JPS6168499A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I (n is 0 or 1; R is Pro, Pro-Ile-Pro). USE:An analgesic, or hypnotic. PREPARATION:Cesium salt shown by the formula Boc-L-Pro (Boc is t- butyloxycarbonyl) is dissolved in DMF, etc., reacted with chloromethyl polystyrene resin, the amino-protecting group is removed, and an amino acid whose amino group is protected according to the amino acid sequence of the peptide is reacted in the presence of a condensation agent of a carbodiimide, to remove the amino-protecting group. These operations are repeated to give a peptide resin. The resin is treated with a solution of m-cresol, trifluoromethanesulfonic acid, and thioanisole in trifluoroacetic acid, elimination of protecting groups from the peptide resin and removal of the whole protecting groups are carried out to give a compound shown by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide that can be expected to be used as a medicinal agent such as analgesic and hypnotic agent.

A peptide with opioid activity (morphine-like activity), β-casimorphin 7, has been isolated from milk zein peptone (Hoppe-8eyler's Z, P
hysiol, Chem, 360.1211 and 121), 1979), but no reports regarding peptides having opioid activity as in the present invention have been found.

The development and production of peptides that can be used as medicines such as analgesics and hypnotics is expected.

The present inventor has succeeded in newly synthesizing peptides having the general formula % and their amide and ester derivatives, and these new peptides have opioid activity and are suitable for use in the above-mentioned medicines. We have discovered something promising, and have completed the present invention based on this discovery.

In the novel peptide of the present invention, the N-terminus, C-terminus, and side chain functional groups may be protected with protecting groups commonly used in peptide synthesis. Furthermore, the constituent amino acids may be either L-unit or D-unit.

The novel peptide of the present invention is produced based on the Examples described below, and further using a conventional peptide synthesis method (see, for example, Izumiya et al., Synthetic Chemistry Series "Peptide Synthesis" published by Maruzen Co., Ltd., 1975). be able to. The same applies to the method of protection using a protecting group or the method of removing it.

Among the novel peptides of the present invention, amide derivatives can be synthesized by conventional methods, such as a similar peptide synthesis method using the amide in place of the C-terminal amino acid, or a method in which the corresponding peptide ester is amidated by treatment with ammonia. can be manufactured.

When the novel peptide of the present invention is used as an analgesic or hypnotic agent as an active ingredient, it can be used as an in-form or as a pharmaceutically acceptable non-toxic salt or acid addition salt.

In the present invention, the pharmaceutically acceptable non-toxic salts include commonly used organic and inorganic acid addition salts, such as addition salts with hydrochloric acid, sulfuric acid, sulfonic acid, citric acid, phosphoric acid, and benzoic acid. do it.

Furthermore, on the other hand, alkali metal salts such as Na and nitric acid salts and ammonium salts are included.

The novel peptide of the present invention is effective as an analgesic or hypnotic agent for humans and mammals, and can be used to relieve various pains such as gallstone colic, renal stone colic, cancer pain, and post-operative pain. It is also effective as a hypnotic drug due to its hypnotic action.

For administration, they may be formulated in tablets, capsules, or elixirs for oral administration, or in sterile solutions or suspensions for parenteral administration.

In addition, physiologically recognized Vexil, carriers, excipients,
They may, of course, be incorporated and administered in unit dosage form as required by generally accepted pharmaceutical practices, along with binders, preservatives, stabilizers, flavoring agents, and the like.

The amount of active substance used in these compositions or preparations is such that a suitable dose within the indicated range will be obtained.

The dosage of the active ingredient is determined by considering the severity of the disease, weight and age of the patient, and other factors.

The abbreviations used in this specification are as follows.

Tyr: Tyrosine, Pronibroline, Phe: Phenyl 7? nin, Val: valine, Glu: glutamic acid, Ile: isoleucine, Boa: t-butyloxycarbonyl, 0Bzl: penciloxy, Cl2-Bzl: 2,8-19clk pen') 7, T
FA:) Lifluoroacetic acid, ODS: octadecylsilane, HEPES: N-2-hydroxyethylpiperazine-
N'-2-ethanesulfurefonic acid

Example I Tyr-Pro-Phe-Val-Glu
-Pr.

Ascending Tyr-Pro-Phe-Val-Glu-Pr
Synthesis of o-11e-Pro 1, Introduction of Boc-L-Pro into chloromethyl resin 2
After adding and dissolving ethanol (12 theory 2) and 4 ml of water to Boc-L-Pro (g), the mixture was neutralized with an aqueous cesium bicarbonate solution and dried to obtain a cesium salt. This is 85m
7.2 g of chloromethyl polystyrene resin (1,28+eq, CI/g
) and stirred overnight at 50°C. After the reaction was completed, the resin was washed with 300 mN dimethylformamide, 300 m, i! of 90% dimethylformamide, 300ml of dimethylformamide and 600J! Boa-L-Pro tM fat (0.86 mmol)
Pro/g) was obtained.

2. Extension of peptide chain on Boa-L-Pro resin 1.6 g of the above Boa-L-Pro resin was added to 40 ml of debo
After removing the Boa group by boiling for 30 minutes at room temperature in an a-forming agent (55% TFA, 5% anisole, 40% methylene chloride mixture, each v/v%), 60 ml of 33% dioxane and then 67% chloride were added. Wash with methylene mixture, 20ml
The mixture was neutralized with a mixture of 10% triethylamine and 90% methylene chloride, and washed with 100 d of methylene chloride. 13.8-mol of dicyclohexylcarbodiimide, which corresponds to 10 times the equivalent of Pro resin, was added to 13.8 mmol of Boc-
Dissolve L-Ile in 16 ml of methylene chloride and
It was added to the resin and allowed to stand overnight at room temperature.

After the reaction was completed, the resin was washed with methylene chloride, and it was confirmed that there was no unreacted amine 7 group in the ninhydrin reaction.
OBzl), Boc-L-Val, Boc-L-P
heS Boa-L-Pro, Boa-L-Tyr (C
lz-B zl) were coupled in this order to obtain an octapeptide-resin.

In addition, Boa-L-G resin is separately added to Boa-L-Pro resin.
1u (OBzl), Boa-L-Val, Boa-L
-Phe.

Boa-L-Pro Boa-L-Tyr(C1,-
Bzl) were combined in this order to obtain a hexapeptide resin.

3. Cleavage of peptide from resin and removal of protective groups To 1.25 g of the above octapeptide resin was added 1M tri7fluoromethanesulfonate solution of 1,1a+N m-cresolcyto10@1 at 0°C for 30 minutes, and then After reacting for 2 hours at room temperature, 190+J! of ether was added and centrifuged. The obtained precipitate was subjected to the same operation as above and then filtered with suction to obtain a filtrate. To this, 190 mN of ether was added to precipitate and collect the peptide, which was dissolved in water, neutralized with ammonia, and then freeze-dried. Similar treatment was performed for hexapeptide.

4. Purification of peptide The peptide obtained as described above was subjected to gel filtration using a Biogel P-2 column equilibrated with 0.1M ammonia acetate to obtain the earliest eluting fraction.

These two fractions were transferred to an ODS column (Cosmosil 5 C
+s) by reverse phase chromatography with 0.1% T
Elution was performed with an acetonitrile gradient containing FA. 280n for both octapeptide and hexapeptide
One main peak with absorption at m was obtained, each with 2
Eluted with 9% and 26.5% acetonitrile. When these peaks were dried by concentrating centrifugation and subjected to amino acid analysis, each showed the predicted amino acid composition.

Yield: Octaveptide 300mg, Hexapeptide 25
0mg.

5. Preparation of pentapeptide by carboxypeptidase treatment of hexapeptide
1 of 100mM HEPES buffer (pH 7.
0) and 0.5B of carboxypeptidase Y (manufactured by Oriental Yeast Co., Ltd., 130 units/mg) was added, and after 5 minutes, the gradient was developed at 100"C. The peak eluted with 24% acetolyl was T
yr:Pro:Phe:Val:Glu=
It had an amino acid composition of 1:1:1:1:1 and was the desired pentapeptide.

6. Physicochemical properties T r-Pro-Phe-Val-Glu specific light intensity [α
]t, = -49,3° (C = 0.3, methanol) U
V A a+ax=277, 5nms Ex”y.s
nm=20.2 (methanol) Amino acid composition (6N H C 1, 110°C 24 hour hydrolysis) 2G lu(1) 0.98, Pro(2) 2.
04, Val(1) 1.00, Tyr(1) 0.9f, Phe(1) 1.03ODS column (Cosmos
il 5 C+s, 4,6X150mm, made by Gyudon Kagaku)
Elution from: 0-50% acetonitrile linear graphene containing 1% TFA) 150 m, l! Tr-Pro-Phe-Vat Glu Pr eluted with 29% acetonitrile.

Specific luminosity [ff]. = −64.0” (C =0.
3, methanol) UVλ-ax=277, 5nm,
]11g'47.1nm=17. ．． 6 (methanol) Amino acid composition (hydrolyzed under the same conditions as above) G lu (1
) 0.99, Pro (2) 2.02, Val (1)
1,001 T yr (1) 0,99, P he (1) 1
．． Elution from the 02ODS column: Elution with 31% acetonitrile under the same conditions as above.Tr-Pro-Phe-Val-Glu-Pro-
I le-Pr.

Specific light intensity [a]l) = -72.1° (C=0.3, methanol) UVλ-ax-277, 5nmq Ez?
t. s=13, F(methanol) amino acid composition (hydrolyzed under the same conditions above) G lu (1) 0.98, P r
o(3)2,95, V al(1)1.00, I le
(1)1,02,Tyr(1)0,98,Phe(
1) Elution from the 1.02 ODS column: Elution with 35% acetonitrile under the same conditions as above Example 2 Measurement of rat brain obioid receptor assay (1) Experimental method (Snyder) et al.'s method (Proe, N.
atl.

Acad, Sci, USA, 70, 2243 (1
973). ).

Cerebrum of a male Wistar rat (100-200g) (1
, 1 to 1.3 g), and using a Potter homonizer, 10 m, l! 50mM) Lysu salt RvL solution (pH 7,4) was homogenized under O'C. This was diluted to 100 times the weight of the layer with the same buffer solution, and then centrifuged (1,000 rpm, 5 minutes, 0°C) to remove the precipitate.

Add the sample or morphine hydrochloride (
(manufactured by Takeda Pharmaceutical Company, Ltd.) and incubated at 35°C for 5 minutes. Subsequently, [3H]-naloxone (NEN,
37,7Ci/+110101) at a final concentration of 1 nM (
34,000c, p, m,) and again 3
Incubated for 155 minutes at 5°C.

Glass filter (Whatman GF/B
Vacuum filtration was carried out using a 2.4-cm filter to retain the membrane components with receptors on the filter, and the filter was quickly washed four times with 4 ml of buffer (owning time 3
0 seconds), put this filter in the planning tool, and
111 of 10% sodium dodecyl sulfate (SDS) was added and left to stand for 30 minutes or more. Then, 10− PSC(A
Marsham Co., Ltd.) was added thereto, shaken well, and measured using a liquid scintillation counter. However, the amount of specific binding was determined by subtracting the amount of binding observed in the presence of a large excess of non-radioactive naloxone. The activity of the sample is [3H1
- Expressed as the temperature of the sample required to inhibit the specific binding of naloxone by 50% (ICs).

(2) Results - IC, oμM pentapeptide 600 hexapeptide
540 octapeptide 1
300 or more, the novel peptide of the present invention has mild morphine-like analgesic activity and is expected to be used as a pharmaceutical.

Claims (1)

[Claims] 1) A peptide represented by the general formula Tyr-Pro-Phe-Val-Glu-(R)n, and its amide and ester derivatives. However, in the formula, n is 0 or 1, R is Pro or P
ro-Ile-Pro, respectively.