JPH1160598A - Opioid peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject new peptide composed of an opioid peptide having a specific amino acid sequence, exhibiting agonistic activity on endomorphine and antagonistic action on tachykinin receptor and useful as an analgesic agent, an agent for the prevention and treatment of nervous diseases, etc. SOLUTION: This new opioid peptide is expressed by the formula I [X and Y are each an amino acid residue which may be a non-natural type residue, a peptide residue, single bond (X and Y are not single bond at the same time); the amino acid residues of Pro, Phe, X or Y and one or more amino acid residues in a peptide residue of X or Y may be D-isomers] [excluding the peptide expressed by the formula II or the formula III (all amino acid residues in the peptide residue are L-isomers)] or its salt and is useful as an analgesic agent, an agent for the prevention or treatment of various nervous diseases, etc. The peptide can be produced by the successive condensation reaction of amino acids from the peptide carboxyl terminal by a liquid-phase peptide synthesis or a solid-phase peptide synthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel neurotransmitter peptide having excellent pharmacological action and a salt thereof. In particular, the present invention relates to a peptide having an agonistic activity on an endomorphin receptor and an antagonistic activity on a tachykinin receptor.

[0002]

2. Description of the Related Art Morphine has been conventionally known as an active substance of an opioid receptor. It is also used clinically for severe pain such as cancer pain. Although morphine has such a powerful analgesic action, it has drawbacks such as resistance formation and development of dependence.

Recently, endomorphin was discovered as an endogenous binding substance for opioid receptors (Nature, 386, 499).
-502 (1997)). Endomorphin is a tetrapeptide found as an endogenous substance having an activity at a mu opioid receptor, and has an analgesic effect. This peptide is expected as a compound that overcomes the disadvantages of morphine such as resistance formation and dependence.

[0004] On the other hand, antagonists of tachykinin receptors such as neurokinin-2 (NK-2) receptor also have an analgesic effect, for example, Japanese Patent Application Laid-Open No. 6-73093 and WO9605.
193 is known. However, these do not provide sufficient analgesic activity and are not finally analgesics.

[0005]

Means for Solving the Problems The present inventor has found a peptide having both an antagonistic activity on a tahikinin receptor and an activity on an opioid receptor. This peptide has substance P because it has both activities.
Inhibits the action of endogenous pain transmitters, such as those, and exhibits an analgesic effect, and at the same time, acts on opioid receptors to exhibit a morphine-like analgesic effect. Accordingly, the peptide of the present invention is useful as an analgesic and as a preventive or therapeutic agent for various neurological diseases.

The present invention is the following invention relating to this peptide or a salt thereof. A peptide represented by the following formula (1) (however, excluding peptides represented by the following formulas (2) and (3)), or a salt thereof.

[0007]

Embedded image Tyr-Pro-X-Phe-Y-NH ₂ (1) Tyr-Pro-Phe-Phe-NH ₂ (2) Tyr-Pro-Trp-Phe-NH _2.・ (3)

[In the formula (1), X and Y each represent an amino acid residue, a peptide residue or a single bond which may be non-natural type (however, X and Y are not single bonds at the same time) An amino acid residue which is Phe, X or Y;
And one or more amino acid residues in the peptide residue of X or Y may be D-form. The amino acid residues in the peptide residues represented by the formulas (2) and (3) are all L-forms. ]

The peptide represented by the formula (2) is endomorphin-2, and the peptide represented by the formula (3) is endomorphin-1, both of which are known.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the following description, an amino acid refers to a naturally-occurring L-form amino acid unless otherwise specified. Unless otherwise specified, a peptide refers to a natural L-form in which two or more amino acids are linked. The naturally-occurring amino acids refer to α-amino acids that are components of naturally occurring peptides and proteins. Those in which a three-letter symbol of a natural amino acid is suffixed with D indicate the D-form of the corresponding amino acid. In addition, the amino acid of D form is also hereafter called D-amino acid. In the notation of peptides, the left end indicates the N-terminal and the right end indicates the C-terminal in accordance with general rules.

In the peptide of the present invention represented by the formula (1), the amino acid residue of Pro, Phe, X or Y, and one or more amino acid residue in the peptide residue of X or Y are It may be D-form, and it is particularly preferable that at least one amino acid residue of these is D-form. Among them, at least one of X and Y is preferably a D-amino acid residue or a peptide containing at least one D-amino acid residue. Note that X and Y do not simultaneously form a single bond.

X is an amino acid residue or the number of amino acid residues 3
The following peptide residues are preferred. Among them, X is preferably an amino acid residue, particularly preferably L- or D-form Phe or Trp. Y is preferably a peptide residue or an amino acid residue having 5 or less (especially 3 or less) amino acid residues. More preferred Y is His-Leu-M from the amino terminal side.
It is preferably a peptide residue having the sequence et or a peptide residue or amino acid residue having a sequence obtained by removing any one or two amino acid residues from this sequence.
Of these, His and His in the peptide residue having His are preferably in the D-form. Most preferred Y is a peptide residue having the sequence DHis-Leu-Met.

The most preferred peptide of the present invention represented by the formula (1) is a peptide represented by the following formulas (A) to (1D).

[0014]

Embedded image Try-Pro-Phe-Phe-DHis-Leu-Met-NH ₂ (1A) Try-Pro-DPhe-Phe-DHis-Leu-Met-NH ₂ (1B) Try- Pro-Trp-Phe-DHis- Leu-Met-NH 2 ··· (1C) Try-Pro-DTrp-Phe-DHis-Leu-Met-NH 2 ··· (1D)

The salt of the peptide represented by the formula (1) includes an acid addition salt derived from the peptide and an inorganic acid or an organic acid. Such salts include, for example, hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, p-toluenesulfonate, oxalate, tartrate, citrate, maleic acid Salts, fumarate, succinate, lactate, glutarate, acetate, trifluoroate, various amino acid salts and the like.

The salt of the peptide represented by the formula (1) includes a salt formed from the peptide and a base. Such salts include, for example, alkali metals (eg, sodium, potassium), alkaline earth metals (eg, calcium, magnesium), ammonium and substituted ammonium (eg, dimethylammonium,
Triethylammonium) and the like.

The peptide represented by the formula (1) is, for example,
It can be produced by a general peptide synthesis method as described in "Basic and Experimental Peptide Synthesis" (Maruzen Co., Ltd.). That is, the peptide represented by the formula (1) can be produced by sequentially performing the amino acid condensation reaction from the peptide carboxyl terminal by the peptide liquid phase synthesis method or the peptide solid phase synthesis method. Alternatively, after producing a fragment of the target peptide by a peptide liquid phase synthesis method or a peptide solid phase synthesis method, these are used to produce the peptide represented by the formula (1) by a peptide liquid phase synthesis method or a peptide solid phase synthesis method. be able to.

As the amino acid raw material to be used, those in which the amino group of the amino acid is protected with a protective group such as a t-butyloxycarbonyl group (Boc) or a 9-fluorenylmethyloxycarbonyl group (Fmoc). preferable. Further, it is preferable that the side chain functional groups of the amino acid raw materials used are each protected with a preferable protecting group.
Examples of these amino acid side chain protecting groups include t-butyloxycarbonyl group (Boc) and t-butyl group (tB
u), a trityl group (Trt), a benzyl group (Bzl),
And a 2,2,5,7,8-pentamethylchromansulfonyl group (Pmc).

Examples of the condensing agent for forming a peptide bond include N, N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (WSC), and 1H-benzotriazole. -1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP), 1H-benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (pyBOP), 2- (1H- Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBT
U), 2- (1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate and the like. Further, N-hydroxybenzotriazole (HOBt) and the above-mentioned condensing agent can be mixed and used in a preferable ratio.

For the formation of the peptide bond, a method of activating the carboxyl terminus may be used. Examples of the activating agent include N-hydroxysuccinimide and p-hydroxysuccinimide.
-Nitrophenyl esters and pentafluorophenyl esters. Examples of a base used for forming a peptide bond include triethylamine, diisopropylethylamine (DIEA) and the like. Examples of the solvent used for the peptide bond formation reaction include chloroform, methylene chloride, acetonitrile, N, N-dimethylformamide (DMF), and dimethyl sulfoxide.

Boc and Fmoc, protecting groups for the amino terminal amino group of a peptide or amino acid, can be removed with trifluoroacetic acid (TFA) or piperidine, respectively. Protecting groups for the side chain functional groups of the amino acid residues of the peptide include, for example, TFA, hydrogen fluoride (H
F), trifluoromethanesulfonic acid or the like.

In the peptide solid phase synthesis method, as a method for releasing a peptide having a protecting group on a side chain functional group of a peptide or an amino acid residue from a peptide solid phase synthesis resin, for example, TFA may be used. Can be. The elimination of the peptide from the peptide solid phase resin and the elimination of the protecting group of the side chain functional group of the amino acid residue can be performed simultaneously in the same reaction system. Alternatively, they can be performed independently. Examples of the peptide solid phase synthesis resin for peptide solid phase synthesis include 4-hydroxymethyl-3
-Methoxyphenoxybutyric acid-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin and oxime resin can be used.

The desired peptide or its intermediate can be isolated by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, recrystallization, extraction, and fractional crystallization. Purification can be performed. Further, the peptide represented by the formula (1), which is the target compound thus obtained,
Each salt can be converted by a conventional method.

The target compound of the present invention, the peptide represented by the formula (1) and a salt thereof are novel and have an activity on opioid receptors and an antagonistic activity on tachykinin receptors.

When the compound of the present invention is used for the prevention or treatment of an analgesic or a nervous system disease, the compound of the present invention is used as an active ingredient, and is preferably an organic or inorganic solid or a solid suitable for oral administration, parenteral administration or external use. It is used in the form of a conventional pharmaceutical preparation containing the compound as a mixture with a pharmaceutically acceptable carrier such as a liquid excipient. The pharmaceutical preparation may be in the form of tablets, granules, powders, solids such as capsules,
It may be a liquid, suspension, syrup, emulsion, or lemonade. If necessary, auxiliaries, stabilizers, wetting agents and other conventional additives may be added to the above-mentioned preparations, for example, lactose, citric acid, tartaric acid, stearic acid, magnesium stearate, clay, sucrose, corn starch, talc, gelatin, Agar, pectin, peanut oil, olive oil, cocoa oil, ethylene glycol and the like may be blended.

The dose of the target compound of the present invention varies depending on the age of the patient, the condition, the type of disease or medical condition, the compound of the present invention to be applied, and the like. A dose in the range of 500 mg, or even higher, may be administered to the patient. In treating various diseases, the average dose of the compound of interest of the present invention was 0.05 m
g, 0.1 mg, 0.25 mg, 0.5 mg, 1 mg,
What is necessary is just to use as 20 mg, 50 mg, 100 mg, etc.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples. In addition, the composition ratio (% etc.) of the mixed solvent used in the following examples
Represents a capacity ratio.

[0028]

【Example】

[Example 1] Tyr-Pro-Phe-Phe-DHis-Leu-
Synthesis of acetate salt of Met-NH _2.

(Synthesis Example 1) Tyr (tBu) -Pro-Phe-Phe-DHis
Synthesis of (Trt) -Leu-Met-amide resin.

The 4-substituted compound having a substitution rate of 0.49 mmol / g
About 0.5 g of (2,4-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamide-norleucylaminomethyl resin was used as the amide resin. 20
% Of piperidine (hereinafter referred to as 20% piperidine / DMF) in 20 ml of Fm of the amide resin.
oc removal was performed. Hereinafter, amino acids were sequentially condensed on this amide resin. 20% piperidine / DMF was used for removing Fmoc, and PyB was used for peptide bond formation reaction.
op 166 mg (0.5 mmol), HOBt 19
mg (0.125 mmol), DIEA 172 μl
(1 mmol) was used. The Fmoc amino acids used are shown in Table 1 in the order used for the condensation.

Finally, the amino-terminal Fmoc was removed with 20 ml of 20% piperidine / DMF, and then the solvent was removed to obtain the desired product, Tyr (tBu) -Pro-Phe.
-Phe-DHis (Trt) -Leu-Met-amide resin was obtained.

[0032]

[Table 1]

(Synthesis Example 2) Tyr-Pro-Phe-Phe-DHis-Leu-
Synthesis of TFA salt of Met-NH _2.

Tyr (tBu)-obtained in Synthesis Example 1
Pro-Phe-Phe-DHis (Trt) -Leu
-After thoroughly drying the Met-amide resin, TFA:
Mixed solution 3 of triisopropylsilane: water = 94: 2: 4
0 ml was added, and the mixture was reacted at room temperature for about 1 hour. After removing the solid, the remaining solution was distilled off under reduced pressure, and about 30 ml of diethyl ether was added to the residue obtained.
g was obtained. This solid was dissolved in a 20% aqueous acetic acid solution and dissolved in 0.1%.
% TFA aqueous solution, 0.1% TFA aqueous solution / acetonitrile mixed solvent was used for purification by C18 column reverse phase chromatography, and the target substance, Tyr-Pro-P
T of he-Phe-DHis-Leu- Met-NH 2
130 mg of FA salt was obtained.

(Synthesis Example 3) Tyr-Pro-Phe-Phe-DHis-Leu-
Met-NH ₂ acetate (hereinafter referred to as peptide salt a)
Synthesis of

Tyr-Pro-Ph obtained in Synthesis Example 2
e-Phe-DHis-Leu- Met-NH 2 of the TF
A salt (130 mg) is dissolved in a 5% acetic acid aqueous solution, and 20
The solution was replaced with an acetate salt by a strong base exchange resin column that had been treated with a 10% aqueous acetic acid solution. The obtained acetic acid solution was lyophilized to give Tyr-P, the target compound of Example 1,
ro-Phe-Phe-DHis-Leu-Met-N
120 mg of acetate of H ₂ were obtained.

Example 2 Tyr-Pro-Phe-Phe-DHis-Leu-
Identification of Met-NH ₂ acetate by amino acid analysis.

The Tyr-Pro-Phe obtained in Example 1
Some of the acetic acid salt of -Phe-DHis-Leu-Met- NH 2 140 ℃ with 6N HCl, after hydrolysis with 4 hours and analyzed for its amino acid composition. Table 2 shows the results.

[0039]

[Table 2]

Example 3 Tyr-Pro-DPhe-Phe-DHis-Leu
Synthesis of acetate -Met-NH _2.

(Synthesis Example 4) Tyr (tBu) -Pro-DPhe-Phe-DHi
Synthesis of s (Trt) -Leu-Met-amide resin.

4- having a substitution rate of 0.49 mmol / g
About 0.5 g of (2,4-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamide-norleucylaminomethyl resin was used as the amide resin. 20
% Piperidine / DMF 20 ml
moc removal was performed. Hereinafter, on this amide resin,
Amino acid condensation was performed. 20% piperidine / DMF is used for removing Fmoc, and Py is used for peptide bond formation reaction.
Bop 166 mg (0.5 mmol), HOBt19
mg (0.125 mmol), DIEA 172 μl
(1 mmol) was used. The Fmoc amino acids used are shown in Table 3 in the order used for the condensation.

Finally, the amino-terminal Fmoc was removed with 20% piperidine / DMF (20 ml), and then the solvent was removed to obtain the desired product, Tyr (tBu) -Pro-DPh.
e-Phe-DHis (Trt) -Leu-Met-amide resin was obtained.

[0044]

[Table 3]

(Synthesis Example 5) Tyr-Pro-DPhe-Phe-DHis-Leu
Synthesis of TFA salt of -Met-NH _2.

Tyr (tBu)-obtained in Synthesis Example 4
Pro-DPhe-Phe-DHis (Trt) -Le
After thoroughly drying the u-Met-amide resin, TF
A: 30 ml of a mixed solution of 94: 2: 4 of triisopropylsilane: water was added and reacted at room temperature for about 1 hour. After the solid was removed, the remaining solution was distilled off under reduced pressure, and about 30 ml of diethyl ether was added to the obtained residue to remove 15% of a white solid.
0 mg was obtained. This solid was dissolved in a 20% acetic acid aqueous solution and purified by reverse phase chromatography on a C18 column using a 0.1% TFA aqueous solution, a 0.1% TFA aqueous solution / acetonitrile mixed solvent, and the target substance, Tyr-Pr
o-DPhe-Phe-DHis-Leu-Met-N
The TFA salt of H ₂ to give 100 mg.

(Synthesis Example 6) Tyr-Pro-DPhe-Phe-DHis-Leu
-Met-NH ₂ acetate (hereinafter, the peptide referred salt b) Synthesis of.

Tyr-Pro-DP obtained in Synthesis Example 5
T of he-Phe-DHis-Leu- Met-NH 2
Dissolve 130 mg of FA salt in 5% aqueous acetic acid
It was replaced with an acetate type by a strong base exchange resin column which had been treated with a 0% acetic acid aqueous solution. The obtained acetic acid solution was freeze-dried to give Tyr-
Pro-DPhe-Phe-DHis-Leu-Met
Acetate of -NH ₂ to give 97 mg.

Example 4 Tyr-Pro-DPhe-Phe-DHis-Leu
Identification by amino acid analysis of -Met-NH ₂ acetate.

Tyr-Pro-DPh obtained in Example 3
A part of e-Phe-DHis-Leu-Met-NH ₂ acetate was hydrolyzed with 6N HCl at 140 ° C. for 4 hours, and the amino acid composition was analyzed. Table 4 shows the results.

[0051]

[Table 4]

Example 5 Tyr-Pro-Trp-Phe-DHis-Leu-
Synthesis of Met-NH ₂ acetate.

(Synthesis Example 7) Tyr (tBu) -Pro-Trp (Boc) -Phe
-Synthesis of DHis (Trt) -Leu-Met-amide resin.

4-substituted with a substitution rate of 0.49 mmol / g
About 0.5 g of (2,4-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamide-norleucylaminomethyl resin was used as the amide resin. 20
% Piperidine / DMF 20 ml
moc removal was performed. Hereinafter, on this amide resin,
Amino acid condensation was performed. 20% piperidine / DMF is used for removing Fmoc, and Py is used for peptide bond formation reaction.
Bop 166 mg (0.5 mmol), HOBt 1
9 mg (0.125 mmol), DIEA 172 μl
(1 mmol) was used. The Fmoc amino acids used are shown in Table 5 in the order used for the condensation.

Finally, the amino-terminal Fmoc was removed with 20% piperidine / DMF (20 ml), and then the solvent was removed to remove the target compound, Tyr (tBu) -Pro-Trp.
(Boc) -Phe-DHis (Trt) -Leu-M
An et-amide resin was obtained.

[0056]

[Table 5]

(Synthesis Example 8) Tyr-Pro-Trp-Phe-DHis-Leu-
Synthesis of Met-NH ₂ TFA.

The Tyr (tBu)-obtained in Synthesis Example 7
Pro-Trp (Boc) -Phe-DHis (Tr
After sufficiently drying the t) -Leu-Met-amide resin, TFA: triisopropylsilane: water = 94: 2:
30 ml of the mixed solution of No. 4 was added, and the mixture was reacted at room temperature for about 1 hour. After removing the solid, the remaining solution was evaporated under reduced pressure, and about 30 ml of diethyl ether was added to the obtained residue to obtain 160 mg of a white solid. This solid was dissolved in a 1N hydrochloric acid solution and freeze-dried to obtain about 150 mg of a white powder. This powder was dissolved in a 20% aqueous acetic acid solution, and purified by reverse phase chromatography on a C18 column using a 0.1% aqueous TFA solution and a 0.1% aqueous TFA / acetonitrile mixed solvent to obtain the target substance, Tyr-Pro-Trp. -Phe
_{-DHis-Leu-Met-NH 2} TFA 130
mg.

(Synthesis Example 9) Tyr-Pro-Trp-Phe-DHis-Leu-
Synthesis of Met-NH ₂ acetate (hereinafter referred to as peptide salt c).

The Tyr-Pro-Tr obtained in Synthesis Example 8
p-Phe-DHis-Leu-Met-NH ₂ TF
A130mg was dissolved in 5% acetic acid aqueous solution, and 20%
It was replaced with an acetate type by a strong base exchange resin column that had been treated with an acetic acid aqueous solution. The obtained acetic acid solution was lyophilized to give Tyr-Pr, the target compound of Example 5,
o-Trp-Phe-DHis-Leu-Met-NH
₂ acetate was obtained 115 mg.

Example 6 Tyr-Pro-Trp-Phe-DHis-Leu-
Identification of Met-NH ₂ acetate by amino acid analysis.

The Tyr-Pro-Trp obtained in Example 5
A portion of -Phe-DHis-Leu-Met-NH ₂ acetate was hydrolyzed with 6N HCl at 140 ° C. for 4 hours, and the amino acid composition was analyzed. Table 6 shows the results.

[0063]

[Table 6]

Example 7 NK-2 Receptor Binding Activity Test The compound of the present invention (the above-mentioned peptide salts a to c) was prepared using Chinese Hamster Ovary cells expressing human NK-2 receptor. Was measured for its affinity for the receptor. Affinity was determined by a binding competition test using [ ¹²⁵ I] neurokinin-A.
Table 7 shows the results. In addition, the numerical value is the concentration of peptide salt 1.
The inhibition rate (%) at 0 × 10 ⁻⁶ M is shown.

[0065]

[Table 7]

[0066]

Industrial Applicability The peptide of the present invention is an opioid-like peptide having a structure showing an agonistic activity on endomorphin and an antagonistic activity on a tachykinin receptor.

Claims (4)

[Claims] 1. A peptide represented by the following formula (1) (however, excluding peptides represented by the following formulas (2) and (3)), or a salt thereof: Embedded image Tyr-Pro-X-Phe-Y-NH ₂ (1) Tyr-Pro-Phe-Phe-NH ₂ (2) Tyr-Pro-Trp-Phe-NH _2. -(3) [In the formula (1), X and Y each represent an amino acid residue, a peptide residue or a single bond (however, X and Y are not a single bond at the same time) Pro,
The amino acid residue which is Phe, X or Y, and one or more amino acid residues in the peptide residue which is X or Y may be D-form. The amino acid residues in the peptide residues represented by the formulas (2) and (3) are all L-forms. ] 2. The peptide or salt thereof according to claim 1, wherein X is an amino acid residue and Y is an amino acid residue or a peptide having 3 or less amino acid residues. (3) X is Phe or Trp (provided that X is
The peptide or a salt thereof according to claim 1 or 2, wherein Y is His-Leu-Met (where His is D-form) from the amino terminal side. 4. A medicament comprising the peptide according to claim 1, 2 or 3 or a salt thereof as an active ingredient.