt thereof. (4) Following formula (II): Cys-Tyr-Cys-Asn-Asp-Met-T
hr-Asp-Glu-Glu-Cys-Leu-As
n-Phe-Cys-His-Gln-Asp-Val
-Ile-Trp---(II) A saraphotoxin derivative or a pharmaceutically acceptable salt in which Met at position 6 is substituted with homoserine in the oligopeptide represented by (II). (5) A sarafotoxin derivative of formula (II) according to claim 4, in which the amino group of the N-terminal Cys is acetylated, or a pharmaceutically acceptable salt thereof. (6) A blood pressure regulating drug containing one or more of the sarafotoxin derivatives according to claims 1 to 5 as an active ingredient. (7) The blood pressure regulating drug according to claim 6, which is in a form suitable for parenteral administration. (8) The blood pressure regulating drug according to claim 6, which is in a form suitable for oral administration. (9) The blood pressure regulating drug according to claim 6, which is in a form suitable for local administration.