JP3137349B2 - Peptide derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to novel peptide derivatives. For details, please refer to the new vasoactive intestinal peptide (Va
soactive Intestinal Peptide).

[0002]

2. Description of the Related Art VIP is a kind of physiologically active peptide called a brain-intestinal peptide, and is a peptide consisting of 28 amino acids having a blood flow promoting and blood pressure lowering action extracted from pig intestine in 1970. Since the amino acid sequence is similar to secretin, glucagon and the like, it is considered to be a peptide belonging to the glucagon family. VIP
Due to its strong vasodilation and blood flow promoting effects, it is expected to have effects such as ulcers, morbidity, impotence, hair growth or hair growth.

[0003]

SUMMARY OF THE INVENTION However, VIP
Is low in the body, difficult to obtain in large quantities by extraction, and difficult to produce by genetic recombination because the C-terminal is amidated. Production by peptide synthesis using a liquid phase method or a solid phase method is also expensive.

On the other hand, VI consisting of 29 to 31 amino acid residues having bronchodilating, hypotensive or hair-growth effects
P derivatives are known (JP-A-62-246595 and JP-A-64-83012).

[0005] The present invention aims to enhance or reduce the specific action of known VIPs.

[0006]

SUMMARY OF THE INVENTION The present invention provides a compound of the formula His Ser As
p Ala Val Phe Thr Asp Asn Tyr Thr Arg LeuArg AG
lnB Ala Val CD Tyr Leu Asn Ser Ile Leu Asn
(Wherein A, C and D represent Lys or Arg, at least one of which represents Arg and B represents Met or Leu) or a VIP derivative comprising a salt thereof. In VIP, the amino acids at positions 15, 20, and 21 are Lys, whereas the VIP derivative of the present invention has at least one of the amino acids at the corresponding positions.
Arg.

In the present specification, the following abbreviations are used for protecting groups, solvents and the like. Aoc; t-amyloxycarbonyl Boc; t-butoxycarbonyl Bzl; benzyl OBzl; benzyl ester Tos; p-toluenesulfonyl Z; carbobenzoxy DCC; dicyclohexylcarbodiimide DCM; dichloromethane DIEA; diisopropylethylamine DMF; dimethylformamide DMSO; HMPA; hexamethylphosphoric triamide HOBt; 1-hydroxybenzotriazole HOSu; N-hydroxysuccinimide MBHA; benzhydrylamine TFA; trifluoroacetic acid THF; tetrahydrofuran

[0008] The peptide of the present invention can be synthesized according to a conventional method for synthesizing a known peptide. For example, "The Peptides", Vol. 1 (1966) [Schreder
andLuhke, Academic Press, New York, USA],
Alternatively, according to the method described in “Peptide Synthesis” [Izumiya et al., Maruzen Co., Ltd. (1975)], for example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester Method (p-nitrophenyl ester method, N-hydroxysuccinimide ester method,
Cyanomethyl ester method) method using Woodward's reagent K, carboimidazole method, redox method, DCC-
The additive (HOBt, HOSu) method can be exemplified. In the above, both the solid phase synthesis method and the liquid phase synthesis method can be applied. In the present invention, according to the above-described general polypeptide synthesis method, for example, a so-called stepwise method in which amino acids are sequentially condensed one by one with terminal amino acids, or a method in which a fragment is coupled into several fragments for coupling. Is done.

More specifically, for example, the solid phase synthesis method is a method of Merrifield (RB) [Solid phasepep
tide synthesis. J. Amer. Chem. Soc., 85,2149-215
9 (1963)] as follows. That is, a C-terminal amino acid (an amino-protected one) is bound to an insoluble carrier by its carboxyl group, and after removing the amino-protecting group, the amino-protected amino acid is successively added according to the amino acid sequence represented by the general formula.
It is produced by coupling by a condensation reaction with the reactive amino group and the reactive carboxyl group, synthesizing step by step, synthesizing the entire sequence, and removing the peptide from the insoluble carrier. The insoluble carrier used here may be any of various types having a binding property with a reactive carboxyl group, and for example, an MBHA-based resin or the like can be used.

In the above various methods, each amino acid having a side chain functional group such as His, Tyr, Thr, Lys,
Asp, Arg and Ser preferably have their side-chain functional groups protected, which are protected by usual protecting groups, and after the reaction is completed, the protecting groups are eliminated. Also, the functional groups involved in the reaction are usually activated. Each of these reaction methods is known, and the reagents and the like used therefor can be appropriately selected from known ones.

The protecting groups for amino acids include, for example, Z, B
oc, Aoc, isobonyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl, etc. No.

Examples of the protecting group for the imino group of His include Tos, Bzl, Z, and trityl. Se
The hydroxyl groups of r and Thr can be protected, for example, by esterification or etherification, but need not be. Suitable groups for this esterification include lower alkanoyl groups such as acetyl, aroyl groups such as benzoyl, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl. Examples of a group suitable for etherification include Bzl, tetrahydropyranyl, tert-butyl group and the like.

Examples of the hydroxyl-protecting group of Tyr include Bzl, Cl ₂ -Bzl, Z, acetyl, Tos group and the like. The protecting group of the amino groups of Lys, for example _{Z, Cl-Z, Cl 2} -Bzl, Boc, etc. Tos group. Examples of the protecting group for the guanidino group of Arg include Tos, nitro, Z, and Aoc groups.

Examples of the carboxyl-protecting group include alkyl esters (eg, methyl, ethyl, propyl, butyl, tert-butyl), benzyl esters, p-nitrobenzyl esters, methylbenzyl esters, p-chlorobenzyl esters, and benzhydrido. Ester, carbobenzoxyhydrazide, tert-butyloxycarbonylhydrazide, tritylhydrazide and the like. As
The protection of the carboxyl group of p is performed, for example, by esterification with benzyl alcohol, methanol, ethanol, tert-butanol and the like.

Examples of the activated carboxyl group include, for example, corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, ester with N-hydroxy-5-norbornene-2,3-dicarboximide) and the like. In some cases, the peptide bond type reaction can be carried out in the presence of a condensing agent, for example, a carbodiimide reagent such as DCC or carbodiimidazole, or tetraethylpyrophosphate.

In the above method, a condensation reaction between a reactive amino group and a reactive carboxyl group (peptide bond forming reaction)
Can be performed in the presence of a solvent. Various solvents known to be usable for peptide bond formation,
For example, anhydrous or hydrous DMF, DMSO, pyridine,
Chloroform, dioxane, DCM, THF, ethyl acetate, N-methylpyrrolidone, HMPA or a mixed solvent thereof can be used.

The peptide of the present invention and a salt thereof are usually processed into a pharmaceutical composition together with a pharmaceutical carrier when used as a blood flow improving agent. Is preferred.

When prepared as an injection, the resulting preparation is preferably sterile and isotonic with blood. When molding into an injection form, a diluent commonly used in this field can be used.
For example, water, physiological saline and the like can be mentioned. In this case, a sufficient amount of salt or glycerin for preparing an isotonic solution may be contained in the preparation in the form of an injection. The above-mentioned preparations can also contain ordinary buffers, soothing agents, preservatives and the like, and if necessary, coloring agents, preservatives, flavors, flavors, sweeteners and other pharmaceuticals. The above-mentioned active ingredient can be used by dissolving it in distilled water for injection at the time of use and dissolving it in a solubilizer for business use.

The preparations prepared as described above are administered by a method according to the form. In the case of an injection, it is administered intravenously either alone or as a mixture with a normal replacement fluid such as amino acids.

The dose of the active ingredient is appropriately determined depending on the purpose of use, symptoms, etc., but is usually 1 ng to 1 mg / day per person.
It is preferably used in the range of about Kg, and is preferably administered in divided doses 2 to 4 times a day.

[0021]

The present invention will be described in more detail with reference to the following Examples and Reference Examples, which do not limit the present invention.

(1) Protected peptide: (Boc) His (Tos) S
er (Bzl) Asp (OBzl) Ala Val Phe Thr (Bzl) Asp (OBzl) Asn Tyr (Bzl) Thr (Bzl) Arg (Tos)
Leu Arg (Tos) Arg (Tos) Gln Met Ala Val Arg (Tos) Arg (Tos) Tyr (Bzl) Leu Asn
Production of Ser (Bzl) Ile Leu Asn-MBHA resin

Solid phase synthesis was performed using a 430-A peptide synthesizer manufactured by Applied Biosystems as a solid phase synthesizer. First, 0.694 mg of MBHA resin (manufactured by Applied Biosystems, amino group 0.72 mmol / g) was placed in a reaction vessel for peptide solid phase synthesis, and 8 ml of DCM (4 times, 1 for each)
Min), 8 ml DCM container containing 60% TFA (20 min), D
DMF containing 4 ml of CM (3 times, 15 seconds each) and 1 ml of DIEA
3 ml of solution (2 times, 1 minute each), 8 ml of DMF (6 times, 40 minutes each)
Seconds) in the order of the argon gas stream while stirring, and after each treatment, filtration was performed.

On the other hand, the 28th amino acid sequence (Boc)
Dissolve 2 mmol of Asn-OH in 4 ml of DCM and add 3 ml of DCC (0.5M-DCM solution) in the amino acid activation vessel.
And 4 ml of HOBt (0.5M-DCM solution)
The reaction was performed for 0 minutes. Filter the reaction and transfer to a concentration vessel,
To this was added 3 ml of DMF, and DCM was distilled off under a stream of argon gas. To this, 3 ml of DMF was added, transferred to the above-mentioned reaction vessel and reacted for 30 minutes. Then 8 ml of DCM (6 times,
(20 seconds each). Further, (Boc) -Asn-O
H2 mmol was dissolved in 4 ml of DCM, and the so-called double coupling method in which the same operation was repeated in an amino acid activation vessel was carried out, followed by filtration and (Boc) -Asn-MBH
A resin was obtained.

Next, the obtained Boc-Asn-MBHA
The resin was washed in a reaction vessel with 8 ml of DCM (4 times, 1 minute each) and filtered. To this, a DCM solution 8 containing 60% TFA was added.
ml (20 minutes), 4 ml of DCM (3 times, 15 seconds each), DIE
A1 ml DMF solution 3 ml (2 times, 1 minute each), DMF8
In the order of ml (6 times, 40 seconds each), the mixture was stirred and treated in an argon gas stream, and filtered after each treatment.

Further, the amino acid sequence of the 27th amino acid sequence (Bo)
c) 2 mmole of -Leu-OH is dissolved in 4 ml of DCM and DCC (0.5M-DCM solution) in an amino acid activation vessel
1.5 ml was added and reacted for 7 minutes. The reaction solution was filtered and transferred to a concentration vessel, to which 3 ml of DMF was added, and DCM was distilled off under a stream of argon gas. To this was added 3 ml of DMF,
It was transferred to the reaction vessel and reacted for 30 minutes. Then D
Wash with 8 ml of CM (6 times, 20 seconds each), filter (Bo
c) -Leu-Asn-MBHA resin was obtained.

The 26th to 1st amino acids were sequentially coupled using the following protected amino acids. Amino-protected amino acid Amount used Acid order (mmol) 26 Boc-Ile-OH 225 Boc-Ser (Bzl) -OH 224 Boc-Asn-OH 2 × 223 Boc-Leu-OH 222 Boc-Try (Bzl) -OH221Boc-Arg (Tos) -OH2x220 Boc-Arg (Tos) -OH2x219 Boc-Val-OH218 Boc-Ala-OH217 Boc-Leu-OH216Boc -Gln-OH 2x2 15 Boc-Arg (Tos) -OH 2x2 14 Boc-Arg (Tos) -OH 2x2 13 Boc-Leu-OH2 12 Boc-Arg (Tos) -OH 2x2 11 Boc-Thr (Bzl) -OH 2 10 Boc-Thr (Bzl) -OH 29 Boc-Asn-OH 2 × 28 Boc- Asp (OBzl) -OH27Boc-Thr (Bzl) -OH26Boc-Phe-OH25Boc-Val-OH24Boc-Ala-OH23Boc-Asp (OBzl) -OH22Boc -Ser (Bzl) -OH21Boc-His (Tos) -OH2x2

In the above solid phase synthesis, Asn, Arg,
When Gln and His were used, double coupling was performed. In this way, 2.76 g of the indicated resin was obtained.

(2) Production of peptide described in Sequence Listing Protected peptide obtained in (1) -MBHA resin 2.76
50 ml of DMF and 3 ml of thioanisole were added to the resulting mixture, and the mixture was stirred at room temperature for 2 hours, washed several times with ether, filtered and dried. 5 ml of anisole was added thereto, and 25 ml of anhydrous hydrogen fluoride was further added thereto, followed by stirring at 0 ° C. for 1 hour. After the reaction,
After distilling off anhydrous hydrogen fluoride under reduced pressure, the residue was washed with ether, and 100 ml of 0.1 M acetic acid was added thereto to extract the peptide.

After the extract was stirred for 15 minutes with 20 ml of an anion exchange resin of Amberlite IR-410, the insoluble resin was removed by filtration. The obtained solution was filtered through a 0.22 μm Millipore filter and freeze-dried to obtain 823 mg of a white powder. Next, a CM-cellulose column (2.5 × 30
AcONH ₄ with a linear gradient of 0.5M from 0.05M subjected to cm), eluting with (pH 7.0) performs (10ml / fraction), to obtain a peptide 382mg of interest Fractions 69-82. This was further purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (55 × 300 mm) Solvent: 0.1% TFA-acetonitrile (linear gradient gradient changing acetonitrile from 20% to 40%) Flow rate: 10 ml / min 63 mg from partially purified peptide 100 mg by the high performance liquid chromatography was gotten.

The peptide map and amino acid value of the obtained pure peptide were determined to confirm that the substance was the substance of the present invention, and the physical properties were determined such as the degree of light emission and the retention time of high performance liquid chromatography. Amino acid analysis conditions are 6
110 ° C. in N-HCl (containing 0.1% phenol),
After performing a hydrolysis reaction for 20 hours, analysis was performed using Hitachi Amino Acid Analysis Model L-8500. Asp (5) 5.00, Thr (2) 1.72, Ser (2) 1.61, Glu (1) 1.1
0, Ala (2) 2.00, Val (2) 2.00, Ile (1) 1.02, Leu (4) 4.
15, Tyr (2) 1.86, Phe (1) 0.92, His (1) 0.95, Arg (5)
4.75

The conditions for high performance liquid chromatography were analyzed as follows. Column: TSK GEL ODS-120T (4.6 ×
Solvent: 0.1% TFA-acetonitrile (linear gradient gradient changing acetonitrile from 20% to 50%) Flow rate: 1 ml / min Retention time 26.2 minutes The light intensity measuring device and measuring conditions are as follows. Like. Equipment: SEPA-200 (Horiba, Ltd.) Conditions: Na lamp 589 nm Temperature 20 ° C. Cell 100 nm Integration time 5 seconds Concentration 1% (0.1 M in acetic acid) [α] _D 52.5

Test Example The effects of the VIP derivative of the present invention shown in the above Examples on blood flow and blood pressure were compared with those of a known VIP. Male Wistar rats weighing 220-250 g were anesthetized by intraperitoneal administration of pentobarbital (25 mg / kg) and fixed in the dorsal position. For blood pressure, insert an artery cannula into the carotid artery,
Connected to a blood pressure transducer (DX-300 Nihon Kohden) via a dome kit (SCK-512 Nihon Kohden) filled with heparinized saline (10 U / ml), and a strain pressure amplifier (AP-601G Nihon Kohden) Was measured by The blood flow was measured by a blood flow measurement probe (FR) in the right femoral artery.
-030T Nihon Kohden) and an electromagnetic blood flow meter (MFV)
-3200 Nihon Kohden).

[0035]

[Table 1]

[0036]

As is clear from the above test examples, the VIP derivative of the present invention enhances the activity of the peptide, enables to exhibit a blood flow improving effect at a low dose, and further has a side effect of lowering blood pressure. Has few features.

[Sequence List] SEQ ID NO: 1 Sequence length: 28 Sequence type: amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Characteristic symbol: peptide Location: 15, 20, 21 Characteristic determined Method: E sequence His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn 20 25 28

Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C07K 7/08 ZNA A61K 38/00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] [1] His Ser Asp Ala Val Phe Thr Asp Asn
Tyr Thr Arg Leu Arg Arg Gln Leu Ala Val Arg Arg Ty
r Leu Asn Peptide represented by Ser Ile Leu Asn (SEQ ID NO: 1) or a salt thereof. 2. A blood flow improving agent containing the peptide according to claim 1 or a salt thereof.