JPH0692991A - New active peptide - Google Patents

Abstract

PURPOSE:To obtain a new physiologically active peptide showing smooth muscle relaxing activity, having bronchodilatory action by possession of increasing action on blood flow of blood vessel. CONSTITUTION:A peptide of the following general formula His-Ser-Asp-Ala-X1- Phe-Thr-X2-X3-X4-X5-X6-Leu-X7-X8-X9-X10-Ala-X11-X12-Lys-X13-Leu-X14-Se r-X15- X16-X17-Y [e.g. His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys- Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Ser-Arg-Thr-Se r-Pro- Pro-Pro-NH2] (VIP is omitted) and its pharmaceutically acceptable salt.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel physiologically active peptide which exhibits smooth muscle relaxing activity and has a blood flow increasing action of blood vessels, thereby having a bronchodilating action.

[0002]

2. Description of the Related Art VIP (Vasoactive Intes isolated from porcine or human intestine as a natural peptide having a relaxing activity on bronchial smooth muscle)
(Telonal Polypeptide) and herodermin (Helodermi) isolated from the venom of venom lizards
n) is known. In particular, regarding the relaxing activity of VIP on the bronchial smooth muscle, p. J. Burns (P.J.
Barnes) et al. [P.
J. PJ Barnes, “The Journal of Allergy and Clinical Iminology (The Journal of Alle
rgy and Clinical Immunolo
gy) ", Vol. 79, No. 2, pp. 285-295 (1987).
reference〕.

According to this, VIP is acetylcholine,
It has a very good effect of relaxing smooth muscles contracted by stimulating substances such as histamine. This VI
The relaxing action of P is different from the relaxing action of general bronchodilators that act via the β2-receptor of adrenaline,
It is not suppressed by β-blockers. Therefore, VIP and VIP derivatives are expected to exert remarkable effects on asthma attacks in which β-blockers do not act effectively [Uchida, Hasegawa, "Medical Ayumi," Vol. 143, No. 3, 148 (1987). )reference〕.

Based on such an idea, novel VIP derivatives have been reported in JP-A No. 62-246595 and US Pat. No. 4,605,641. However, all of them are satisfactory as a bronchodilator. Not not.

VIP is a peptide having a structure having a sequence of 28 amino acids shown in the following formula with the final N-terminal written at the leftmost end of the formula (however, the C-terminal carboxyl group is an amide type). .

His-Ser-Asp-Ala-Val
-Phe-Thr-Asp-Asn-Tyr-Thr-
Arg-Leu-Arg-Lys-Gln-Met-A
la-Val-Lys-Lys-Tyr-Leu-As
n-Ser-Ile-Leu-Asn-NH _{2 In} addition, herodermin is a peptide having a structure having a sequence of 35 amino acids shown in the following formula with the final N-terminus written at the leftmost end of the formula.

His-Ser-Asp-Ala-Ile
-Phe-Thr-Gln-Gln-Tyr-Ser-
Lys-Leu-Leu-Ala-Lys-Leu-A
la-Leu-Gln-Lys-Tyr-Leu-Al
a-Ser-Ile-Leu-Gly-Ser-Arg
-Thr-Ser-Pro-Pro- Pro-NH 2

[0008]

The reason why VIP having such a specific smooth muscle relaxing activity cannot be sufficiently satisfied as a bronchodilator with the natural amino acid sequence of its original nature is that VIP has. There are various physiological activities and instability in vivo.

On the other hand, helodermin, which is a peptide consisting of 35 amino acids, is known to have a very similar amino acid sequence to VIP and to show very similar properties in terms of physiological activity. The physiological activity of herodermin, like VIP, has a blood flow increasing action and a smooth muscle relaxing action. Comparing these actions by the strength of the activity, V
Although said to be slightly weaker than the activity of IP, the duration of action is known to be much stronger than that of VIP. This is because the amino acid sequence of herodermin is VI
It is suggested that it has a structure capable of expressing the same physiological activity as P and sustaining the medicinal action of herodermin or resisting the degradation of peptides.

[0010]

Therefore, the present inventors have proposed a novel VIP derivative which can effectively utilize the amino acid sequence of VIP exhibiting a specific physiological activity, and exhibit a more selective smooth muscle relaxing activity. Further, the study was conducted for the purpose of providing a VIP derivative as a novel physiologically active peptide capable of exhibiting a long-lasting smooth muscle relaxing action. In this research, the present inventors designed a novel VIP derivative by modifying the amino acid sequence of VIP with reference to the amino acid sequence of helodermin.

As a result of the above studies by the present inventors, the first type (called A type) of the first type (A type) obtained by combining and binding a part of the C-terminal side of the amino acid sequence of helodermin to the C-terminal of the VIP amino acid sequence. A second peptide prepared by partially replacing one or more amino acid residues within the VIP amino acid sequence with amino acid residues of another species
A novel peptide of the type (B type) and a C-terminal portion of the peptide of the second type (B type) described above, which is formed by combining and binding a part of the amino acid sequence of herodermin on the C terminal side. A novel peptide of the type (C type) and a V obtained by deleting one or more amino acid residues near the C-terminal side in the amino acid sequence of VIP
We succeeded in synthesizing a novel peptide of the fourth type (D type), which was prepared by linking a part of the amino acid sequence of herodermin on the C-terminal side to the IP-truncated peptide. Moreover, these four types of novel peptides and their pharmaceutically acceptable salts have smooth muscle relaxing activity and, moreover, effectively and selectively depending on the type of causative agent that caused smooth muscle contraction. It has been found that it has selectivity of physiological activity capable of expressing smooth muscle relaxing action, and shows low toxicity or no toxicity to mammals.

The above four kinds of peptides synthesized this time by the present inventors can be generally represented by the following general formula (I).

Therefore, according to the first aspect of the present invention, the following general formula (I):-His-Ser-Asp-Ala-X ₁ -Phe-Th
_{r-X 2 -X 3 -X 4} -X 5 -X 6 -Leu-X 7 -X
_{8 -X 9 -X 10 -Ala-X} 11 -X 12 -Lys-
_{X 13 -Leu-X 14 -Ser-} X 15 -X 16 -X
_17- Y (In the formula, X ₁ is Ile or Val, X ₂ is Asp or Gln, X ₃ is Asn or Gln, X ₄ is Tyr or D-Tyr, X ₅ is Ser or Thr, and _{X 6} is A.
rg or Lys, X ₇ is Arg or Leu, X ₈ is Ala or Lys, X ₉ is Gln or Lys, X
₁₀ is Leu or Met, X ₁₁ is Leu or Va
1, X ₁₂ is Gln or Lys, X ₁₃ is Tyr or D-Tyr, X ₁₄ is Ala or Asn, X ₁₅ is Ile or Pro or a simple bond, and X ₁₆ is Le.
u or Pro or a simple bond, X ₁₇ is Asn,
Gly or Pro or a mere bond, Y is an amino group, or Pro-Pro-Pro-NH ₂ ,
_{Gly-Pro-Pro-Pro-} NH 2, Gly-L
_{ys-Pro-Pro-Pro-} NH 2, Gly-Se
r-Arg-Thr-Ser-Pro-Pro-Pro
-NH ₂ or Gly-Lys-Arg-Thr- S,
A peptide represented by er-Pro-Pro-Pro-NH ₂ (excluding VIP) and a pharmacologically acceptable salt thereof are provided.

In the general formula (I), X ₁
Is the 5th Val from the final N-terminal of VIP, X ₂ is the 8th Asp, X ₃ is the 9th Asn, X ₄ is the 10th Tyr, X ₅ is the 11th Thr, and X ₆ is the 12th A
rg, X ₇ is the 14th Arg, X ₈ is the 15th Ly
s, X ₉ is the 16th Gln, X ₁₀ is the 17th Men
t, X ₁₁ is the 19th Val, and X ₁₂ is the 20th L
ys, X ₁₃ is the 22nd Tyr, X ₁₄ is the 24th Asn, X ₁₅ is the 26th Ile, X ₁₆ is the 27th Leu, X ₁₇ is the 28th Asn, and Y is the amino. In the case of the group (—NH ₂ ), the peptide represented by the general formula (I) in this case has exactly the same amino acid sequence as VIP, so that the peptide represented by the general formula (I) in this case is VIP is excluded from the present invention.

Specific examples of the peptide of the general formula (I) according to the first aspect of the present invention include 21 kinds of peptides having the amino acid sequences shown in Examples 1 to 21 to be described later. The relationship between example peptides and the values of X _{1 to} X ₁₇ and Y in the structural formula of general formula (I) is summarized in Table 1 below for the sake of clarity. For comparison, Table 1 shows which amino acid residue in the amino acid sequence of VIP corresponds to X _{1 to} X ₁₇ and Y in the general formula (I).
It is displayed together with the number in parentheses that represents the amino acid position calculated from the end.

[0016]

[0017]

Furthermore, in the specific peptides of Examples 1 to 21 of the present invention, the entire amino acid sequence of VIP is abbreviated as VIP, and the difference from the type of some amino acid residues in the amino acid sequence of VIP is shown. It is as shown in the following Table 2 when expressed by the simplified formula noted in brackets [].

[0019]

For example, in Table 2, the simplified formula representing the peptide of Example 1 is as follows: the peptide has the formula -Gly-Ser-Arg-Thr- at the end of the entire amino acid sequence of VIP.
It shows that the peptide is formed by binding the amino acid sequence of Ser-Pro-Pro-Pro-NH ₂ (a part of herodermin), and the simplified formula representing the peptide of Example 10 shows that the peptide is a VIP amino acid. N of the array
The 14th Arg from the end is Leu, the 15th Lys is Ala, the 16th Gln is Lys, the 17th Met is Leu, and the 19th Va is
It shows that 1 is a peptide in which Leu is replaced and Lys at the 20th position is replaced by Gln.

The peptides of the general formula (I) according to the first aspect of the present invention are roughly classified into the following four types (A, B, C and D types) of peptides, that is, the formula (Ia), the formula (Ib), It includes the peptides represented by formula (Ic) and formula (Id).

(I) X _{1 to} X ₁₇ in the general formula (I)
Each of which is exactly the same as the amino acid residue at the corresponding position in VIP and Y is not an amino group, that is, a peptide of the above type A, that is, the following formula (Ia) [VIP] -Z-Pro-Pro-Pro- NH ₂ Formula (Ia) (In the formula, [VIP] represents the same amino acid sequence as VIP, and Z represents the amino acid sequence-Gly-Ser-Arg-Th.
r-Ser- or Gly-Lys-Arg-Thr-
Ser- or -Gly-Lys-, or an amino acid residue -Gly- or -Ser-, or a mere bond-).

(Ii) X _{1 to} X in the general formula (I)
A peptide of the type B wherein at least one of ₁₇ is replaced by an amino acid residue of a different species from the amino acid residue at the corresponding position in VIP and Y is an amino group, ie ib) H-His-Ser- Asp-Ala-X 1 Phe-T
_{hr-X 2 -X 3 -X 4} -X 5 -X 6 -Leu-X 7 -
_{X 8 -X 9 -X 10 -Ala-} X 11 -X 12 -Lys
_{-X 13 -Leu-X 14 -Ser-} X 15 -X 16 -
X ₁₇ -NH 2 _... formula (Ib) (wherein, _{X 1} is Ile or Val, _{X 2} is Asp or Gln, _{X 3} is Asn or Gln, _{X 4} is Tyr or D-Tyr, _{X 5} is Ser or Thr , X ₆ is A
rg or Lys, X ₇ is Arg or Leu, X ₈ is Ala or Lys, X ₉ is Gln or Lys, X
₁₀ is Leu or Met, X ₁₁ is Leu or Va
1, X ₁₂ is Gln or Lys, X ₁₃ is Tyr or D-Tyr, X ₁₄ is Ala or Asn, X ₁₅ is Ile or Pro, X ₁₆ is Leu or Pro, X
₁₇ represents Asn, Gly or Pro].

(Iii) X _{1 to} X in the general formula (I)
A peptide of the above type C wherein at least one of ₁₇ has been replaced by an amino acid residue of a different species from the amino acid residue at the corresponding position in VIP and Y is not an amino group, namely the following formula (Ic ) H-His-Ser-Asp-Ala-X ₁ Phe-T
_{hr-X 2 -X 3 -X 4} -X 5 -X 6 -Leu-X 7 -
_{X 8 -X 9 -X 10 -Ala-} X 11 -X 12 -Lys
_{-X 13 -Leu-X 14 -Ser-} X 15 -X 16 -
_{X 17 -Z-Pro-Pro-} Pro-NH 2 ... formula (I
c) (wherein, X ₁ is Ile or Val, X ₂ is Asp or Gln, X ₃ is Asn or Gln, X ₄ is Tyr or D-Tyr, X ₅ is Ser or Thr, and X ₆ is A.
rg or Lys, X ₇ is Arg or Leu, X ₈ is Ala or Lys, X ₉ is Gln or Lys, X
₁₀ is Leu or Met, X ₁₁ is Leu or Va
1, X ₁₂ is Gln or Lys, X ₁₃ is Tyr or D-Tyr, X ₁₄ is Ala or Asn, X ₁₅ is Ile or Pro, X ₁₆ is Leu or Pro, X
₁₇ represents Asn, Gly or Pro, and Z has the same meaning as in formula (Ia)].

(Iv) X _{1 to} X in the general formula (I)
Each of ₁₄ is exactly the same as the amino acid residue at the corresponding position in VIP, but at least one of X _{15 to} X ₁₇ represents a mere bond and Y is not an amino group. That is, the following formula (Id) VIP (1 to m) -Z-Pro-Pro-Pro-NH
₂ ... Formula (Id) [In formula, VIP (1-m) is N of the amino acid sequence of VIP.
A peptide represented by a sequence of m amino acids from the 1st amino acid residue to the mth amino acid residue from the end, and Z has the same meaning as in formula (Ia)].

A preferred compound example of the peptide of the general formula (I) according to the first aspect of the present invention belongs to the above-mentioned type C peptide, that is, the peptide of formula (Ic), and the following Example 1 is given.
Peptides having the amino acid sequences obtained at 0, 12 and 16, which are the peptides described in claims 2, 3 and 4 of the present application.

The pharmacologically acceptable salts of the peptide of formula (I) of the present invention include acid addition salts at the amino group or other basic group of the peptide, for example, drugs such as hydrochloric acid, sulfuric acid and phosphoric acid. A physically acceptable inorganic acid, or acetic acid,
Included are acid addition salts with pharmacologically acceptable organic acids such as malic acid and maleic acid, as well as metal salts at the carboxylic acid group present or possible in the peptide, such as sodium salt, potassium salt, and the like. It

The peptides of general formula (I) according to the invention are
It can be chemically synthesized by a conventional solid-phase method or a liquid-phase method.

For example, the novel peptide of the general formula (I) of the present invention can be chemically synthesized by a conventional method by the known active ester method or mixed acid anhydride method described in many chemical textbooks and papers. However, the amino acid that comes to the final C-terminus of the peptide to be synthesized can be attached at its carboxyl group,
Then, by the solid phase method using a resin having an amino functional group capable of fixing the peptide chain being synthesized, the final C-terminal of the peptide of the general formula (I) to be synthesized (the final right end of the formula (I))
The desired peptide by the stepwise method in which the required type and number of amino acids are sequentially condensed to the His group located at the final N-terminal (the first left end of the formula (I)) starting from the amino acid located at It is convenient to chemically synthesize.

A preferred method for synthesizing the peptide of the general formula (I) of the present invention is, for example, an automated peptide synthesizer ABI 430A manufactured by Applied Biosystems by the solid phase method.
The method is as follows using.

That is, an N-protected form of an amino acid located at the final C-terminal of the peptide to be synthesized [t-butyloxycarbonyl group (abbreviated as Boc) as an amino-protecting group]
The preferred form is a styrene-based resin carrier such as benzhydrylamine resin or p-methylbenzhydrylamine resin. Next, the amino-protecting group of the amino acid bound and fixed to the resin, such as the Boc group, is eliminated with an acid to leave the amino group of the amino acid at the C-terminal first position bound to the resin in an unprotected form. Furthermore, in the next step, the amino acid N-protected form of the second C-terminal amino acid of the peptide to be synthesized is condensed with the first amino acid to form a peptide bond. At this time, 2 to 5 times the amount of N-protected amino acid is used, and dicyclohexylcarbodiimide (abbreviated as DCC) or a mixture of DCC and 1-hydroxybenztriazole (abbreviated as HOBt) is used as a condensing agent. The end of the reaction is confirmed by using ninhydrin at the point where the reaction of the free amino group becomes negative, that is, the end point of the reaction. Thus, an amino acid whose N-terminal and, if necessary, side chain functional groups have been protected, can be prepared by using the peptide of the formula (I) of the present invention.
Condensation is sequentially carried out from the final C-terminal according to the order of the amino acid sequence in (3) to finally obtain a conjugate of the peptide of the present invention with a protected functional group and N-terminal and a resin.
Finally, the peptide of the present invention is treated with hydrogen fluoride to remove the protecting group and to remove the peptide from the resin. At this time, anisole and dimethyl sulfide are added for the purpose of preventing side reactions. After removing the residual hydrogen fluoride, the resulting crude peptide product may be purified using ion exchange chromatography using CM-cellulose or the like or preparative reverse phase high performance liquid chromatography. The purity and structure of the obtained peptide of the present invention may be confirmed by using high performance liquid chromatography, amino acid analysis method and the like.

Hereinafter, the chemical synthesis of the novel peptide of the present invention will be described with reference to Examples. Indications of amino acids, amino acid residues, protected amino acids, amino-protecting groups, hydroxyl-protecting groups, active groups, etc. are as follows: IUPAC-IUB
commissioning on Biological N
The abbreviations based on the homcureture or the abbreviations commonly used in the peptide synthesis field are followed.

The abbreviations used in the following examples are as follows.

Bzl: benzyl Cl-Z: 2-chlorobenzyloxycarbonyl Tos: 4-toluenesulfonyl Br-Z: 2-bromobenzyloxycarbonyl TFA: trifluoroacetic acid DCM: dichloromethane DMF: dimethylformamide HOBt: 1-hydroxybenzo Triazole DCC: Dicyclohexylcarbodiimide DIEA: Diisopropylethylamine Boc: Tertiary butyloxycarbonyl AcONH ₄ : Ammonium acetate AcOH: Acetic acid CH ₃ CN: Acetonitrile (Example 1) Formula (Ia-1): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Gly-
Ser-Arg-Thr-Ser-Pro-Pro-P
Synthetic peptides of ro-NH _2.

0.74 g of paramethylbenzhydrylamine resin (amino group content) was placed in a reaction vessel of an automatic peptide synthesizer ABI 430A manufactured by Applied Biosystems.
0.68 meg / g: manufactured by Peptide Institute Co., Ltd. was put in first, and this reaction vessel was attached to an automatic peptide synthesizer,
The peptide of formula (Ia-1) was synthesized by the following method.

This para-methylbenzhydrylamine resin was stirred for 2 hours with DCM in the reactor of the peptide automatic synthesizer to swell the resin.

(A) Next, Boc-Pro-OH, which is an N-Boc protected derivative of proline, which is the first amino acid from the C-terminal of the peptide of formula (Ia-1) to be synthesized, is applied to the resin by DCC. The reaction was carried out by the method to bond them. The Boc-Pro-resin conjugate thus produced is washed 5 times with DCM.

Further, in order to successively condense the amino acid to be bound next time to the amino acid already bound to the resin or the peptide under synthesis, the following operation cycle was performed.

1) 80% binding of amino acid (or peptide under synthesis) and resin together with 33% TFA-DCM solution.
2) Stir for 2 seconds, then filter and wash; 2) Stir and filter with 50% TFA-DCM solution for 18.5 minutes to remove Boc of N-protecting group; 3) Amino acid (or peptide during synthesis) with DCM. ) And the resin-bound product are washed 3 times; 4) Stirred with 10% DIEA-DMF solution for 1 minute, washed, and then treated by filtration (× 2); 5) Washed 5 times with DMF; 6) As an amino acid to be condensed next time, Boc-Pro-OH activated by DCC (430 mg: 2.0 mmo
l) Symmetrical anhydride is added and stirred for 26 minutes to react; 7) The reaction product is washed 5 times with DCM.

Thereafter, the Boc-amino acid condensation cycle is repeated in the same manner to obtain Boc-Pro at the C-terminal 1-position.
Starting from -OH, Boc-Pro-OH, B in sequence
oc-Pro-OH, Boc-Ser (Bzl) -O
H, Boc-Thr (Bzl) -OH, Boc-Arg
(Tos) -OH, Boc-Ser (Bzl) -OH,
Boc-Gly-OH, Boc-Asn-OH, Boc
-Leu-OH, Boc-Ile-OH, Boc-Se
r (Bzl) -OH, Boc-Asn-OH, Boc-
Leu-OH, Boc-Tyr (Br-Z) -OH, B
oc-Lys (Cl-Z) -OH, Boc-Val-O
H, Boc-Ala-OH, Boc-Met-OH, B
oc-Gln-OH, Boc-Lys (Cl-Z) -O
H, Boc-Arg (Tos) -OH, Boc-Lys
(Cl-Z) -OH, Boc-Leu-OH, Boc-
Arg (Tos) -OH, Boc-Thr (Bzl)-
OH, Boc-Tyr (Br-Z) -OH, Boc-A
sn-OH, Boc-Asp (OBzl) -OH, Bo
c-Thr (Bzl) -OH, Boc-Phe-OH,
Boc-Val-OH, Boc-Ala-OH, Boc
-Asp (OBzl) -OH, Boc-Ser (Bz
1) -OH and Boc-His (Tos) -OH.
0 mmol was sequentially condensed.

However, Boc-Arg (Tos) -OH,
At the time of condensation of Boc-Asn-OH and Boc-Gln-OH, Boc-amino acid (2.0 mmol) converted into HOBt ester by DCC was added in step 6) and stirred for 26 minutes. Further, recondensation was repeated by repeating steps 5) to 7).

(B) Boc-His thus obtained
(Tos) -Ser (Bzl) -Asp (OBzl)-
Ala-Val-Phe-Thr (Bzl) -Asp
(OBzl) -Asn-Tyr (Br-Z) -Thr
(Bzl) -Arg (Tos) -Leu-Arg (To
s) -Lys (Cl-Z) -Gln-Met-Ala-
Val-Lys (Cl-Z) -Lys (Cl-Z) -T
yr (Br-Z) -Leu-Asn-Ser (Bzl)
-Ile-Leu-Asn-Gly-Ser (Bzl)
-Arg (Tos) -Thr (Bzl) -Ser (Bz
l) -Pro-Pro-Pro-NH-resin (hereinafter referred to as protected peptide-resin) was subjected to the next step.

That is, 1.11 g of the protected peptide-resin that had been sufficiently dried under reduced pressure was placed in an HF (hydrogen fluoride) treatment apparatus (manufactured by Peptide Institute Co., Ltd.) to give anisole 1.
2 ml, ethyl methyl sulfide 0.2 ml and HF1
2 ml was added and stirred at 0 ° C. for 1 hour to remove the peptide from the resin and the protecting group. After removing HF by suction from the device, the resulting mixture of crude peptide and resin was thoroughly washed with diethyl ether. After drying this mixture under reduced pressure, the crude peptide was extracted by stirring in a 1M aqueous acetic acid solution and then in a 50% aqueous acetic acid solution. Next, these extracts were combined and lyophilized to give 467 mg of crude peptide.
Got

(C) Next, 400 mg of this crude peptide was equilibrated with 0.025 M AcONH ₄ aqueous solution to obtain a cation exchange chromatography column (Whatmann CM).
52) (3.2 cmφ × 20 cmL), added to 0.0
17 after eluting with 25M AcONH ₄ aqueous solution for 10 minutes
Elution was performed by applying a linear concentration gradient up to 0.20M over 6 minutes, and then eluted with a 0.20M AcONH ₄ aqueous solution.
It eluted over a period of minutes. Flow rate is 1.5 ml / min, 3 m
Fractions were separated by l and detected by absorption at 280 nm and high performance liquid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and lyophilized to obtain 107 mg of the intermediate purified peptide.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 28/7
2 Flow rate: 8 ml / min Detection wavelength: 280 nm 34 mg of the purified peptide TFA salt was obtained from 107 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

34 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
30 ml of purified peptide acetate
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed by the present inventors on the peptides obtained in this example, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and Rf values are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.28 (5), Thr 3.0
3 (3), Ser 3.50 (4), Glu 1.11.
(1), Gly 1.06 (1), Ala 2.14
(2), Val 1.67 (2), Met 0.78
(1), Ile 0.94 (1), Leu 3.30
(3), Tyr 1.86 (2), Phe 0.96
(1), Lys 2.88 (3), His 0.98
(1), Arg 3.17 (3), Pro 3.10
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in () to obtain a single peak of the target peptide at the elution position at 30.6 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 128.1 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.58 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 2 The following formula (Ia-2): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Gly-
Lys-Arg-Thr-Ser-Pro-Pro-P
Synthetic peptides of ro-NH ^2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Asn-Ser (Bz
l) -Ile-Leu-Asn-Gly-Lys (Cl
-Z) -Arg (Tos) -Thr (Bzl) -Ser
1.75 g of (Bzl) -Pro-Pro-Pro-NH-resin (hereinafter referred to as a protected peptide-resin) was treated with HF (hydrogen fluoride) to obtain 779 mg of a crude peptide.

Next, 650 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL) and added over 200 minutes to 0.
Elute with a linear concentration gradient up to 40 M
Elution was carried out with 0M AcONH ₄ aqueous solution over 400 minutes. Flow rate is 2.0 ml / min, fractionated every 16 ml,
Intermediate purified peptide 145 m was obtained by collecting the fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 275 nm and by high performance liquid chromatography.
g was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 26/7
4 Flow rate: 6 ml / min Detection wavelength: 275 nm 42 mg of the purified peptide TFA salt was obtained from 145 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

42 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
28 ml of purified peptide acetate
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.11 (5), Thr 2.9
1 (3), Ser 2.64 (3), Glu 1.05
(1), Gly 1.04 (1), Ala 2.14
(2), Val 1.83 (2), Met 0.98
(1), Ile 1.02 (1), Leu 3.28
(3), Tyr 2.07 (2), Phe 1.02
(1), Lys 3.83 (4), His 1.00
(1), Arg 3.10 (3), Pro 2.97
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in () to obtain a single peak of the target peptide at the elution position of 24.1 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 125.2 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.53 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 3 The following formula (Ia-3): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Gly-
Synthesis of the peptide _{Pro-Pro-Pro-NH 2} .

Boc-obtained in the same manner as in Example 1.
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Bz-Z) -Leu-Asn-Ser (Bz
l) -Ile-Leu-Asn-Gly-Pro-Pr
1.94 g of o-Pro-NH-resin was treated with HF (hydrogen fluoride) to obtain 660 mg of crude peptide.

Next, 650 mg of this crude peptide was added to 0.0
Cation exchange chromatographic column (Whatmann CM52) equilibrated with 25M AcONH ₄ aqueous solution
(3.2 cmφ × 20 cmL), 0.025M
After elution with an AcONH ₄ aqueous solution for 10 minutes, elution was performed with a linear concentration gradient up to 0.25M over 300 minutes, and further with 0.25M AcONH ₄ aqueous solution for 150 minutes. Flow rate is 2.0 ml / min, 3 ml
Each fraction was fractionated and detected by absorption at 280 nm and high performance liquid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and lyophilized to obtain 281 mg of the intermediate purified peptide.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 30/7
0 Flow rate: 8 ml / min Detection wavelength: 280 nm 42 mg of the purified peptide TFA salt was obtained from 151 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

42 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 36 m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After performing a hydrolysis reaction in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.22 (5), Thr 2.0
1 (2), Ser 1.76 (2), Glu 1.07
(1), Gly 1.06 (1), Ala 2.14
(2), Val 1.68 (2), Met 0.78
(1), Ile 0.93 (1), Leu 3.38
(3), Tyr 1.86 (2), Phe 0.93
(1), Lys 2.89 (3), His 1.02
(1), Arg 2.08 (2), Pro 3.11
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 26.0 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 123.7 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.59 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 4 The following formula (Ia-4): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Pro-
Synthesis of the peptide Pro-Pro-NH _2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Asn-Ser (Bz
l) -Ile-Leu-Asn-Pro-Pro-Pr
1.71 g of o-NH-resin was treated with HF (hydrogen fluoride) to obtain 774 mg of crude peptide.

Then, 704 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL), and added over 100 minutes to 0.
Elute with a linear concentration gradient up to 30 M, then 0.3
Elution was carried out with 0M AcONH ₄ aqueous solution over 400 minutes. Flow rate is 2.0 ml / min, fractionated every 14 ml,
Fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 275 nm and high performance liquid chromatography, were collected, lyophilized, and intermediately purified peptide 184 m
g was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (5.5 cmφ × 60 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 28 /
72 Flow rate: 35 ml / min Detection wavelength: 214 nm 41 mg of TFA salt of the purified peptide was obtained from 184 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

41 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 35 m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.20 (5), Thr 2.0
2 (2), Ser 1.77 (2), Glu 1.06
(1), Ala 2.16 (2), Val 1.67
(2), Met 0.77 (1), Ile 0.94
(1), Leu 3.30 (3), Tyr 1.87.
(2), Phe 0.92 (1), Lys 2.91.
(3), His 1.05 (1), Arg 2.05
(2), Pro 3.14 (3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 26.2 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 143.6 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.59 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 5 The following formula (Ia-5): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Gly-
Synthesis of the peptide Lys-Pro-Pro-Pro- NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Asn-Ser (Bz
l) -Ile-Leu-Asn-Gly-Lys (Cl
-Z) -Pro-Pro-Pro-NH-resin 1.56
g with HF (hydrogen fluoride) to give 661 mg of crude peptide
Got

Next, 620 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL) and added over 200 minutes to 0.
Elution was carried out by applying a linear concentration gradient up to 40 M, and further, 0.
Elution was performed with a 40M AcONH ₄ aqueous solution for 450 minutes. Flow rate is 2.0 ml / min, fractionated every 16 ml,
Fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 275 nm and high performance liquid chromatography, were collected and freeze-dried to obtain 202 m of the intermediate purified peptide.
g was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 27/7
3 Flow rate: 6 ml / min Detection wavelength: 275 nm 84 mg of purified peptide TFA salt was obtained from 202 mg of the intermediate purified peptide by the high performance liquid chromatography and freeze-drying.

84 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 71 m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After hydrolysis in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.20 (5), Thr 2.0
2 (2), Ser 1.76 (2), Glu 1.05
(1), Gly 1.07 (1), Ala 2.12.
(2), Val 1.67 (2), Met 0.84
(1), Ile 0.93 (1), Leu 3.23
(3), Tyr 1.97 (2), Phe 0.92
(1), Lys 3.86 (4), His 1.01
(1), Arg 2.06 (2), Pro 3.12
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 25.5 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 129.2 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.54 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 6 The following formula (Id-1): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Pro-Pro- Pro-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Bz-Z) -Leu-Asn-Ser (Bz
l) -Pro-Pro-Pro-NH-resin at 1.54
g with HF (hydrogen fluoride) to give crude peptide 645 mg
Got

Next, 600 mg of this crude peptide was added to 0.0
Cation exchange chromatographic column (Whatmann CM52) equilibrated with 25M AcONH ₄ aqueous solution
(3.2 cmφ × 20 cmL), 0.025M
After elution with an AcONH ₄ aqueous solution for 20 minutes, elution was performed with a linear concentration gradient up to 0.25M over 270 minutes, and further with 0.25M AcONH ₄ aqueous solution for 170 minutes. Flow rate is 2.0 ml / min, 3 ml
Each fraction was fractionated and detected by absorption at 280 nm and high performance liquid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and freeze-dried to obtain 107 mg of the intermediate purified peptide.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 25/7
5 Flow rate: 8 ml / min Detection wavelength: 214 nm 45 mg of TFA salt of the purified peptide was obtained from 107 mg of the intermediate purified peptide by the high performance liquid chromatography and freeze-drying.

45 mg of this purified peptide TFA salt was equilibrated with an aqueous solution of 0.1 M AcOH to obtain an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
Collected ml, freeze-dried and purified peptide acetate 42m
g was obtained.

With respect to the obtained purified peptide acetate, the amino acid analysis value was obtained, and it was confirmed that it was the peptide of the present invention, and further it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 4.15 (4), Thr 2.0
1 (2), Ser 1.78 (2), Glu 1.04
(1), Ala 2.13 (2), Val 1.64
(2), Met 0.84 (1), Leu 2.29.
(2), Tyr 1.99 (2), Phe 0.93
(1), Lys 2.80 (3), His 1.02
(1), Arg 2.09 (2), Pro 3.11
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in () to obtain a single peak of interest at the elution position of 19.3 minutes, and it was confirmed that the present peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 149.5 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.54 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 7 The following formula (Ic-1): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Ala-V
al-Lys-Lys-Tyr-Leu-Asn-Se
r-Ile-Leu-Asn-Gly-Lys-Pro
Synthetic peptides of -Pro-Pro-NH _2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Leu-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Asn-Ser (Bz
l) -Ile-Leu-Asn-Gly-Lys-Pr
1.46 g of o-Pro-Pro-NH-resin was HF
Treatment with (hydrogen fluoride) gave 662 mg of crude peptide.

Next, 650 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL), and added over 100 minutes to 0.
Elute with a linear concentration gradient up to 30 M, then 0.3
Elution with a M AcONH ₄ aqueous solution was performed for 500 minutes.
Flow rate is 2.0 ml / min, fractionated every 20 ml, 27
Fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 5 nm and high performance liquid chromatography, were collected and freeze-dried to obtain 134 mg of the intermediate purified peptide.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (5.5 cmφ × 60 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 28/7
2 Flow rate: 40 ml / min Detection wavelength: 214 nm 36 mg of the purified peptide TFA salt was obtained from 117 mg of the intermediate purified peptide by the high performance liquid chromatography and freeze-drying.

Anion exchange chromatography column (Amberlite IRA) in which 36 mg of the TFA salt of the purified peptide was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
32 ml of collected peptide and freeze-dried to obtain purified peptide acetate
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After carrying out a hydrolysis reaction in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.09 (5), Thr 2.0
1 (2), Ser 1.64 (2), Glu 1.02
(1), Gly 0.96 (1), Ala 2.14
(2), Val 1.74 (2), Ile 0.93
(1), Leu 4.25 (4), Tyr 2.01.
(2), Phe 0.94 (1), Lys 3.86
(4), His 0.93 (1), Arg 2.12.
(2), Pro 3.16 (3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 26.2 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 132.8 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.55 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 8 The following formula (Ic-2): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Le
u-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Gly-
Synthesis of the peptide _{Pro-Pro-Pro-NH 2} .

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Leu-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Asn-Ser (Bz
l) -Ile-Leu-Asn-Gly-Pro-Pr
The o-Pro-NH-resin was treated with 2.01 g HF (hydrogen fluoride) to give 664 mg of crude peptide.

Next, 640 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL) and added over 200 minutes to 0.
Elute with a linear concentration gradient up to 30 M, then 0.3
Elution was carried out with 0M AcONH ₄ aqueous solution over 400 minutes. Flow rate is 2.0 ml / min, fractionated every 20 ml,
The fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 275 nm and high performance liquid chromatography, were collected and lyophilized to obtain the intermediate purified peptide 222m.
g was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 35/6
5 Flow rate: 6 ml / min Detection wavelength: 275 nm 57 mg of TFA salt of the purified peptide was obtained from 222 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

52 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 45 m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.20 (5), Thr 2.0
0 (2), Ser 1.89 (2), Glu 1.06
(1), Gly 1.07 (1), Ala 2.08
(2), Val 1.61 (2), Ile 0.88
(1), Leu 4.12 (4), Tyr 2.15.
(2), Phe 0.90 (1), Lys 2.90.
(3), His 1.07 (1), Arg 2.04
(2), Pro 3.02 (3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position at 27.0 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 133.3 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.57 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 9 The following formula (Ic-3): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Le
u-Ala-Val-Lys-Lys-Tyr-Leu
-Asn-Ser-Ile-Leu-Asn-Pro-
Synthesis of the peptide Pro-Pro-NH _2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Leu-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Asn-Ser (Bz
l) Ile-Leu-Asn-Pro-Pro-Pro
-NH-resin 1.50 g was treated with HF (hydrogen fluoride) to obtain 672 mg of crude peptide.

Next, 650 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL) and added over 200 minutes to 0.
Elute with a linear concentration gradient up to 40 M
Elution was performed with 0M AcONH ₄ aqueous solution over 300 minutes. Flow rate is 2.0 ml / min, fractionated every 20 ml,
Intermediate purified peptide 246m obtained by absorption at 275 nm and detection by high performance liquid chromatography, collecting fractions in which the target peptide was eluted at a high concentration, and freeze-drying.
g was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 28 /
72 Flow rate: 6 ml / min Detection wavelength: 275 nm By the high performance liquid chromatography and lyophilization, 40 mg of TFA salt of the purified peptide was obtained from 175 mg of the intermediate purified peptide.

40 mg of this purified peptide TFA salt was equilibrated with an aqueous solution of 0.1 M AcOH to obtain an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 34 m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After hydrolysis in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.11 (5), Thr 2.0
1 (2), Ser 1.74 (2), Glu 1.03
(1), Ala 2.12 (2), Val 1.66
(2), Ile 0.92 (1), Leu 4.35.
(4), Tyr 2.04 (2), Phe 0.94
(1), Lys 2.80 (3), His 0.97
(1), Arg 2.04 (2), Pro 3.09
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position at 27.0 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 126.1 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.58 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 10 The following formula (Ib-1): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Leu-Ala-Lys-Le
u-Ala-Leu-Gln-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Leu-A
la-Lys (Cl-Z) -Leu-Ala-Leu-
Gln-Lys (Cl-Z) -Tyr (Br-Z) -L
eu-Asn-Ser (Bzl) -Ile-Leu-A
The sn-NH-resin was treated with 2.01 g HF (hydrogen fluoride) to obtain 1019 mg of crude peptide. Next, 219 mg of this crude peptide was equilibrated with an aqueous solution of 0.1 M AcOH for gel filtration chromatography (Sephadex column).
G-25) (3.5 cmφ × 46 cmL),
Elution was performed with 0.1M AcOH aqueous solution. Fractions were collected every 4.5 ml and detected by absorption at 280 nm and high performance liquid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and lyophilized to give an intermediate purified peptide 1.
37 mg was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 37 /
63 Flow rate: 8 ml / min Detection wavelength: 214 nm 22 mg of the purified peptide TFA salt was obtained from 137 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

22 mg of this purified peptide TFA salt was equilibrated with an aqueous solution of 0.1 M AcOH to obtain an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
20 ml of purified peptide acetate
g was obtained.

With respect to the obtained purified peptide acetate, an amino acid analysis value was determined, and it was confirmed that it was the peptide of the present invention, and further it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.15 (5), Thr 2.0
1 (2), Ser 1.86 (2), Glu 1.08
(1), Ala 3.01 (3), Val 0.82
(1), Ile 0.90 (1), Leu 6.16
(6), Tyr 2.11 (2), Phe 0.88
(1), Lys 2.04 (2), His 0.99
(1), Arg 0.99 (1). Reversed phase high performance liquid chromatography was performed under the conditions shown below in () to obtain a single peak of the target peptide at the elution position of 21.3 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 30/70 → 30/70 (5 min)
→ 50/50 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ =- 69.4 ° (c = 0.1,
0.1 M acetic acid aqueous solution) TLC: Rf value = 0.67 (cellulose plate) n-butanol: acetic acid: water: pyridine = 15: 3: 1
2:10).

Example 11 The following formula (Ib-2): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-S
er-Lys-Leu-Leu-Ala-Lys-Le
u-Ala-Leu-Gln-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Se
r (Bzl) -Lys (Cl-Z) -Leu-Leu-
Ala-Lys (Cl-Z) -Leu-Ala-Leu
-Gln-Lys (Cl-Z) -Tyr (Br-Z)-
Leu-Asn-Ser (Bzl) -Ile-Leu-
Treatment with Asn-NH-resin 1.96 g HF (hydrogen fluoride) gave 648 mg of crude peptide.

Next, 610 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL), and added over 100 minutes to 0.
Elution was performed by applying a linear concentration gradient up to 30 M, and further elution was performed with a 1 M AcONH ₄ aqueous solution containing 8 M urea for 200 minutes. The flow rate was 2.0 ml / min, fractionated every 14 ml, detected by absorption at 275 nm and high performance liquid chromatography, and the fractions in which the target peptide was eluted at a high concentration were collected. It was purified by high performance liquid chromatography.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 34.5
/65.5 Flow rate: 6 ml / min Detection wavelength: 275 nm By the high performance liquid chromatography and lyophilization, 24 mg of the purified peptide TFA salt was obtained.

24 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution to form an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
18 ml of purified peptide acetate
g was obtained.

With respect to the obtained purified peptide acetate, an amino acid analysis value was determined, and it was confirmed that it was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After hydrolysis in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.11 (5), Thr 1.1
1 (1), Ser 2.31 (3), Glu 1.15
(1), Ala 3.05 (3), Val 1.11
(1), Ile 0.90 (1), Leu 6.21
(6), Tyr 1.97 (2), Phe 0.95
(1), Lys 3.17 (3), His 0.76
(1). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 19.3 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -58 .9 ° (c = 0.1,
0.1 M acetic acid aqueous solution) TLC: Rf value = 0.64 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 12 The following formula (Ib-3): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Leu-Ala-Lys-Le
u-Ala-Lys-Gln-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Leu-A
la-Lys (Cl-Z) -Leu-Ala-Lys
(Cl-Z) -Gln-Lys (Cl-Z) -Tyr
(Br-Z) -Leu-Asn-Ser (Bzl) -I
2.74 g of le-Leu-Asn-NH-resin was HF
Treatment with (hydrogen fluoride) gave 830 mg of crude peptide.

Next, 711 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL), and added over 100 minutes to 0.
Elute with a linear concentration gradient of up to 50 M, then add 0.5
Elution was carried out with 0M AcONH ₄ aqueous solution over 400 minutes. Flow rate is 2.0 ml / min, fractionated every 20 ml,
The fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 275 nm and high performance liquid chromatography, were collected, lyophilized, and the intermediate purified peptide 217m
g was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (5.5 cmφ × 60 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 30/7
0 Flow rate: 35 ml / min Detection wavelength: 214 nm 90 mg of TFA salt of the purified peptide was obtained from 175 mg of the intermediate purified peptide by the high performance liquid chromatography and lyophilization.

90 mg of this purified peptide TFA salt was equilibrated with an aqueous solution of 0.1 M AcOH to obtain an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 76 m
g was obtained.

With respect to the obtained purified peptide acetate, an amino acid analysis value was obtained, and it was confirmed that it was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was carried out at 110 ° C. for 24 hours in 6N hydrochloric acid (containing 0.1% phenol), L850 was added.
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.02 (5), Thr 1.8
9 (2), Ser 1.57 (2), Glu 1.04
(1), Ala 3.15 (3), Val 0.99
(1), Ile 1.03 (1), Leu 5.23
(5), Tyr 2.11 (2), Phe 1.01
(1), Lys 3.00 (3), His 0.94
(1), Arg 1.02 (1). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 29.2 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ x 25 cmL) eluent: A / B: 2
0/80 → 20/80 (5 min) → 40/60 (30
min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -77.2 ° (c = 0.1,
0.1 M acetic acid aqueous solution) TLC: Rf value = 0.61 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 13 The following formula (Ic-4): His-Ser-Asp-Ala-
Ile-Phe-Thr-Gln-Gln-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Le
u-Ala-Val-Lys-Lys-Tyr-Leu
-Ala-Ser-Ile-Leu-Gly-Ser-
ArgThr-Ser-Pro-Pro-Pro-NH
Synthesis of _two peptides.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Ile-Phe-Thr (Bzl) -G
In-Gln-Tyr (Br-Z) -Thr (Bzl)
-Arg (Tos) -Leu-Arg (Tos) -Ly
s (Cl-Z) -Gln-Leu-Ala-Val-L
ys (Cl-Z) -Lys (Cl-Z) -Tyr (Br
-Z) -Leu-Ala-Ser (Bzl) -Ile-
Leu-Gly-Ser (Bzl) -Arg (Tos)
-Thr (Bzl) -Ser (Bzl) -Pro-Pr
2.10 g of o-Pro-NH-resin was treated with HF (hydrofluoric acid) to obtain 365 mg of a crude peptide.

Next, 340 mg of this crude peptide was added to 0.1
Cation-exchange chromatographic column (Whatmann CM52) equilibrated with M AcONH ₄ aqueous solution
(1.5 cmφ × 30 cmL), followed by elution with a linear concentration gradient up to 0.30 M over 187 minutes, and further elution with a 0.30 M AcONH ₄ aqueous solution over 30 minutes. Flow rate is 2 ml / min, fractionated every 7 ml, and
The fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 80 nm and high performance liquid chromatography, were collected, freeze-dried and 150 mg of the intermediate purified peptide.
Got

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 30 /
70 Flow rate: 8 ml / min Detection wavelength: 214 nm TFA salt 3 of purified peptide from 83 mg of intermediate purified peptide by the high performance liquid chromatography and freeze-drying.
1 mg was obtained.

An anion exchange chromatography column (Amberlite IRA) in which 31 mg of the TFA salt of this purified peptide was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
Collected ml, freeze-dried and purified peptide acetate 27m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After hydrolysis in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 1.03 (1), Thr 2.8
7 (3), Ser 3.76 (4), Glu 3.06
(3), Gly 1.07 (1), Ala 3.08
(3), Val 0.86 (1), Ile 1.84
(2), Leu 4.24 (4), Tyr 2.19.
(2), Phe 0.92 (1), Lys 2.93.
(3), His 0.99 (1), Arg 3.09
(3), Pro 3.07 (3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 27.5 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific optical rotation: [α] _D ²⁵ = -143 0.0 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.56 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 14 The following formula (Ic-5): His-Ser-Asp-Ala-
Ile-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Le
u-Ala-Val-Lys-Lys-Tyr-Leu
-Ala-Ser-Ile-Leu-Gly-Ser-
Arg-Thr-Ser-Pro-Pro-Pro-N
Synthesis of H ₂ peptide.

Boc-obtained in the same manner as in Example 1.
His (Tos) -Ser (Bzl) -Asp-Ile
-Phe-Thr (Bzl) -Asp (OBzl) -A
sn-Tyr (Br-Z) -Thr (Bzl) -Arg
(Tos) -Leu-Arg (Tos) -Lys (Cl
-Z) -Gln-Leu-Ala-Val-Lys (C
1-Z) -Lys (Cl-Z) -Tyr (Br-Z)-
Leu-Ala-Ser (Bzl) -Ile-Leu-
Gly-Ser (Bzl) -Arg (Tos) -Thr
(Bzl) -Ser (Bzl) -Pro-Pro-Pr
2.20 g of o-NH-resin was treated with HF (hydrogen fluoride) to obtain 857 mg of crude peptide.

Next, 705 mg of this crude peptide was added to 0.0
Cation-exchange chromatographic column (Wharmann CM52) equilibrated with 25M AcONH ₄ aqueous solution
(3.2cmφ × 20cmL) and add 0.025M
After elution with an AcONH ₄ aqueous solution for 30 minutes, a linear concentration gradient up to 0.25 M was applied over 180 minutes to elute,
Further, it was eluted with a 0.25 M AcONH ₄ aqueous solution for 14 hours. The flow rate is 1.2 ml / min. Fractions are collected every 20 ml and detected by absorption at 280 nm and high performance fluid chromatography. Fractions in which the target peptide is eluted at a high concentration are collected, lyophilized and the intermediate purified peptide 2
93 mg was obtained.

Further, it was purified by high performance fluid chromatography under the following conditions.

Column: TSK GEL-120T (2.
15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 30/7
0 Flow rate: 8 ml / min Detection wavelength: 254 nm TFA salt 1 of purified peptide from 125 mg of intermediate purified peptide by the high-performance liquid chromatography and freeze-drying.
6 mg was obtained.

16 mg of the TFA salt of this purified peptide was equilibrated with an aqueous solution of 0.1 M AcOH to obtain an anion exchange chromatography column (Amberlite IRA).
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
Collected ml, freeze-dried and purified peptide acetate 15m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was performed in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 240 hours, L85 was added.
Amino acid analysis values were measured by a 00 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp3.07 (3), Thr2.77
(3), Ser3.20 (4), Glu1.08
(1), Gly1.09 (1), Ala3.19
(3), Val1.02 (1), Ile2.01
(2), Leu4.22 (4), Tyr2.06
(2), Phe1.01 (1), Lys3.10
(3), His0.94 (1), Arg3.10.
(3), Pro3.13 (3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 27.1 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cm × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN, 0.1% TFA B: H ₂ O, 0.1% TFA Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ =- 122.4 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.57 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 15 The following formula (Ib-4): His-Ser-Asp-Ala-
Val-Phe-Thr-Gln-Gln-Tyr-S
er-Lys-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1.
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -G
ln-Gln-Thr (Br-Z) -Ser (Bzl)
-Lys (Cl-Z) -Leu (Tos) -Lys (C
1-Z) -Gln-Met-Ala-Val-Lys
(Cl-Z) -Lys (Cl-Z) -Leu-Asn-
1.59 g of Ser (Bzl) -Ile-Leu-Asn-NH-resin was treated with HF (hydrogen fluoride) to obtain 836 mg of crude peptide.

Then, 661 mg of this crude peptide was added to 0.0
Cation-exchange chromatographic column (Wharmann CM52) equilibrated with 25M AcONH ₄ aqueous solution
(3.2 cmφ × 20 cmL), 0.025M
After elution with an AcONH ₄ aqueous solution for 10 minutes, elution was performed with a linear concentration gradient up to 0.25M over 120 minutes, and further with a 0.25M AcONH ₄ aqueous solution for 15 hours. The flow rate is 2.0 ml / min. Fractions were collected every 14 ml and detected by absorption at 280 nm and high performance fluid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and freeze-dried to obtain 109 mg of the intermediate purified peptide.

Further, it was purified by high performance fluid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 31/6
9 Flow rate: 8 ml / min Detection wavelength: 254 nm TFA salt 1 of purified peptide from 109 mg of intermediate purified peptide by the high-performance liquid chromatography and freeze-drying.
0 mg was obtained.

An anion exchange chromatography column (Amberlite IRA) in which 10 mg of the TFA salt of this purified peptide was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 7.4 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml, collected, lyophilized and purified peptide acetate 9 mg
Got

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance fluid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After the hydrolysis reaction was performed in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 240 hours, L85 was added.
Amino acid analysis values were measured by a 00 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp2.99 (3), Thr0.97
(1), Ser2.58 (3), Glu2.90
(3), Ala1.97 (2), Val1.77
(2), Met 0.97 (1), Ile 1.01
(1), Leu 3.17 (3), Tyr 2.10
(2), Phe0.97 (1), Lys3.89
(3), His0.91 (1), Arg1.01
(3). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 25.4 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -107.2 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.56 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 16 The following formula (Ib-5): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Leu-Ala-Lys-Le
u-Ala-Val-Lys-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Thr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Leu-A
la-Lys (Cl-Z) -Leu-Ala-Val-
Lys (Cl-Z) -Lys (Cl-Z) -Tys (B
r-Z) -Leu-Asn-Ser (Bzl) -Ile
1.61 g of -Leu-Asn-NH-resin was treated with HF (hydrogen fluoride) to obtain 775 mg of crude peptide.

Then, 405 mg of this crude peptide was added to 0.0
Cation-exchange chromatographic column (Wharmann CM52) equilibrated with 25M AcONH ₄ aqueous solution
(3.2 cmφ × 20 cmL), 0.025M
Elute with AcONH ₄ aqueous solution for 20 minutes, then elute with a linear concentration gradient up to 0.25M over 100 minutes, then with 0.25M AcONH ₄ aqueous solution for 120 minutes, followed by 0.5M AcONH aqueous solution for 160 minutes. Was eluted. The flow rate is 1.5 ml / min. Fractions were collected every 10 ml and detected by absorption at 280 nm and high performance fluid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and freeze-dried to obtain 136 mg of the intermediate purified peptide.

Further, it was purified by high performance fluid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 35/6
5 Flow rate: 8 ml / min Detection wavelength: 214 nm 25 mg of TFA salt of the purified peptide was obtained from 136 mg of the intermediate purified peptide by the high performance liquid chromatography and freeze-drying.

An anion exchange chromatography column (Amberlite IRA) in which 25 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 7.4 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
24 ml of purified peptide acetate
g was obtained.

With respect to the obtained purified peptide acetate, an amino acid analysis value was determined, and it was confirmed that it was the peptide of the present invention, and further it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After hydrolysis in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.01 (5), Thr 1.9
9 (2), Ser 1.88 (2), Ala 2.99.
(3), Val 1.83 (2), Ile 0.98
(1), Leu 5.09 (5), Tyr 2.17.
(2), Phe 1.02 (1), Lys 2.91
(3), His 1.01 (1), Arg 0.99
(1). Reversed phase high performance liquid chromatography was performed under the conditions shown below in () to obtain a single peak of the target peptide at the elution position of 25.8 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 25/75 → 25/75 (5 min)
→ 45/55 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -110.1 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.65 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 17 The following formula (Ib-6): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Leu-Gln-Lys-Tyr-Leu
Synthesis of the peptide -Asn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1.
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Leu-Gln-Lys (Cl-Z) -Tys (Br
-Z) -Leu-Asn-Ser (Bzl) -Ile-
1.82 g of Leu-Asn-NH-resin was treated with HF (hydrogen fluoride) to obtain 982 mg of crude peptide.

Next, 499 mg of this crude peptide was added to 0.0
Cation exchange chromatographic column (Whatmann CM52) equilibrated with 25M AcONH ₄ aqueous solution
(3.2 cmφ × 20 cmL), 0.025M
After elution with an AcONH ₄ aqueous solution for 10 minutes, elution was performed with a linear concentration gradient up to 0.25M over 120 minutes, and further with a 0.25M AcONH ₄ aqueous solution for 15 hours.
Subsequently, elution was performed with a 1M AcONH ₄ aqueous solution for 200 minutes. The flow rate was 1.5 ml / min, fractionated every 10 ml, detected by absorption at 280 nm and high performance liquid chromatography, and the fractions in which the target peptide was eluted at a high concentration were collected, freeze-dried and subjected to intermediate purification. Peptide 13
7 mg was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 32/6
8 Flow rate: 8 ml / min Detection wavelength: 254 nm TFA salt 1 of purified peptide from 104 mg of intermediate purified peptide by the high-performance liquid chromatography and freeze-drying.
5 mg was obtained.

An anion exchange chromatography column (Amberlite IRA) in which 15 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
12 ml of purified peptide acetate
g was obtained.

With respect to the obtained purified peptide acetate, the amino acid analysis value was determined, and it was confirmed that it was the peptide of the present invention, and further it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After hydrolysis in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.11 (5), Thr 1.9
5 (2), Ser 1.83 (2), Glu 2.05
(2), Ala 2.11 (2), Val 0.82
(1), Met 1.00 (1), Ile 0.89
(1), Leu 4.08 (4), Tyr 2.16.
(2), Phe 0.90 (1), Lys 2.06
(2), His 1.05 (1), Arg 2.01
(2). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 29.9 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 25/75 → 25/75 (5 min)
→ 45/55 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -81.9 ° (c = 0. 1,
0.1 M acetic acid aqueous solution) TLC: Rf value = 0.62 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 18 The following formula (Ib-7): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Me
t-Ala-Val-Lys-Lys-Tyr-Leu
Synthesis of peptides -Ala-Ser-Ile-Leu- Gly-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-Tyr (Br-Z) -Th
r (Bzl) -Arg (Tos) -Leu-Arg (T
os) -Lys (Cl-Z) -Gln-Met-Ala.
-Val-Lys (Cl-Z) -Lys (Cl-Z)-
Tyr (Br-Z) -Leu-Ala-Ser (Bz
l) -Ile-Leu-Gly-NH-resin 1.58 g
Was treated with HF (hydrogen fluoride) to obtain 799 mg of a crude peptide.

Next, 505 mg of this crude peptide was added to 0.1
Cation-exchange chromatographic column (CM Toyopearl 650S) flattened with M AcONH ₄ aqueous solution (3.2
cmφ × 10 cmL), 0.1M AcONH
Elute with ₄ aqueous solution for 10 minutes, then 0.2
Elute with a linear concentration gradient up to 5 M, then 0.25
58 minutes with M AcONH ₄ aqueous solution, followed by 0.3M
Elution was performed with an AcONH ₄ aqueous solution for 60 minutes. The flow rate is 7.0 ml / min. Fraction every 14 ml, 280 nm
The fractions in which the target peptide was eluted at a high concentration were collected and freeze-dried to obtain 112 mg of the intermediate purified peptide.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL DOS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 30/7
0 Flow rate: 8 ml / min Detection wavelength: 214 nm 62 mg of the purified peptide TFA salt was obtained from 104 mg of the intermediate purified peptide by the high performance liquid chromatography and freeze-drying.

Anion exchange chromatography column (Amberlite IRA) prepared by flattening 48 mg of this purified peptide TFA salt with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
Collected ml, freeze-dried and purified peptide acetate 42m
g was obtained.

With respect to the obtained purified peptide acetate, the amino acid analysis value was determined, the peptide of the present invention was confirmed, and further it was confirmed to be a pure product by reverse phase high performance liquid chromatography. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After carrying out a hydrolysis reaction in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 3.00 (3), Thr 1.9
1 (2), Ser 1.79 (2), Glu 1.03
(1), Gly 1.03 (1), Ala 2.98
(3), Val 1.82 (2), Met 0.97
(1), Ile 0.99 (1), Leu 3.14
(3), Tyr 2.11 (2), Phe 1.00
(1), Lys 2.95 (3), His 0.99
(1), Arg 2.00 (2). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 27.7 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -99.1 ° (c = 0. 1,
0.1 M acetic acid aqueous solution) TLC: Rf value = 0.63 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 19 The following formula (Ib-8): His-Ser-Asp-Ala-
Val-Phe-Thr-Asp-Asn-D-Tyr
-Thr-Arg-Leu-Arg-Lys-Gln-
Met-Ala-Val-Lys-Lys-Tyr-L
eu-Asn-Ser-Ile-Leu-Asn-NH
Synthesis of _two peptides.

Boc-obtained in the same manner as in Example 1.
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-D-Tyr (Br-Z)-
Thr (Bzl) -Arg (Tos) -Leu-Arg
(Tos) -Lys (Cl-Z) -Gln-Met-A
la-Val-Lys (Cl-Z) -Lys (Cl-
Z) -Tyr (Br-Z) -Leu-Asn-Ser
(Bzl) -Ile-Leu-Asn-NH-Resin 1.
Treatment of 97 g with HF (hydrogen fluoride) to give crude peptide 990
mg was obtained.

Next, 590 mg of this crude peptide was added to 0.1
Cation exchange chromatograph column (CM Toyopearl 650S) equilibrated with M AcONH ₄ aqueous solution (2.0
cmφ × 50 cmL), and added over 100 minutes to 0.
Elute with a linear concentration gradient up to 3M, then 0.30
Elution was performed with a M AcONH ₄ aqueous solution over 650 minutes.
Liquid speed is 2.0 ml / min. Fraction every 30 ml, 28
Fractions in which the target peptide was eluted at a high concentration, which was detected by absorption at 0 nm and high performance liquid chromatography, were collected and freeze-dried to obtain 75 mg of the intermediate purified peptide.

Further, it was purified by high performance fluid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 28/7
2 Flow rate: 7 ml / min Detection wavelength: 275 nm TFA salt 4 of purified peptide from 75 mg of intermediate purified peptide by the high performance liquid chromatography and lyophilization.
5 mg was obtained.

Anion-exchange chromatography column (Amberlite IRA) in which 14 mg of the TFA salt of this purified peptide was equilibrated with an aqueous 0.1 M AcOH solution.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
12 ml of purified peptide acetate
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Further, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After carrying out a hydrolysis reaction in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.16 (5), Thr 2.0
0 (2), Ser 1.82 (2), Glu 1.12.
(1), Ala 2.13 (2), Val 1.68
(2), Met 0.98 (1), Ile 0.89
(1), Leu 3.09 (3), Tyr 2.00
(2), Phe 0.92 (1), Lys 2.97.
(3), His 1.20 (1), Arg 2.04
(2). Reversed phase high performance liquid chromatography was performed under the conditions shown below in () to obtain a single peak of the target peptide at the elution position of 28.0 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -110.6 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.52 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

Example 20 The following formula (Ib-9): His-Ser-Asp-Ala-
Val-Phe-Asp-Asn-Tyr-Thr-A
rg-Leu-Arg-Lys-Gln-Met-Al
a-Val-Lys-Lys-D-Tyr-Leu-A
Synthetic peptides of sn-Ser-Ile-Leu- Asn-NH 2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (OBzl) -Asp (OB
zl) -Ala-Val-Phe-Asp (OBzl)
-Asn-Thy (Br-Z) -Tyr (Bzl) -A
rg (Tos) -Leu-Arg (Tos) -Lys
(Cl-Z) -Gln-Met-Ala-Val-Ly
s (Cl-Z) -Lys (Cl-Z) -D-Tyr (B
r-Z) -Leu-Asn-Ser (Bzl) -Ile
1.81 g of -Leu-Asn-NH-resin was treated with HF (hydrogen fluoride) to obtain 801 mg of crude peptide.

Next, 743 mg of this crude peptide was added to 0.0
Cation exchange chromatographic column (CM Toyopearl 650S) equilibrated with 25M AcONH ₄ aqueous solution
(3.2 cmφ × 10 cmL), 0.025M
After elution with an AcONH ₄ aqueous solution for 10 minutes, a linear concentration gradient up to 0.25 M was applied over 77 minutes to elute,
Further, it was eluted with a 0.25M AcONH ₄ aqueous solution for 68 minutes. The flow rate is 7.0 ml / min. Fractions were collected every 14 ml and detected by absorption at 280 nm and high performance liquid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected, freeze-dried and the intermediate purified peptide 7
7 mg was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 29/7
1 Flow rate: 8 ml / min Detection wavelength: 254 nm TFA salt 3 of purified peptide from 77 mg of intermediate purified peptide by the high performance liquid chromatography and lyophilization.
3 mg was obtained.

An anion exchange chromatography column (Amberlite IRA) in which 33 mg of this purified peptide TFA salt was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
ml was collected, lyophilized and purified peptide acetate 29 m
g was obtained.

With respect to the obtained purified peptide acetate, the amino acid analysis value was determined, and it was confirmed that it was the peptide of the present invention, and further it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After performing a hydrolysis reaction in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that the target peptide of the present invention was obtained.

Asp 5.10 (5), Thr 2.0
0 (2), Ser 1.80 (2), Glu 1.09
(1), Ala 2.11 (2), Val 1.71
(2), Met 1.02 (1), Ile 0.92
(1), Leu 3.06 (3), Tyr 2.06
(2), Phe 0.92 (1), Lys 2.95.
(3), His 1.18 (1), Arg 2.08
(2). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 27.3 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -106.7 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.55 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

(Example 21) The following formula (Ib-10): His-Ser-Asp-Ala
-Val-Phe-Thr-Asp-Asn-D-Ty
r-Thr-Arg-Leu-Arg-Lys-Gln
-Met-Ala-Val-Lys-Lys-D-Ty
r-Leu-Asn-Ser-Ile-Leu-Asn
Synthesis of peptide -NH _2.

Boc-obtained in the same manner as in Example 1
His (Tos) -Ser (Bzl) -Asp (OBz
l) -Ala-Val-Phe-Thr (Bzl) -A
sp (OBzl) -Asn-D-Tyr (Br-Z)-
Thr (Bzl) -Arg (Tos) -Leu-Arg
(Tos) -Lys (Cl-Z) -Gln-Met-A
la-Val-Lys (Cl-Z) -Lys (Cl-
Z) -D-Tyr (Br-Z) -Leu-Asn-Se.
The crude peptide 1 was prepared by treating 2.63 g of r (Bzl) -Ile-Leu-Asn-NH-resin with HF (hydrogen fluoride).
099 mg was obtained.

Next, 1000 mg of this crude peptide was added to 0.
Cation exchange chromatographic column (CM Toyopearl 650S) equilibrated with 025M AcONH ₄ aqueous solution
(3.2 cmφ × 10 cmL), 0.025M
After elution with an AcONH ₄ aqueous solution for 10 minutes, a linear concentration gradient up to 0.25 M was applied over 77 minutes to elute,
Further, it was eluted with a 0.25M AcONH ₄ aqueous solution for 68 minutes. The flow rate is 7.0 ml / min. Fractions were collected every 14 ml and detected by absorption at 280 nm and high performance liquid chromatography. Fractions in which the target peptide was eluted at a high concentration were collected and lyophilized to give the intermediate purified peptide 1.
61 mg was obtained.

Further, it was purified by high performance liquid chromatography under the following conditions.

Column: TSK GEL ODS-120
T (2.15 cmφ × 30 cmL) Eluent: CH ₃ CN / 0.1% TFA aqueous solution = 29/7
1 Flow rate: 8 ml / min Detection wavelength: 254 nm By the high performance liquid chromatography and lyophilization, 25 mg of the TFA salt of the purified peptide was obtained from 100 mg of the intermediate purified peptide.

An anion exchange chromatography column (Amberlite IRA) in which 20 mg of the TFA salt of this purified peptide was equilibrated with an aqueous 0.1 M AcOH solution was used.
No. 410) (0.83 cmφ × 3.7 cmL) and eluted with 0.1 M AcOH aqueous solution. Eluent first liquid 5
Collected ml, freeze-dried, and purified peptide acetate 19 m
g was obtained.

Amino acid analysis values of the obtained purified peptide acetate were determined, and it was confirmed that the peptide was the peptide of the present invention. Furthermore, it was confirmed by reverse phase high performance liquid chromatography that it was a pure product. Further, the specific optical rotation and the Rf value were measured. The results of amino acid analysis performed on the substance obtained by the present inventor by the present inventor, the results of reverse phase high performance liquid chromatography, the results of measurement of specific optical rotation and the Rf value are shown below.

After carrying out a hydrolysis reaction in 6N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 24 hours, L850
Amino acid analysis values were measured by a 0 type Hitachi amino acid analyzer. As shown below, the measured values were in good agreement with the theoretical values, and it was confirmed that this was the target substance of the present invention.

Asp 5.15 (5), Thr 1.9
8 (2), Ser 1.82 (2), Glu 1.11.
(1), Ala 2.16 (2), Val 1.67
(2), Met 1.01 (1), Ile 0.91
(1), Leu 3.06 (3), Tyr 2.02
(2), Phe 0.90 (1), Lys 2.94.
(3), His 1.21 (1), Arg 2.06
(2). Reversed phase high performance liquid chromatography was performed under the conditions shown below in (), and a single peak of the target peptide was obtained at the elution position of 26.8 minutes, and it was confirmed that this peptide was highly pure.

Column: TSK GEL ODS-120
T (0.46 cmφ × 25 cmL) Eluent: A / B: 20/80 → 20/80 (5 min)
→ 40/60 (30 min) A: CH ₃ CN B: 0.1% TFA aqueous solution Flow rate: 1 ml / min Detection wavelength: 214 nm Specific rotation: [α] _D ²⁵ = -110.8 ° (c = 0.
1,0.1 M acetic acid aqueous solution) TLC: Rf value = 0.55 (cellulose plate) (n-butanol: acetic acid: water: pyridine = 15: 3: 1)
2:10).

The novel peptide of the general formula (I) according to the present invention has a carboxylic acid group of the C-terminal amino acid of amide type, but has a physiological activity of relaxing mammalian smooth muscle, especially tracheal smooth muscle. It has only a very low toxicity to mammals.

Therefore, the peptide of the present invention is useful as a tracheal dilator. The smooth muscle relaxant activity of the novel peptide of the present invention is particularly important in the cause of tracheal smooth muscle contraction LTD.
4 has a characteristic that it can selectively suppress smooth muscle contraction and relax smooth muscle during contraction.

Therefore, according to the second aspect of the present invention, the peptide represented by the above general formula (I) (excluding VIP) or a pharmaceutically acceptable salt thereof is contained as an active ingredient. A tracheal dilator is provided.

The bronchodilator according to the second aspect of the present invention may contain a non-toxic liquid carrier or solid carrier in admixture with the active ingredient peptide. The bronchodilator of the present invention can be in the form of a solution prepared by dissolving the peptide of the present invention of general formula (I) in physiological saline, and can be administered in the form of an aerosol.

[0254] Preferable formulation examples of the bronchodilator of the present invention are
The novel peptides of general formula (I) above, preferably Example 16
It is an aqueous solution obtained by dissolving the peptide of 5) in physiological saline at a concentration of 5 to 500 μg / ml, preferably 50 μg / ml. This solution can be atomized in the form of an aerosol by a commercially available ultrasonic inhaler and administered by inhalation into the bronchi. Alternatively, alternatively, the peptide solution may be housed with or without a conventional propellant under pressure or under pressure in a vessel equipped with a spray nozzle.

The peptide of formula (I) of the present invention can be administered by inhalation in an amount of 0.2 to 20 mg / adult per day.

Next, test examples for evaluating the physiological activity of the peptide of the general formula (I) of the present invention will be described.

Test Example 1 This example is a test exemplifying the relaxing activity of the peptide of the present invention on tracheal smooth muscle.

The guinea pig was killed by exsanguination from the femoral artery,
The trachea and bronchi were removed. Except the respiratory epithelium, the trachea muscle was traversed to a width of 2-3 mm to prepare a tracheal muscle sample. The bronchial muscle was a helical specimen. The prepared tracheal muscle specimen and bronchial muscle specimen were previously kept at 32 ° C. and aerated with Locke-
Ringer's solution (154, 0 mM NaCl, 5.
6 mM KCl, 2.2 mM CaCl ₂ , 2.1
mM MgCl ₂ , 5.9 mM NaHCO ₃ , 2.
Organ ba filled with 8 mM glucose)
Suspended during th. Under the static tension of 1g for the tracheal muscle specimen and 0.5g for the bronchial muscle specimen, org every 15-20 minutes.
Equilibration was performed for about 1 hour while replacing the solution in an bath. Each specimen was treated by adding and dissolving the contractile agent shown below in the bath several times, and then allowed to stand until the muscle contraction reaction became constant. After that, as a test peptide,
VIP and the peptide of the present invention were cumulatively added to the bath and dissolved, and the relaxation reaction of smooth muscle which was affected by the test peptide was recorded isometrically.

Muscle preparation Contractile concentration Constrictor concentration Tracheal muscle Histamine 10 ⁻⁵ M Carbachol 10 ⁻⁶ M Substance P 10 ⁻⁶ M Leukotriene D ₄ 3 × 10 ⁻⁹ g / ml Bronchial muscle Leukotriene D ₄ 3 × 10 ⁻⁹ g / Ml.

Leukotriene D ₄ was used after being dissolved in methanol and diluted with distilled water. All other drugs were dissolved in distilled water and used in the experiment.

VIP (comparative) and the tested peptides of the present invention relaxed tracheal muscle and bronchial muscle contracted by each contractile drug in a concentration-dependent manner. From the obtained recording curve of relaxation reaction,
The pIC ₅₀ value, which is the negative logarithm of the peptide concentration required to obtain a relaxation rate of 50% of the maximum relaxation response, and the maximum relaxation rate were determined. Based on these values, the potency of VIP and each test peptide were compared. Considering the comparison result, VI
Table 3 shows the determination results of the overall relaxing effect of the peptides of the present invention as compared with P. Table 4 shows the relative relaxation effect of the peptide of the present invention when the relaxation activity of VIP on a muscle sample contracted with carbachol is set to "1".

[0262]

(Note) ++: Indicates that the effect is equivalent to VIP ++: Indicates that the effect is lower than VIP +: Indicates that the effect is much lower than VIP −: No effect Indicates.

[0264]

When the results of Table 4 above were examined, it was revealed that the peptide of the present invention has the advantage of having the selectivity of selectively suppressing the contraction of smooth muscle by LTD _{4 as} compared with VIP.

Test Example 2 In this example, the acute toxicity of the compound of Example 16 was evaluated as a typical example of the peptide of the present invention. The compound of Example 16 was dissolved in physiological saline, and 20 mg / kg of this test compound was intravenously administered (iv) to one group of 3 male ddY mice (5 weeks old). It was observed for 8 days. The result is as follows. 1. Death and LD ₅₀ : No deaths were observed. Therefore, the LD ₅₀ of this test compound is estimated to be 20 mg / kg or more. 2. General condition: Sedation and ptosis were observed in 2 of 3 cases immediately after administration or 10 minutes after administration, but recovered 1 hour later. No abnormalities were observed during subsequent observations. After administration of the test compound, no particular abnormality in body weight was observed.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takafumi Kawamoto 13-28-303 Kawaguchi, Tsuchiura City, Ibaraki Prefecture (72) Inventor Hitoshi Kimura 1-14-14 Sengen, Tsukuba City, Ibaraki Prefecture (72) Naomi Nagata Meiji Seika Co., Ltd. Pharmaceutical Research Laboratory, 760 Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture

Claims (5)

[Claims] 1. A general formula (I): - His-Ser -Asp-Ala-X 1 -Phe-Th
_{r-X 2 -X 3 -X 4} -X 5 -X 6 -Leu-X 7 -X
_{8 -X 9 -X 10 -Ala-X} 11 -X 12 -Lys-
_{X 13 -Leu-X 14 -Ser-} X 15 -X 16 -X
_17- Y (In the formula, X ₁ is Ile or Val, X ₂ is Asp or Gln, X ₃ is Asn or Gln, X ₄ is Tyr or D-Tyr, X ₅ is Ser or Thr, and X ₆ is A.
rg or Lys, X ₇ is Arg or Leu, X ₈ is Ala or Lys, X ₉ is Gln or Lys, X
₁₀ is Leu or Met, X ₁₁ is Leu or Va
1, X ₁₂ is Gln or Lys, X ₁₃ is Tyr or D-Tyr, X ₁₄ is Ala or Asn, X ₁₅ is Ile or Pro or a simple bond, and X ₁₆ is Le.
u or Pro or a simple bond, X ₁₇ is Asn,
Gly or Pro or a mere bond, Y is an amino group, or Pro-Pro-Pro-NH ₂ ,
_{Gly-Pro-Pro-Pro-} NH 2, Gly-L
_{ys-Pro-Pro-Pro-} NH 2, Gly-Se
r-Arg-Thr-Ser-Pro-Pro-Pro
-NH ₂ or Gly-Lys-Arg-Thr- S,
er-Pro-Pro-Pro- NH except peptide (although VIP indicated ₂ in the representative)) and a pharmacologically acceptable salt thereof. 2. In the general formula (I), X ₁ is Val or X.
₂ is Asp, X ₃ is Asn, X ₄ is Tyr, X ₅ is Th
r, X ₆ is Arg, X ₇ is Leu, X ₈ is Ala, X ₉
I but _{Lys, X 10} is _{Leu, X 11} is _{Leu, X 12} is _{Gln, X 1 3} is _{Tyr, X 14} is _{Asn, X 15} is
_le, salt _{X 16} is, _{Leu, X 17} is Asn, Y is acceptable peptides, and its pharmacologically claim 1 is amino group (-NH _2). 3. In the general formula (I), X ₁ is Val or X.
₂ is Asp, X ₃ is Asn, X ₄ is Tyr, X ₅ is Th
r, X ₆ is Arg, X ₇ is Leu, X ₈ is Ala, X ₉
I but _{Lys, X 10} is _{Leu, X 11} is _{Lys, X 12} is _{Gln, X 1 3} is _{Tyr, X 14} is _{Asn, X 15} is
The peptide according to claim 1, wherein le, X ₁₆ is Leu, X ₁₇ is Asn, and Y is an amino group (—NH ₂ ).
And a pharmacologically acceptable salt thereof. 4. In the general formula (I), X ₁ is Val or X.
₂ is Asp, X ₃ is Asn, X ₄ is Tyr, X ₅ is Th
r, X ₆ is Arg, X ₇ is Leu, X ₈ is Ala, X ₉
I but _{Lys, X 10} is _{Leu, X 11} is _{Val, X 12} is _{Lys, X 1 3} is _{Tyr, X 14} is _{Asn, X 15} is
The peptide according to claim 1, wherein le, X ₁₆ is Leu, X ₁₇ is Asn, and Y is an amino group (—NH ₂ ).
And a pharmacologically acceptable salt thereof. 5. A tracheal dilation characterized by containing a peptide represented by the general formula (I) according to claim 1 (excluding VIP) or a pharmacologically acceptable salt thereof as an active ingredient. Agent.