JPS6049200B2 - peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide that acts on mast cells and exhibits a degranulation effect.

Many peptides have been known that exhibit mast cell degranulation activity, but most of them exhibit smooth muscle contraction activity.

Recently, Granule Liberine-R was announced by Nakajima et al.
(Granuliberin-R) is isolated from the skin of the Japanese frog, and its structure is H-Phe-Gly-P.
he-Leu-Pro-ne-Tyr-Arg-Arg
-Pro-Ala-Ser-Ni, which is a physiologically preferred peptide that exhibits mast cell degranulation effects but no effect on smooth muscle.

The present inventors learned the chemical structure necessary for the degranulation action of Granulelibellin-R, and pursued the main body of the action with the aim of searching for a lower molecular weight active substance, and found that the active center is C-
We discovered that it exists on the terminal side, synthesized various peptides based on this, and as a result of further investigation, we completed the present invention.

That is, the present invention provides the formula H-ne-Tyr-Arg-A
rg-X-R(I) [wherein X is Pro) Pro-Al
a or Pro-Ala-Ser, and R is OH or NH.

, respectively. ] There is a peptide represented by In this specification, indications regarding amino acids, peptides, protecting groups, etc. are based on IUPAC-IUBCommissi
-ononBiologicalNomenclatu
It is based on the abbreviation re or common abbreviations in the field, examples of which are shown below.

■e: Isoleucine Tyr: Tyrosine Arg: Arginine Pro: Proline A1a: Alanine Ser: Serine z: Carbobenzoxy group BOC: Tert-butyloxycarbonyl group NO2
: Nitro group OMe: Methyl ester OB21: Benzyl ester HONE: N-hydroxy 5-norbornene-2,
3-Dicarboximide ONB: Same ester ■°C: N,N'-dicyclohexylcarbodiimide Also, R of thin layer chromatography (silica gel plate)
The following solvent system was used for the f value.

Rfl: Chloroformrimethanol: Glacial acetic acid
(9:1:0.5) Rf2: Ethylnipyridine acetate diglacial acetic acid: water (60:20:6:10) Rf3:
Ethyl acetate diglacial acetic acid: n-butanol dihydrate (
1:1:1:1) Rf4: n-butanol pyridine diglacial acetic acid: water
(30:20:6:24) Compound (1) of the present invention can be produced by appropriately using a conventional method of peptide condensation reaction and a suitable means for protecting a functional group.

Such peptide condensation reactions and protection measures are, for example,
hePeptides, Volume 1 (1966), Sch
rOderandLubke, AcademicPr
ess. sNewYOrk. sU. S. A or '6
Peptide synthesis by Izumiyaya, Maruzen Co., Ltd. (197 master)
More specifically, condensation reactions include, for example, the azide method, chloride method, acid anhydride method, DC
C method, active ester method, Woodward method, carbodiimidazole method, redox method, DCC/Atiteb (HON
B. ．． HOBt. ．． Examples include the HOSu) method. Further, as a method for protecting a functional group, for example, as a protecting group for an α-amino group, carbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2- Examples include chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, 0-nitrophenylsulfenyl, diphenylphosphinothioyl, and the like.

Although a protecting group for the guanidino group of arginine is not necessarily required, it may be protected with, for example, a nitro group, tosyl group, carbobenzoxy, isopolyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzelsulfonyl, or the like. In addition, carboxyl groups are, for example, metal salts (e.g., sodium, potassium, lithium salts, etc.), t-
Alkylamine salts (e.g., triethylamine, N-methylmorpholine, N-ethylmorpholine, etc.) or esters (e.g., methyl, ethyl, propyl, t-butyl, t-
-amyl, benzyl, p-nitrobenzyl and other esters). Furthermore, it is not necessary to protect both the hydroxyl group of serine and the hydroxyl group of tyrosine, but in solid phase synthesis, they may be protected with ethers such as benzyl, t-butyl, 2-chlorobenzyl, etc., but in liquid phase synthesis, In some cases, the above-mentioned ethers and the like are used as protecting groups. During the production process of this peptide (1), the condensation reaction is carried out in the presence of a solvent.

The solvent may be appropriately selected from those known to be usable in the condensation reaction. For example, anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane,
Examples include tetrahydrofuran, ethyl acetate, and appropriate mixtures thereof. The reaction temperature is generally about -30°C to 50°C, which is generally used for peptide bond-forming reactions.
The temperature is appropriately selected from a range of about 60°C. The bond-forming reaction is mainly carried out by activating the carboxyl group of the raw material. , p-nitrophenol, cyanomethyl alcohol, N-hydroxy 5-norbornene-2,3-dicarboximide,
In addition to N-hydroxysuccinimide, N-hydroxyphthalimide, esters with N-hydroxybenztriazole), dehydrating agents such as dicyclohexylcarboimide and carbodiimidazole are also used. After the condensation reaction is completed, the product generally has a protecting group, which is removed by a conventional method.

Such a conventional method is carried out, for example, by subjecting the compound (1) having a protecting group to a reduction reaction or solvent decomposition (solpolysis). Reduction reactions include hydrogenation using catalysts such as palladium black, palladium-carbon, and platinum, reduction with metallic sodium in liquid ammonia, and solpolysis using trifluoroacetic acid, hydrogen fluoride,
Examples include solpolysis using strong acids such as methanesulfonic acid.

Peptide (1) produced as described above can be collected from the reaction mixture by peptide separation means, extraction, distribution, column chromatography, etc.

Since peptide (1) has an arginine residue, it is generally collected in the form of a salt.

Acids which can form salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, or for example formic acid, acetic acid, propionic acid, lactic acid, citric acid, cylic acid, maleic acid, fumaric acid. Examples include organic acids such as acid and tartaric acid. The peptide (1) of the present invention acts on animal mast cells and has the effect of causing the mast cells to release granules, and along with this, releases histamine, so it can be used as an antihistamine, anti-allergic agent, etc. It can be used as a reagent for searches such as

Specific examples of use as the reagent are listed below.

That is, mast cells of mammals are separated by the method specifically explained in the reference example, and about 1 x 1σ of these mast cells are suspended in a suitable medium, and the total volume is about 1 ml, and 1 ml is added. Three types of reagent solutions are prepared: one without, one containing two compounds (1), and one containing three compounds (1) and an appropriate amount of the compound to be subjected to screening. Each of these is heated at 3 rC for 1 to 2 minutes, and after the reaction is completed, it is immersed in ice water to stop the reaction. Centrifuge and heat the supernatant at 100°C for 10 minutes.
After centrifuging again, the histamine content in the supernatant is measured and compared to determine the antihistamine release activity of the compound subjected to screening. Regarding the amino acid residues constituting the peptide (1), if an optically active form is possible, L-integrated forms are preferable from the viewpoint of action and effect.

In the following Examples and Reference Examples, regarding amino acids, the optically active form is assumed to be an L-unit unless otherwise specified.

Example 1 Production of H-■e-Tyr-Arg-Arg-PrO-0H (1) BOC-Tyr-Ar-g(NO2)-Ar-
g(NO2)-PrO-0CH3Z-Arg(NO2)
-Arg(NO2)-PrO-0CH3(R.Schw
yzerandH. KappeIerlHeIveti
cahlmlCaACta. ．． Outside 1550 (1963
)) Dissolve 3.7 Da in 10 ml of 25% hydrogen bromide, monoglacial acetic acid, and stir at room temperature.

100 ml of dry ether is added to this, and the resulting precipitate is collected by filtration and dried. Add this powder to 6 m of dimethylformamide.
1 and 1.4 mt of triethylamine and BO under ice cooling.
Add 2.2y of C-Tyr'-ONB and stir at room temperature for 1 full hour. Insoluble matters are removed by filtration, the solvent is distilled off under reduced pressure, ethyl acetate is added to form a powder, and the powder is collected by filtration. This is purified by reprecipitation from aqueous dimethylformamide and further reprecipitation from methanol-ethyl acetate. Yield 3.43y (86
%), melting point 144-146 Sei C1 [α] 0-40.7
(c=0.4, methanol), Rfl=0.0cmRP=
Calculated value as 0.61 elemental analysis C32H5OOl2N2.H2O: C47.28; H6.45; N2O. 68 actual value: C47.54; H6.33; N2O. 52(2)
BOC-11e-Tyr-Arg(NO2)-Arg(
NO,)-PrO-0cH3B0C-Tyr-Arg(
NO2)-Arg(NO2)-PrO-0CH, 2.0
y is anisole 1.35wL1 and trifluoroacetic acid 10
Dissolve in ml and stir at room temperature.

Trifluoroacetic acid was distilled off under reduced pressure, and ether was added to the residue to form a powder, which was collected by filtration. After drying, it was dissolved in 15 mL of dimethylformamide, and 0.35 ml of triethylamine and BOC-11e-ONBl. Add 2g and stir for m hours. The solvent is distilled off, ethyl acetate is added, the powder is collected by filtration, and the powder is reprecipitated with water-containing methanol. Yield 1.9f (84%), melting point 120°C (decomposition), [
α〕? -43.0 ((c=0.methanol), Rf2
=0. Legend RP=0.60 Elemental analysis C38H6lOl3N
Calculated value as l3・H2O: C49.29; H6.86
;Nl9.67 actual value:C49.54;H6.87;N
l9.98(3) BOC-11e-Tyr-Arg(N
O2)-Ar'g(NO2)-PrO-0HB
0C-11e-Tyr-Arg(NO2)-Ar'g(
NO2) -PrO-0CH31.8y in methanol 4T
Dissolve fLt in 2 ml of water, add 4.7 ml of 1N sodium hydroxide under ice-cooling, stir for 3 minutes, and further stir for 4 hours and 7 at room temperature.

Cool on ice and neutralize by adding 4.7 mt of 1N hydrochloric acid, filter the precipitate, wash with water, and dry. 1.3q0 of this was mixed with ethyl acetate-pyridine-acetic acid monohydrate (106:30:9
:15), poured into a silica gel column (4×4 Crft) filled with the same solvent, and eluted with the same solvent.

74 to 1 L of elution fraction was collected, the solvent was distilled off under reduced pressure,
Add ether and filter as a powder.

Yield 1.08y1 Melting point 143C (decomposition), Rf2=0
．． 511[α]Ba0-35.8=0. (Methanol) Elemental analysis Calculated value as C37H59Ol3・H2O: C4
8.73: H6.74; Nl9.97 Actual value: C48.
8O; H6.8O; Nl9.94 (4) H-■e-Ty
r-Arg-Arg-PrO-0HB0C-11e-T
yr-Arg(NO2)-Ar-g(NO2)-PrO
450 mg of -0H is dissolved in 100 ml of glacial acetic acid and catalytically reduced using palladium black as a catalyst.

After 8 hours of hydrogenation, the catalyst is filtered off, the filtrate is dried under reduced pressure, dissolved in a small amount of water, and freeze-dried.

Yield 410WL9. 50mg of this product is mixed with 2 parts of trifluoroacetic acid.
After 15 minutes at room temperature, the residue was dried under reduced pressure, and the residue was purified with a column (1.0 x 91.5 cff1) of Cephadex LH-20 (manufactured by Pharmacia Fine Chemical Co., Ltd.).
Gel filtrate with normal acetic acid water. 29-40m
Fractions of L are collected and lyophilized to obtain the desired product 28m9.

Rf4=0.57 (cellulose plate), [α] Bas 0
-18.1=(c=0.32, 1N acetic acid water) Example 2 H-11e-Tyr-Arg-Ar'g-PrO-NH
2BOC-11e-Tyr-Arg(NO2)-Arg
900m9 of (NO2)-PrO-0H is dissolved in 10ml of dimethylformamide, HONB2OOm9 is added thereto, and 250mg of DCC is added under ice-cooling.

After stirring for 2 hours under ice-cooling and 8 hours at room temperature, 1.5 ml of 1N ammonia-dioxane was added under ice-cooling and stirred for 1 full hour. The insoluble matter was filtered off and dried under reduced pressure, dissolved in ethyl acetate-pyridine-acetic acid monowater (106:30:9:15), and added to a silica gel column (4Cr11X) filled with the same solvent.
5C71) and elutes with the same solvent. 85-175
A fraction of m1 was collected and dried under reduced pressure, and the powder was collected and filtered with ether.

Yield 620mg. Rf2=0.62 Dissolve 50mg of this product in 100ml of glacial acetic acid, use palladium black as a catalyst,
Time contact reduction.

The catalyst was filtered off, dried under reduced pressure, added with 2 ml of trifluoroacetic acid, left to stand at room temperature, dried again, and dried in a cephadec.
A column of 3N monoacetic acid (1.0 x 91°G) was coated with 3N monoacetic acid. A portion of 30-40 mL is collected and freeze-dried to obtain 24m9 of the desired product.

Rf4=0.60 (cellulose plate), [α]? −
1&4f (c=0.5.1N-acetic acid water) Example 3H-
Ne-Tyr-Arg-PrO-Ala-Ser-0H
Production (1) BOC-Ala-Ser-0Bz1H-S
er-0Bz1●Balatoluenesulfonate 17.7y
mixed solvent of dioxane and ethyl acetate (1:1) 150
ml and add 7 mL of triethylamine.

Add 7.5 g of BOC-Ala-CNBl to this and
Stir at room temperature. Distill the solvent and add ethyl acetate
00mt, washed with 01N hydrochloric acid water and 5% sodium bicarbonate water, dried over anhydrous sodium sulfate, and dried under reduced pressure. The residue is crystallized from ethyl acetate and petroleum ether and filtered off. ) Yield 15.3 Da. melting point 50-52,
C1 [α] 0-27.3 = 1.0 ton methanol), R
Calculated value as fl=0.71 elemental analysis Cl8H26O6N2: C59. OO;H7. l5; N7.65 actual value:
C58.96; H7. O3; N7.6O gate (2) BOC
-11e-Tyr-Arg(NO2)-Arg(NO2
)-PrO-Ala-Ser-0Bz1B0C-Ala
-Ser-0Bz11.8y was dissolved in 3 ml of 6°N hydrochloric acid monodioxane and left to stand at room temperature.

Ether was added and the precipitated crystals were collected by filtration and dried.
Yield 1.5y (100%). 515mg of this product and BOC-e-T2pT-Arg(NO2)-Arg(NO2)
-PrO-0H1.07y with dimethylformamide 5n
Dissolve in t and add 0.24 mt of triethylamine (5H0N
Add B432wI9, cool to 0℃, and heat to ■℃397Tn.
Add 9 and stir for 4 hours, then let it cool to room temperature for 1'2! IF!
Stir for a while. After filtration, the filtrate was evaporated under reduced pressure, and the residue was washed with 2% aqueous sodium bicarbonate and then with 1% aqueous acetic acid. After drying, it is purified by dissolving it in dimethylformamide and reprecipitating it by adding ethyl acetate. Yield 1.2y0 Melting point 140°C (decomposed), [α] Bass 0-35.7. (c=0.
75, glacial acetic acid), Rf2=0.82(3)H-11e-
Tyr'-Arg-, Arg-PrO-Ala-Ser
10H above BOC-■e-Tyr''-Ar'g(NO
2)-Arg(NO2)-PrO-Ala-Ser-0
Bz11y was dissolved in 200 ml of glacial acetic acid, catalytically reduced using palladium black as a catalyst for 8 hours, the catalyst was filtered off, and the mixture was dried under reduced pressure.

The residue was dissolved in 10 ml of water and applied to a column of carboxymethyl cellulose (2.5 x 36 cTn) at a concentration of 0.0
% normal and 0.2 normal acetic acid ammonia water (700 mt1
Gradient elution is performed at 700 mt). 355-475
The mL elution fractions are collected and lyophilized.

Yield 650m9. 32m9 of this product to 2m of trifluoroacetic acid
1, leave at room temperature for 10 minutes, and dry under reduced pressure.

The residue was dissolved in 3N acetic acid water, and Sephadex LH-
20 columns (1.0 x 92 CTf1) with 3N acetic acid water. The elution fraction of 32-40 ml is collected and lyophilized to obtain 18 ml of target product.

[α] 6 minutes-50.5 dry (c=0.2, N-acetic acid), R
r4=0.50 (cellulose plate) Amino acid analysis value: Ar'G2. O8, SerO. 98, Alal. O
O. , PrOl. O2, 11EO. 97, TyrO. 9
9 Example 4H-11e-Tyr-Arg-Arg-Pr
O-. Production of Ala-Ser-NFI2 (1) Z-Al
a-Ser-NH2 Z-Ser-NH22.4y is dissolved in 200 mL of methanol and catalytically reduced using palladium black as a catalyst for 4 hours.

The catalyst was filtered off and dried under reduced pressure. The residue was dissolved in 30 mt of dimethylformamide, 3.8 mt of Z-Ala-ONB was added, and the mixture was stirred at room temperature for an hour. The solvent is distilled off under reduced pressure, ether is added, the powder is collected by filtration, and it is recrystallized from methanol. Yield 2.0y0 Melting point 212-214-C0 [α]
? -10.5. (c=1.1, methanol), Rfl=
0.2, Rf2=0.75 Elemental analysis Cl4Hl9O5N
Calculated value as 3: C54.36; H6. l9; Nl3.59 Actual value: C54.26: H6.23; Nl3.55 (2) B
OC-11e-Tyr-Arg- (NO2)-Arg
(NO2)-PrO-Ala-Ser-NH2Z●Al
A-Ser-NH2l3Omg was dissolved in 50ml of methanol and catalytically reduced using palladium black as a catalyst for 4 hours.

Filter, dry the filtrate under reduced pressure, and dissolve the residue in 7 ml of dimethylformamide. To this ■℃−11e−Tyr−
Arg(NO2)-Arg(NO2)-PrO-0H3
Add 13m9 and 26mg of HONBl and cool to 0°C.
Add 87 mg of DCC and stir for 4 hours. After further stirring at room temperature for w hours, filter and add ether. The resulting precipitate is collected by filtration and reprecipitated with methanol and ethyl acetate. Yield 196m9. Melting point 157-160°C (decomposition),
[α]? 5-48.3. (c=0.48, methanol)
, Rf2=0.3 DenRP=0.73 Elemental analysis C43H7
Calculated value as OO5Nl6・CH3OH-H2O: C4
7.99:H6.96;N2O. 35 Actual value: C47.
58; H6.68; Nl9.98 (3) H-11e-T
yT'-Arg-. Ar'g-PrO-Ala-Ser
-NH2BOC-11e-Tyr-Arg(NO2)-
Ar-g(NO2)-PrO-Ala-Ser-Nll
Dissolve 289 mg in 1 ml of trifluoroacetic acid containing 0.06 mL of anisole and leave at room temperature for 3 times.

Trifluoroacetic acid is distilled off, ether is added, and the powder is collected by filtration, 767F! 9. This was dissolved in 20 ml of acetic acid and catalytically reduced using palladium black as a catalyst. The catalyst was filtered off and dried under reduced pressure. The residue was dissolved in 1N aqueous acetic acid and applied to a column of Sephadex LH-20 (1.0 x 100ml) with 1N monoacetic acid. When the elution fraction of 31 to 3f3ml is collected and freeze-dried, 24m9 of the target product is obtained.

[α] Basa 3-50.5j (c=0.44, 1N monoacetic acid)
, Rf3=0.57 (cellulose plate), Rf4
=0.52 (cellulose plate) amino acid analysis Ar
gl. 92, SerO. 89. Alal. OO, PrO
l. Oll■El. O to Tyrl. O2, NH2l. l
4 Example 5H-11e-Tyr-Arg-Arg-Pr
Production of O-Ala-NH2 (a) BOC-11e-T
yr-Arg(NO2)-Ar-g(NO2)-PrO
-Production of Ala-NH2Z-Ala-NH2llllTn
Dissolve g (0.5 mmol) in 50 ml of methanol,
Reduction is carried out at room temperature for 3 hours using palladium black as a catalyst.

The catalyst was filtered off, methanol was distilled off, and H-Ala-NH
Get 2. This is BOC-e-TyT-Arg (NO
2)-Arg(NO2)-PrO-0H268mg(0
．． 3 mmol) and 8 mg (0.6 mmol) of HONBlO were dissolved in 5 ml of dimethylformamide, and while cooling on ice, 82 mg (0.4 mmol) was added at ■°C and stirred for 4 hours, and further stirred at room temperature for ■ hours. Precipitated urea is removed by filtration, the solvent is distilled off, ether is added to the residue, and the powder is collected by filtration. This is purified by reprecipitation from methanol and ethyl acetate to obtain 174TLg (60%). R
f2O. 42 (b) H-Ne-Tyr-Arg-Ar
Production of g-PrO-Ala-NH2 BOC-11e-T
yr-Arg(NO2)-Ar'g(NO2)-PrO
-Dissolve 296 mg of NH together with 0.07 ml of anisole in L ml of trifluoroacetic acid and place 3 drops at room temperature.

Trifluoroacetic acid is distilled off, ether is added, and the powder is collected by filtration. This was dissolved in 20 ml of acetic acid and reduced for 5 hours using palladium black as a catalyst. The catalyst was removed by filtration, the acetic acid was distilled off, and the residue was purified by Sephadex LH-20 (1.0×
85c7n) column and eluted with 1N monoacetic acid.
Collect 30-38 ml portions and lyophilize to obtain 30 mg.

Rf4 (cellulose) 0.55 amino acid analysis value Argl
．． 96(2), PrOl. O5(1), Alal. O(
1), 11El. α1), Tyr' 0.98 (1), average recovery rate 75% Reference example: Histamine-releasing effect of compound (1)
．． J. Sullivan et al.'s method [Journal of
Immunology (JOUrrl2llOfImmunO
A suspension of mast cells (1×1σ cells/ML) is obtained.

Add compound (1) to 0.1 mt (1 x 1 (P cells) of mast cell suspension, and make the total volume of the reaction solution 1 ml.

[The medium is NaCl 5 Orrl mol, KCl 3.
7mmol, Na2HPO43rTlmol, K2HPO4
3.5 ml mol, CaCl. lmmol, bovine serum albumin 0.1%, gelatin 0.1%, hebalin 10 units/M
pH 6.5-6.6], incubated at 37°C for 15 minutes, immersed in ice water to stop the reaction,
The supernatant was centrifuged for 110 minutes, boiled at 100°C, centrifuged at 700y for 15 minutes, and the histamine in the supernatant was removed using the method of ShOre et al. [Journal of Pharmacology and Experimental ●Therapeutics (JournalOfPharmacOlOgy
andExperimentalTllerapeut
ics) volume 127, page 182, 1959],
0-PhthalaldehydeSpectrOph
It was quantified by OtOmetric method. Compound (1)
The table shows the results of a comparison of the effects of Neurotensin (NeLlr'0-Tensin), which is said to have a specific effect on mast cells.

Claims (1)

[Claims] 1 Formula H-Ile-Tyr-Arg-Arg-X-R [wherein,
X is Pro, Pro-Ala or Pro-Ala-Se
r and R represents OH or NH_2, respectively. ].