JPH0390034A - Production of helodermin and use thereof - Google Patents

Abstract

PURPOSE:To industrially obtain helodermin in high purity and yield by synthesizing a protected helodermin that is a new intermediate in which all the side-chain functional groups of constituent amino acids are protected and then removing the protecting groups. CONSTITUTION:An undecapeptide expressed by formula I (Boc is tert- butyloxycarbonyl; Bzl is benzyl; OcHex is cyclohexyl ester; Br-Z is 2- bromobenzyloxycarbonyl; X is tosyl or H) is condensed with a tetracosapeptide amide expressed by formula II (Cl-Z is 2-chlorobenzyloxycarbonyl; X2 is tosyl or mesitylene-2-sulfonyl) to provide a pentatriacosapeptide amide expressed by formula III. Protecting groups of the resultant pentatriacosapeptide amide are then removed with a strong acid (e.g. hydrogen fluoride or trifluoromethanesulfonic acid) to afford helodermin expressed by formula IV. The helodermin obtained by the aforementioned method is capable of acting not only on bloodstream but also specifically on musculus trachealis and used as a remedy for asthma.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides a pharmaceutically useful peptide, helodermin (
The present invention relates to the pharmaceutical use of Helodern+in) and its manufacturing method.

[Background of the invention/prior art]

Recent advances in research on peptide hormones have revealed that many of the peptide hormones present in the gastrointestinal tract also exist in the brain, and conversely, many hormones discovered in the brain are also found in the gastrointestinal tract. Hormones are called brain-gut peptides, and research on these hormones has been in the spotlight as neuroendocrinology.

1982 G., B., and Laufman (J., P.).

Raufman) et al. (American Journal of Physiology (Am, J, Physiol), 2
42. G470 (1982)) is a poisonous lizard G11a
Monster (Helodermasupectu)
VI, a representative brain-intestinal peptide, was present in the venom of +m).
P (Vasoactive Intestinal P
It was shown that there is a substance that exhibits an activity similar to that of olypeptide. This substance was described in Hoshino et al. (FEBS Lett, 1984), 178.
233 (1984)), it was determined to be a peptide (herodermin) consisting of 35 amino acids and having a molecular weight of 3845.5, and having an amino acid sequence as shown in the following formula (Cl).

In experiments using the pancreas of rats and guinea pigs, this helodermin increases the activity of adenylate cyclase, similar to secretin and VIP, and increases intracellular cAMP (for example, P. Robberecht
), Febs, Letters (FEBSLerrrers)
, Vol. 166, 277 (1984)), and in experiments using dogs, it dose-dependently increased femoral artery blood flow when administered intraarterially under anesthesia. This effect of helodermin is 1/16 weaker than that of VIP, but the duration of action is longer than that of VIP, and its half-life is about 6 times that of VIP. In addition to its effect on increasing blood flow, intravenous administration of helodermin induces a sustained decrease in blood pressure and cardiac tachycardia, and increases carotid blood flow (e.g., Nis, Narce (S),
Naruse et al. (Peptides,
Vol, 7,5upp1.1+237 (1986
)).

As mentioned above, helodergon was previously isolated from the venom of a poisonous lizard, but due to resource issues, it is currently produced by chemical synthesis. As for its production method, P, Robberecht et al.
Peptides), Vol, 7.5uppl.

1, 79 (1986)), Niss, Narce (S, N
(Peptides),
Vol, 7.5upp1. L237 (1986)
) has been reported using a solid phase method.

[Problem to be solved by the invention]

However, these known methods have not been studied much in terms of efficiently producing large amounts of helodermin, and cannot be said to be industrially advantageous methods for synthesizing helodermin. That is, as mentioned above, helodermine is a peptide that structurally consists of 35 amino acids, so in order to industrially obtain helodermin with high purity and high yield, it is necessary to use, for example, a protective group. There are many difficult problems that must be solved, such as what to choose, what to do with the desorption conditions, and what to do with the purification method. Since all of the above-mentioned known methods are solid-phase methods, it is necessary to use extremely difficult purification methods in order to isolate the target Herodel main protein from the synthesized unpurified peptide.

Furthermore, as for the use of helodermin itself, it is not superior to physiologically active peptides such as VIP in terms of its effect on blood flow alone.

[Means to solve the problem]

The present inventors have conducted intensive research on more advantageous, highly active, and industrial methods for synthesizing and purifying helodermine, and have discovered a novel intermediate useful for the synthesis of helodermin. They discovered protected herodermin and found that highly pure synthetic herodermin can be obtained from this protected herodermin in a yield of 15%. At the same time, we established a method for synthesizing this protected helodermin, and discovered that helodermin has a new action that specifically acts on tracheal muscles, leading to the completion of the present invention.

That is, the present invention provides a pentatriacosapeptide amide represented by the following structural formula (n), a method for producing helodermin characterized by removing its protective group, and a new use of helodermin, that is, a method for producing helodermin, which is characterized by removing helodermin as a main component. The objective is to provide a drug for treating asthma.

Boc-His(X+)-Set(Bzl)-Asp(
OcHex)-Ala-11e-Phe-Thr(Bz
l)-Gln-Gln-Tyr(Br-Z)-3er(
Bzl)-Lys (Cl-Z)-Leu-Leu-A
la-Lys (C1-Z)-Leu-Ala-Leu
-Gln-Lys (Cl-Z)-Tyr(Br-Z)
-Leu-Ala-3er(Bzl)-11e-Le
u-Gly-3er(Bzl)-Arg(Xg)-Th
r(Bzl)-3et(Bzl)-Pro-Pro-P
ro-NHz...(II) (In the formula, Boc is a tertiary-butyloxycarbonyl group, Bzl is a benzyl group, 0cRex is a cyclohexyl ester group, Br4
represents a 2-bromobenzyloxycarbonyl group, Cl-Z represents a 2-chlorobenzyloxycarbonyl group, X represents a tosyl (Tos) group or a hydrogen atom, and x2 represents a tosyl group or a mesitylene-2-sulfonyl (Mts) group. ) In this specification, when abbreviations are used for amino acids and peptides, they shall comply with the regulations of IUPAC, IUB, or common symbols in the field, and examples thereof are listed below. Furthermore, if an optical isomer is possible for an anoic acid or the like, only one isomer is shown unless otherwise specified.

Ala: Alanine ^rg: Arginine Asp
: Aspartic acid Gin : Glutamine Gly
Nigericin His: Histidine 11e: Isoleucine Leu: Leucine Lys: Lysine
Phe: Phenylalanine Pro Nibroline Ser: Serine Thr: Threonine Ty
r: Tyrosine As is clear from the above structural formula (II), the feature of the present invention is that the side chain functional group of aspartic acid is a cyclohexyl group, the side chain functional group of serine and threonine is a benzyl group, and the side chain functional group of histidine is a cyclohexyl group. tosyl group, the side chain functional group of arginine to tosyl group or mesitylene-2-sulfonyl group, the side chain functional group of tyrosine to 2-bromobenzyloxycarbonyl group, and the side chain functional group of lysine to 2-chlorobenzyloxycarbonyl group. The point is that protected herodermin was used. Conventionally, there has been no example of synthesizing helodermine by a liquid phase method using such a maximum protection method, and the present invention is characterized in that helodermine can be produced on an industrial scale.

In the present invention, when producing Herodel main chain, the following structural formula (%) is used: (In the formula, Boc is a tertiary-butyloxycarbonyl group, Bzl is a benzyl group, 0cHe is a cyclohexyl ester group, and Br-Z is a 2-bromobenzyloxycarbonyl group, xl represents a tosyl group or a hydrogen atom). Xi represents a tosyl group or a mesitylene-2-sulfonyl group) A pentatriacosabebutidore represented by the following structural formula % Formula % () ) obtained by condensation with a tetracosapebutide ξ-d represented by It is only necessary to remove the protective group of Izumi Do by a suitable method. There are no restrictions on the method of removal, but an example that gives a favorable result is:
An example of this method is to treat with a strong acid such as hydrogen fluoride or trifluoromethanesulfonic acid to remove it all at once at a low temperature in a short period of time.

Herodermin obtained by the method of the present invention can be purified by an extremely simple method, for example as described below, and is a very advantageous method from an industrial perspective. That is, the protecting groups of the above-mentioned protected herodermine were removed all at once with a strong acid such as hydrogen fluoride or trifluoromethanesulfonic acid, and column chromatography using carboxymethyl cellulose with ammonium acetate solutions of different concentrations using a conventional method, and reverse phase liquid chromatography. High purity helodermin can be obtained in good yield by chromatography.

Although various methods can be considered for producing the protected herodermin used in the present invention, the method synthesized by the present inventor is as follows.

That is, in the above-mentioned structural formula (1) of helodermin, the constituent amino acids are numbered from the left N-terminus, but basically there are the following five fragments, that is, 1-
11 protected peptides, specifically Boc-His
(X+)-Ser(Bzl)-Asp(OcHex)-
Ala-11e-Phe-Thr(Bzl)-Gin-
Gill-Tyr(Br-Z)-3er(Bzl)-0
H (L is a hydrogen atom or a tosyl group), a protected peptide of 12-18 as fragment B, specifically Bo
c-Lys (C1-Z) -Leu-Leu-, Al
a-Lys(Cl-Z)-Leu-Ala-:OH, protected peptide of 19-23 as fragment C,
Specifically, Boc-Leu-Gln-Lys (C1-Z
)-Tyr(Br-Z)-Leu-OH.

24-28 protected peptide as fragment D, specifically Boc-^1a-Ser(Bzl)-11
e-Leu-Gly-01 (, 2 as fragment E
9-35 protected peptide, specifically Boc-S
et(Bzl)-＾rg(Xz)-Thr(Bzl)-
Ser(Bzl)-Pro-Pro-Pro-NHz
(Xz is a tosyl group or a methanesulfonyl group), and then the above fragments D and E are condensed to create a new protected peptide of 24-35, followed by fragment C1, then fragment B, and finally fragment I. -A was condensed to synthesize protected herodermin represented by the above structural formula (II). Since the side chains of these or each of the constituent amino acids are completely protected, there are few undesirable side reactions, and the peptides can be processed by condensation and purification methods that are commonly used for peptide processing.

As a result of detailed investigation of the physiological activity of the synthetic helodermin obtained in this way using various isolated organs, it was found that it has a specific action on the tracheal smooth muscle of guinea pigs.

〔Effect of the invention〕

The present invention has made it possible to industrially advantageously synthesize helodermin, a peptide useful as a medicine, and at the same time, it has become clear that helodermin is useful as a therapeutic agent for asthma such as bronchial asthma.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 (Effect on atrial muscle of rats and guinea pigs) Immediately after exsanguination of rats or monopigs, the heart was removed, the atrium and ventricle were separated, and then a Magnus tube filled with Krebs-Henseleit was insufflated with 95% O1 + 5% COt. The atrial muscle was suspended inside.

The tension was recorded on a Polygraph 363 (NEC Sanei) via an FD big amplifier. The test substance was administered cumulatively starting from a low dose, and the change value was expressed as a percentage with the pre-administration value set as 100%.

The results are shown in Figure 1. Also, EDS. The values were calculated and shown in Table 1.

Example 2 (Effect on Guinea Pig Tracheal Muscles) A guinea pig was sacrificed by exsanguination, and the trachea was removed and cut into slices, which were connected to prepare a tracheal chain specimen.

This tracheal chain specimen was suspended in a Magnus tube filled with Tyrode's tube, and the tension was recorded on a Polygraph 363 (Nippon Electric Sanei) via an isotonic transducer. In the experiment, the animals were precontracted with 3x10-'M histamine, and the test substance was cumulatively administered at lower doses to observe the relaxation response. After that, 2X10-''M isoproterenol was administered, and this reaction was defined as a 100% relaxation effect.The EDS (50% effective concentration) value was calculated from the experimental results.The results are shown in Figure 2.
and shown in Table 1.

Example 3 (Effect on guinea pig ileum) After killing the guinea pig by exsanguination, the ileum was removed, and the ileal section of approximately IC− was suspended in a Magnus tube filled with Tyrode's tube.
The tension was recorded on the NEC Sanei). The experiment was conducted in the same manner as in the case of guinea pig trachea, and the results are shown in FIG. Also,
6 bites. The values were calculated and shown in Table 1.

Example 4 (Effect on rabbit thoracic aorta) After killing a rabbit by exsanguination and removing the thoracic aorta by thoracotomy and cutting it into rings.
The pieces were dissected to obtain vascular strips. This is 95% O2 + 5% C
The specimen was suspended in a Magnus tube filled with Krebs-Henseleit gel through which O□ was aerated, and the tension was recorded on a Polygraph 363 (Nippon Electric Sanei) via an FD pickup. In the experiment, after confirming that a vascular endothelial cell-dependent relaxation response was observed using acetylcholine, the effects of the test substance were examined. The obtained data are expressed as % with respect to the relaxation response of 101M norepinephrine as 100%, and are shown in FIG. Also, EDs. The values were calculated and shown in Table 1.

Rat atrial muscle 〉3 Guinea pig atrial muscle 〉3 Guinea pig tracheal muscle 0.08 Guinea pig ileum 〉1 Rabbit thoracic aorta 〉3 Among the organs used, helodermin was used up to 1 to 3A, except for tracheal muscle. ED because the reaction was less than %. Could not calculate value. Also, ED for the tracheal muscles. The value is 0.081
It showed an extremely small value of M, indicating that Herodelion had a specific effect on the tracheal muscles.

Example 5 (Formulation Example) The following formulation examples include a lyophilized injection and a rectal suppository, but are not limited thereto.

Formulation Example 1 Freeze-dried injection helodermin mannitol 00 00 mg Helodermin was dissolved in pyrogen-free distilled water for injection containing 0.001%-/cypolyoxyethylene sorbitan fatty acid ester to a final concentration of 20μ9/-. , and then add mannitol. This solution is dispensed through a sterile membrane filtration system, for example, a 0.22-mm Millex Cut filter (manufactured by Millibore) into each ampoule.
The mixture was frozen at °C, freeze-dried using a freeze dryer, and sealed aseptically using a conventional method.

Formulation Example 2 Rectal formulation Glyceride base (Litepsol H-15) prepared by melting Helodermin 1004 at 40''C 5 to 0.5g
The mixture was mixed to contain 0mM sodium caprate, and then molded into a dosage form according to a conventional method. In addition, other possible formulation bases include polyethylene glycol bases and mono-/di-triglyceride bases.

Example 6 Boa-His-3er (Bzl)-Asp(OcHe
x)-Ala-11e-Phe-Thr(Bzl)-G
tn-Gln-Tyr(Br-Z)-Set(Bzl)
-Lys(Cl-Z) -Leu-Leu-Ala-L
ys (Cl-Z) -Leu-A la -Leu-
G 1n-Lys(Cl-Z)-Tyr(Br-Z)-
Leu-AIa-Ser(Bzl)-11e-Leu-
Gly-Set (BZ l) -Arg (M t
s) -Thr (Bz l) -5er (Bz 1
)-Pro-Pro-Pro-NHt 1.01g (
0.18 mmol) and anisole 1-, 10 mZ of anhydrous hydrogen fluoride is added under cooling with dry ice and isopropyl alcohol. Stir for 1 hour at -2~0℃,
Reduce the pressure by tl. Ether was added to the residue, and the resulting precipitate was collected by filtration. This crude crystal was subjected to reverse phase column chromatography (TS
K-GEL 00S120T (2,15X30cm)
), eluent acetonitrile 35.5% - 0.1% trifluoroacetic acid aqueous solution 64.5%, flow rate 8-/1 lin
The target His-Ser-AspAla-11e-Phe-Th was
r-Gln-Gln-Tyr-Ser-Lys-Leu
-Leu-A Ia-Lys-Leu-A la-Le
u-G 1n-Lys-Tyr-Leu-A 1a-5
er-11e-Leu-Gly-5er-Arg-Th
r-Ser-Pro-Pr.

The eluate corresponding to the peak of Pro-NH was freeze-dried to obtain 102 mg (15%) of the target product.

TLC; 0.27 (C: M:^, 90:10:
1) [α] ”= -5,4' (c=0.2. IM
Ac0H) Amino acid analysis; Ash) 1.05 (1)
, Thr 1.95 (2) Set 4.47 (5),
Gln 3.09 (3) Gly 1.03 (1),
Ala4. IM4)lie 1.89(2), Le
u 6.46 (6) Tyr 2.10 (2), Phe
O,92(1)Lys 3.04(3),His
O,91(1)8rg 1.04(1), Pro
2.94 (3) Example 7 30C-Hts-3er 21 As OcHex -^1a-11e-Phe-Thr Boc-Lys (Cl-Z)-Leu-Leu-Al
a-Lys (Cl-Z) -LeuA Ia-Leu
-Gin-Lys (Cl-Z) -Tyr (Or-
Z)-Leu-Al a-3er(Bz 1)-11
e-Leu-Gly-Set (Bz 1)-Arg
(Mts) -Thr (Bz 1) -Set(Bzl
)-Pro-Pro-Pro-NHz 3. OOg
(0.76 mmol) was dissolved in 30 mZ of trifluoroacetic acid under cooling at O''C and stirred for 3 hours. After the reaction, reduced pressure
After condensation, ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was poured into 84 ml of N-methylpyrrolidone and adjusted to pH 117 with N-methylmorpholine while cooling at 0°C. Boc-His-3er (Bz
l)-Asp(OcHex)-Ala-11e-Phe
-Thr (Bz 1)-Gin-G In-Tyr
(Br-Z)-3er(Bzl)-0H2, 12g (1
1,5 mmol), Hoobto, 28 g (1,74
mmol) and further cooled at 0"C with WSC/HCl
After 0.40 g (2.10 mmol) was added and stirred at room temperature for 5 days, the reaction mixture was concentrated under reduced pressure. 5% aqueous sodium bicarbonate was added to the residue, and the resulting precipitate was washed with water. After drying under reduced pressure, this precipitate was applied to a 5ephadex LFI-20 column chromatograph (3.2 x 96 cm), and the elution solvent was DMF.
It was purified by column chromatography using . 3
The 67th to 76th fractions were collected, concentrated under reduced pressure, ether was added, the precipitate was collected by filtration, and the desired Boc-
)1is-Set(Bzl)-^sp (OcHex)
-Ala-11e-Phe-Thr (Bz l) -
G In-G In-Tyr (Br-Z) -5er
(Bzl)-Lys(Cl-Z)-Leu-Leu-^
1a-Lys(Cl-Z)-Leu-^1a-Leu-
Gln-Lys(Cl-Z)-Tyr(Br-Z)-L
eu-Ala-Ser(Bzl)-I 1e-Leu-
Gly-Set (Bz 1) -Arg (Mts)
-Thr (Bz 1) -Set (Bzl) -Pro
-Pro-Pro-NHz 3.16g (72.6%)
I got it.

Melting point: 268°C (decomposition) TLC; 0.79 (B:A:Py:W, 90:6
:20:24) [α]"--8,0' (c=1.0
+ DMSO) Elemental analysis value Ctss) I4oJnJ7t
HN as ClJrzS l 14EIzO Theoretical value (%) 56.85 6.81 10.93 Actual value (%) 56,73 6.22 10.82Aξ
Noic acid analysis; ^sp 1.00 (1), Thr 1.8
B (2) Set 3.97 (5), Gin 3.0
9(3)Gly 1. IHL), Ala 4.27
(4) lio 1.89 (2), Leu 6.34
(6) Tyr 2.05 (2), Phe O, 99
(1) Lys 3.15(3), I(is 0.88
(1) Arg 1.14(1), Pro, 3.24
(3) Hereinafter, an example of synthesis of the raw material peptide used in the method for producing protected Herodel fruit of the present invention will be shown as a reference example.

Reference example 1 Z-Pro-Pro-OH ゛Fl-Pro-OH 5,18 g (45, Osmol)
Dissolved in 20°C of water and diluted with triethylamine 6°C under cooling at 0°C.
．． 3 m7 (45ms+ol) was added. 10.4 g of Z-Pro-OSu (30, Osmol
) of dioxane was added thereto and stirred overnight at room temperature. After the reaction, add triethylamine to this reaction solution,
After adjusting the pH to 9, it was washed with 100% of ethyl acetate.

After adjusting the aqueous phase to concentrated hydrochloric acid (II) in an O'C cooling field, the deposited precipitate was collected by filtration and washed with water. After drying under reduced pressure, reprecipitation was performed with methanol-ether to obtain the desired Z-Pro-P.
6.85 g (65.9%) of ro-OH was obtained.

Melting point: 169-70°C TLC: 0.40 (COMMA, 90:10:1
)(α) ”=96.6' (c=1.Or M
eoll) Elemental analysis value HN as C+*HzJzOs・N20 Theoretical value (%) 62.57 6.40 7.84 Actual value (%) 62.42 6.40 8.09 Reference example 2 Z-Pro-Pro-Pro -NHZ 1゛Notification Z
-Pro-Pro-OHL, 73g (5,OOmmo
+), 1t-Pro-NHIo, 80g (7,01
mmol) and HOBt 0.92g (6,01+
mmol), THF 15@7-DI'IF 25m
7-DMSo Dissolved in 20 m7 DC under cooling at O''C.
Add 1.50 g (7.25 mmol) of C and place it in a cold room (
+4°C) overnight. After the reaction, the deposited precipitate was collected by filtration, concentrated under reduced pressure, and the residue was dissolved in CthCh150-, washed with 5% sodium bicarbonate solution and water in that order, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, ether-petroleum ether (1:1) was added to the residue, the resulting precipitate was collected by filtration, and the desired Z
-Pro-Pro-Pr.

-Nllz 2.08g (94.0%) was obtained.

Melting point: 188-89°C TLC: 0.31 (C: M:^, 90:10:
1) (α) ”= −16,0” (c=1.0.
MeOH) Elemental analysis value CzJa. N40. as CH
N Theoretical value (%) 62.40 7.21 12.36 Actual value (%) 62.43 6.83 12.66 Reference example 3 Boc-Thr (Bzl -5et (Bzl) -0Pa
c.1 Literature description (Ger, Offen, DE342
Boc-Set (B
zl) -0Pac 8.27g (20,0+III
19.0 g of tosylic acid hydrate (100,Osm
ol) was added and stirred for 8 hours. After the reaction, concentrate under reduced pressure,
Ether was added to the residue, and the resulting precipitate was collected by filtration.

The obtained precipitate was dissolved in DMF 100 aZ, -15
The pH was adjusted to 7 with N-methylmorpholine while cooling at °C.

On the other hand, Boc-Thr(Bzl)-0H7,42g (
24, Osmol),) IOB t3.68g (24
, 0 mmol) was dissolved in a mixed solution of 10 m of DMF and 50 mm of THF, and 5.94 g of DCC (2
Add 20@l of THF solution of 8.8ml iol) and
The mixture was stirred for 30 minutes under cooling. This solution was added to the previous solution and incubated at -15°C for 1 hour and at 0°C for 2 hours in a cold room (+
Stir overnight at 4°C. After the reaction, the precipitate precipitated was collected by filtration, concentrated under reduced pressure, and the residue was dissolved in 300% ethyl acetate, washed with 10% aqueous citric acid, 5% aqueous sodium bicarbonate, and water in this order, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, ether was added to the residue, the resulting precipitate was collected by filtration, and the desired Boc-
Thr(Bzl) 5er(Bzl)-0Pac9.
64 g (79.4%) were obtained.

Melting point, 100'C TLC; 0.76 (C:M, 9 i 1) (α)''
=+7.7' (c=1.0. DMF) Elemental analysis value C5aHa. N*011/ 58 HN as gO Theoretical value (%") 67.08 6.68 4.74 Actual value (%) 67.13 6.69 4.61 Reference example 4 Boa-Ar -Mts -Thr Bzl -5er
Bzl-0Pac's Boc-Thr(Bzl)-S
er(Bzl)-0Pac 6.04g (9,99nv
+ol) in 4N HCl-dioxane 50°C under cooling at 0°C.
After dissolving in @l and stirring for 2 hours, ether was added and the resulting precipitate was collected by filtration. The obtained powder was dissolved in 20d of DMF, and 1.05ad of N-methylmorpholine was added under cooling at 0°C.
(10,3+wmol) was added to adjust to po'y.

Boc-Arg(Mts)-OR-Ac
OEt ・1 / 58z06.24g (12,0+
+■ol), HOBtl, 84g (12,0+u+o
D of n)? Mixed solution of IP10d-THF35-, D
CC2,48g (12,0gaol) of THF30-
solution was added and stirred overnight in a cold room (+4°C). After the reaction, the precipitate precipitated was collected by filtration and concentrated under reduced pressure.
% sodium bicarbonate solution and then water, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, ether was added to the residue, the resulting precipitate was collected by filtration, and the desired Boc-Arg(Mts)-Thr(B
zl)Set (Bzl)-0Pac 8.93g (
94.8%) was obtained.

Melting point near 0-2°C TLC; 0.59 (C:M, 9:1) (α)
”=-1,2° (c=1.0.MeOH)
Elemental analysis value C4, Hi, tNiO+ +S・3 /
41 (IIN as !0 Theoretical value (%) 61.36 6.63 8.85 Actual value (
%) 61,52 6.69 8.78 Amino acid analysis; Thr O,98(1) Set 0.93(1
)Arg 1.08(1) Reference Example 5 Boc-Arg(Mts)-Thr(Bzl)-3et
(Bzl)-0Pac 4.40g (4.67mmo
l) in 35 ml of 4N hydrochloric acid-dioxane solution under cooling at 0°C.
After stirring for 2 hours, ether was added and the resulting precipitate was collected by filtration. The obtained powder was mixed with DMF 30WL1-
The mixture was dissolved in a mixed solution of THF to-, and the pH was adjusted to 7 by adding N-methylmorpholine while cooling at 0°C. In this mixture,
Boc-Set (Bzl)-0t11.65g (5,
59mmol), HOBt 0.86g (5,62n
+ mol) and further 1.29 g of WSC-HCl
(Question 6, 73 o1) was added and stirred for 1 hour in a cold room (+4°C) for 1 hour under cooling at 0°C, followed by condensation under reduced pressure tM,
A 10% aqueous citric acid solution was added to the residue, and the resulting precipitate was collected by filtration and washed successively with 5% aqueous sodium bicarbonate and water. After drying under reduced pressure, it is reprecipitated with methanol-ether-petroleum ether to obtain the desired B.
oc-Ser(Bzl)=Arg(Mts)-Thr
(Bzl)-5et(Bzl)-OPac 5.04g
(96.3%) was obtained.

Melting point: 84-6°C TLC: 0.80 (C:M, 9:1) [α)”
--4,6' (c = 1.0. DMF) Elemental analysis value C*J1. zNiO□S・3/4 ON as auto Theoretical value (%) 61,36 6.63 8.85 Actual value (%) 61,52 6.69 8.78 Anoic acid analysis; Thr 1.01 (1 ) Set 1.89(
2) Arg 1.10(1) Reference Example 6 Boc-3et(Bzl)-Arg(Mts)-Thr
(Bzl)-Ser(Bzl)-0Pac 5. OOg
(4,04ssol) was dissolved in 95% acetic acid aqueous solution, and 7.92g of activated zinc powder (1211Im
ol) was added and stirred vigorously at room temperature for 6.5 hours. After filtering the zinc dust, acetic acid was distilled off under reduced pressure, and ethyl acetate 200117 was added to the residue, which was washed with a 10% aqueous citric acid solution and saturated brine in that order, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, ether-petroleum ether was added to the residue, the resulting precipitate was collected by filtration, and the desired Boc-3et (Bzl)-A was obtained.
rg(Mts)-Thr(Bzl)-5et(Bzl)
-OH 4.07g (100%) was obtained.

Melting point: 81-85°C TLC: 0.53 (C:M, 4:1) [α)”
−+ 12.2” (c = 1-0+ DMF)
Elemental analysis value Cs+HbJLtO□S・7/3t
ON as t and o Theoretical value (%') 58.66 6.92 9.39 Actual value (%) 5B, 69 6,63 9.84 Reference example 7 Boc-Set (Bzl)-^r (Mts)- Thr
(Bzl)-3et(Bzl)-Pr.

22 Emperors 1 Rainbow Festival 44 Z-Pro-Pro-Pro-NHx 2.02g (
4,56a+a+ol) to 25% hydrogen bromide-acetic acid 15-
After stirring at 0°C for 4 hours, ether was added,
The resulting precipitate was collected by filtration. The obtained powder was mixed with DMF 60-
Add N-methylmorpholine under cooling to 0°C to adjust the pH.
Adjusted to 7. Boc-Set (Bzt
)-Arg(Mts)-Thr(Bzl)-Set(B
zl)-0H3,80g (3,79ssol)
,! (OBtO, 71g (4,64m5+ol) was added, and -SC・tlcl 1.05g (5,48ss
After the reaction, the mixture was stirred overnight in a cold room (+4°C), concentrated under reduced pressure, and a 10% aqueous citric acid solution was added to the residue.
The resulting precipitate was collected by filtration and washed successively with 5% sodium bicarbonate solution and water.

After drying under reduced pressure, reprecipitation was performed with DMF-ethyl acetate-ether to obtain the desired Boc-3et (Bzl)-Arg (Mts).
-Thr(Bzl)-3et(Bzl)-Pro-Pr
5.21 g (88.4%) of o-Pro-NHx was obtained.

Melting point: 101-6°C TLC; 0.83 (C:M, 4:1) [α]■
-34,9" (cmo1.0. DJ'lF) Elemental analysis value CM as C1HsvN++0+4S・4HtO
N Theoretical value (%) 58.09 7.16 11.29 Actual value (%) 58,37 6.7? 10.81 Electronoic acid analysis; Thr 1.05 (1) Set 1.
96(2) Arg 1.05(1) Pro 2.9
2(3) Reference Example 8 7゛6TosOH- of Boc-Leu-Gl-OPac
H-Gly-OPac 95. Og (0,26s
sol) was dissolved in DMF350-, and the pH was adjusted to 1 with N-methylmorpholine while cooling at -15°C. on the other hand,
Boc-Leu-OR・H2O64,4g (0,2
6ssol) ,)IOBt 43.5g (0,28
11IIol) to DMF 100 @7-TF! F1
64.4 g of DCC dissolved in a mixed solution of
(0,31 ssol) of THFHF solution 200 times,
The mixture was stirred for 45 minutes while cooling at 0°C.

Add this solution to the above solution and hold at -15°C for 1 hour at 0°C.
The mixture was stirred for 2 hours at room temperature and overnight in a cold room (+4°C). After the reaction,
The precipitate was collected by filtration, concentrated under reduced pressure, and the residue was dissolved in 500% ethyl acetate, washed successively with 10% aqueous citric acid, 5% aqueous sodium bicarbonate, and saturated brine, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, petroleum ether was added to the residue, and the resulting precipitate was collected by filtration to obtain the desired Boc-Lea-Guy-OP.
87.1 g (83.0%) of ac was obtained.

Melting point: 107°C TLC: 0.67 (C:M, 9:1) [α)”
=-28,6 @ (c = 1.0. MeOH) elemental analysis value C! It13°N! CHN as 0° Theoretical value (%) 62.05 7.44 6.89 Actual value (%) 62.06 7.51 6.76 Reference example 9 Boa of Boc-Tie-Leu-Gl-0Pac
-Lea-Gly-OPac 15. Og (37,0
wmol) was dissolved in 4N 1ICl-dioxane 1851117 under cooling at 0°C, stirred for 1 hour, ether was added, and the resulting precipitate was collected by filtration. The obtained powder is DMF
It was dissolved in a mixed solution of 70mj-THF50-, and the pH was adjusted to 7 with N-methylmorpholine while cooling at 0°C. On the other hand, B
oc-I 1eOH・1/2H! 010.7g (44
,5 mmol), HOBt 6.81 g (44,4+
+nol) was dissolved in a mixed solution of DMF 20WLl-TFIP 30-, and DCC11,Og (53
, 2 + wmol) of TI (F solution) was added, and the mixture was heated at O℃.
The mixture was stirred for 1 hour under cooling.

This solution was added to the above solution and stirred overnight in a cold room (+4°C). After the reaction, the precipitate precipitated was collected by filtration and concentrated under reduced pressure. It was washed successively with an aqueous solution, 5% sodium bicarbonate solution, and saturated brine, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, n-hexane was added to the residue, the resulting precipitate was collected by filtration, and the desired Boc-1
1e-Leu-Gly-OPac 19.1g (99
, 9%).

Melting point i 137-8℃ TLC; 0.65 (C:M, 9:1) [α)”
= -25,4@(c -1,0,DMF) Elemental analysis value CHN as Ct7H□N307・1/2nto Theoretical value (%) 61.34 8,01 7.95 Actual value (%") 61.31 7,77 7.98 Reference Example 10 Boc-Ser(Bzl)-11e-Leu-Gl-O
Pac's Boc-11e-Leu-Gly-OPac
53.4g (0,1011ol) was heated at 4N under cooling at 0°C.
After dissolving in 250 m7 of HCl-dioxane and stirring for 3 hours, ether was added and the resulting precipitate was collected by filtration. The obtained powder was dissolved in a mixture of DMF 110 af-THF15-, and the pH was adjusted with N-methylmorpholine while cooling at 0°C.
Adjusted to 7. On the other hand, Boc-5et (Bzl)-08
35.4g (0.12mol), HOBt 18.4
Dissolve g (0.12 mmol) in DMF 100 I17 and cool at 0°C -SC・lIc127.6 g (
A THF solution (30-0.14 mol) of After the reaction was stirred overnight and concentrated under reduced pressure, 5% aqueous sodium bicarbonate was added to the residue to precipitate it, which was then washed with a 10% aqueous citric acid solution and saturated brine.

After drying under reduced pressure, it was reprecipitated with DMF-ether (1:5) to obtain the desired Boc-Set (Bzl)-11e-Leu.
-Gly-OPac 69.7g (100%) was obtained.

Melting point; 137-8°C TLC; 0.81 (C:M, 9:1) [α)”
=-12,2" (c -1,0,DMF) Elemental analysis value CON as C5rHs!N40q Theoretical value (%) 63.77 7,52 8.04 Actual value (%) 63.53 7,53 8. 05 Reference example 1
1 Boc-Ala-3er (Bzl-11e-Leu-
Gl-OPac Boc-5et (Bzl)-11e-
Leu-Gly-OPac 10.5g (15,0nv
ol) was dissolved in a 60% solution of 50% trifluoroacetic acid in methylene chloride while cooling at 0°C, and after stirring for 3 hours, ether was added and the resulting precipitate was collected by filtration.

The obtained powder was dissolved in a mixture of DMF 50d-THP 60- and cooled at 0°C. The pH was adjusted with N-methylphorine.
Adjusted to 7. Add Boa-Ala-OH3 to this mixture.
, 12g (16,5n+s+ol), HOBt 2.
53g (16,5a+mol) of DMF5 mA −
Add a mixture of THF 20- and further add tisc・HC
l 3.45g (18,0011101) of DMF
5 A mixture of at-THF 20- was added and stirred at 0°C for 1 hour and in a cold room (+4°C) overnight. After the reaction, it was concentrated under reduced pressure, and a 5% aqueous citric acid solution was added to the residue, and the resulting precipitate was was collected by filtration, washed with 5% sodium bicarbonate solution and water in that order, and the desired Boc-
Ala-Ser(Bzl)-11e-Leu-Gly-
10.8 g (93.5 mmol) of OPac was obtained.

Melting point: 169-171°C TLC: 0.76 (C: M:^, 90:10:
1) (α) ”−−13,0° (c −1,0,DM
F) Elemental analysis value C4゜H57NSO1゜H! CHN as 0 Theoretical value (%) 61.13 7.57 8.91 Actual value (%) 60,88 7.32 8.99 Reference example 1
2 Boc-Ala-Ser Bzl)-11e-Leu
-Gl-OH BoC-Ala-Ser(Bzl)
-11e-Leu-Gly-OPac 4.77g (6
,01s+5ol) in 90% acetic acid aqueous solution 80-, and 11.8g of activated zinc powder (18111+++
ol) was added and stirred vigorously at room temperature for 4 hours. After filtering the zinc dust, acetic acid was distilled off under reduced pressure, and a 10% aqueous citric acid solution was added to the residue to precipitate it, which was washed with water. After drying under reduced pressure,
Reprecipitate with DMF-ether to obtain the desired Boc-Ala.
-Ser(Bzl)-11e-Leu-Gly-OH3
, 98g (96.7%) was obtained.

Melting point; 20? -9°C (decomposition) TLCio, 30 (COMMA, 9o:io:
1) [α) ”=-15,1” (c 1.0.
DMF) elemental analysis value CatHsiNsOq”
ClN as 3/4LO Theoretical value (%) 57,94 7.98 10.56 Actual value (%) 57,43 7.86 11.06Aξ
Noic acid analysis; Ser O, 92(1), Gly 1.
06(1) Ala O, 98(1), lie 1.
00(1)Leu 1.04(1) Reference Example 13 Literature description of Boc-Tr(Br-Z)-Leu-OPac (Ger, Offen, DE3428942(
Boa-Leu- prepared by the method of (1985))
17.5 g (50.0-mol) of OPac was dissolved in 100-mol of methylene chloride, 43-trifluoroacetic acid was added under cooling at 0°C, and the mixture was stirred for 1.5 hours at 0°C and then at room temperature for 2.5 hours. After the reaction, ether was added to the mixture, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in DMF 100- and adjusted to pH 7 with N-methylmorpholine while cooling at 0°C. On the other hand, Boc-Tyr(Br-Z)-0829
,7g (60,0g*5ol), HOBt 9.22g
(60,0++usol) was dissolved in DMF 50-, and DCC14.9g (72,3+u+ol) was cooled at 0℃.
50 - of THF solution was added, and the mixture was stirred for 40 minutes while cooling at 0°C. This solution was added to the above solution and stirred at 0°C for 4 hours and in a cold room (+4°C) overnight. After the reaction, the precipitate was collected by filtration, concentrated under reduced pressure, and the residue was diluted with 300% ethyl acetate. It was dissolved, washed in this order with a 10% aqueous citric acid solution, a 5% aqueous sodium bicarbonate solution, and water, and dried over anhydrous sodium sulfate. After pressure reduction II'a, ether-n-hexane (1:6) was added to the residue at 350
@l was added, the resulting precipitate was collected by filtration, and the desired Boa-T
yr(Br-Z)-Leu-OPac36.3g (
100%) was obtained.

Melting point: 98-9℃ TLC: 0.92 (C:M, 9:1) [α)”
-5.4 @(c -1-01MeOH) elemental analysis value C5JatNxOJr ・C)I as 3/2HzO
N Theoretical value (%) 57,45 5,89 3.72 Actual value (%) 57.29 5,53 3.85 Reference example 1
4 Boc-L 5(Cl-Z)-Tr(Br-Z)-L
Boc-Tyr(Br-Z)-Leu of eu-OPac
-OPac 2.18g (3,00ssol) at 0℃
Add trifluoroacetic acid 4- while cooling.

The mixture was stirred for 1 hour while cooling at 0°C. After the reaction, n-hexane was added to the mixture, and the resulting precipitate was collected by filtration.

The obtained precipitate was dissolved in TI (F35-), and the pH was adjusted to 7 with N-methylmorpholine while cooling at 0°C.
oa-Lys(Cl-Z)-0H1,49g (3,6
0ssol), HOBtO, 55g (3,59ss
ol) was dissolved in a mixed solution of DMF 5aj-THF 25wJ, and 0.89g of DCC (4.32gm
ol) was added thereto, and the mixture was stirred for 40 minutes while cooling at 0°C. This solution was added to the above solution and kept at 0°C for 1 hour in a cold room (+4°C).
The mixture was stirred overnight. After the reaction, the precipitate was collected by filtration, concentrated under reduced pressure, and the residue was dissolved in 300% ethyl acetate.
It was washed in the following order: citric acid aqueous solution, 5% sodium bicarbonate solution, and water, and dried over anhydrous sodium sulfate. Reduced pressure: a diameter reduction, ether was added to the residue, the resulting precipitate was collected by filtration, and the objective (7) Boc-L
ys(C1-Z)-Tyr(Br-Z)-Leu-OP
2.14 g (69.6%) of ac was obtained.

Melting point: 89-91°C TLC: 0.58 (C:Kun^, 90:10:1) (
α)”--16,2° (C-1,0,DMF) Elemental analysis value C5JssNaOtJr-Cl-Ello as ClN Theoretical value (%) 57,72 5.81 5.39 Actual value (%) 57. 80 5,57 5.47 Reference example 1
5 Boc-Leu-Gln-OH H-Gin-OH6,14g (42,0++ usol
) in water 70@Z and cooled at 0°C to form a THF solution 18-1 of triethylamine 6aZ (42.0 mmol).
Boc-Leu-OSu 11.5g (35,0mmo
Add the THF solution of l) in order of 70°C and store in a cold room (+4°C).
After the reaction was stirred overnight, the reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 200% ethyl acetate, washed successively with a 10% aqueous citric acid solution, a 5% aqueous sodium bicarbonate solution, and water, and dried over anhydrous sodium sulfate.

After concentration under reduced pressure, ether-n-hexane (1:1) was added to the residue.
was added, the resulting precipitate was collected by filtration, and the desired Boc-Leu-
8.58 g (68.2%) of GIn-OH was obtained.

Melting point, 87-90°C TLCi 0.77 (B:^; skin 90:10:
l) [α) tS= -21,2° (c -1,0+
MeOFI) Elemental analysis value CHN as ClJtJxOh Theoretical value (%) 53.47 8.13 11°69 Actual value (%) 53.55 8.46 11.11 Reference example 16 Boa-Leu-Gin-L s C1-Z -Tr
Production of Boc-Lys(Cl-Z)-Tyr(Br-Z)-L
7.15 g (7,00 mmol) of eu-OPac
"c under cooling, trifluoroacetic acid i.

After the reaction, ether was added to the mixture and the resulting precipitate was collected by filtration.The resulting precipitate was dissolved in THF Loaf-DMF 30tIL1, and cooled at 0°C. The pH was adjusted to 7 with N-methylmorpholine.To this solution, Boa-Leu-Gin-OB
3.79g (10.5mmo+), HOBt 1.6
1g (10,5mmol) of DMF 5ml-THF
7-Add the solution and add WSC・IC112, 25g
(11,7m+mol) of DMF 10m1-TIIF
A mixture of 10 and 10% was added, and the mixture was stirred overnight in a cold room (+4°C).

After the reaction, the mixture was concentrated under reduced pressure, a 5% aqueous citric acid solution was added to the residue, and the resulting precipitate was collected by filtration and washed with a 5% aqueous sodium bicarbonate solution and then water. After drying under reduced pressure, reprecipitation is performed with DMF-AcOEt to obtain the desired Boc-Leu-Gln-Lys(Cl-Z)-T.
yr (Br-Z)-Leu-OPac 8.54g
(96.5%) was obtained.

Melting point, 195-96°C TLC; o, ao (C:M:A, 9o: 1o:
1 ”) [α) ”--19,4° (c = 1.0
．． DMF) Elemental analysis value Ch+HtqNrO01C1
-Br-1120 as Cl(N Theoretical value (%) 53,47 8.13 11.69 Actual value (%) 53,55 8.46 11.11 Reference example 17 Boc-Leu, Gin-L s C1-Z -Tr
(Br-Z-Leu-OR of Boc-Leu-Gin-Lys (Cl-Z)-Ty
r(Br-Z)-Leu-OPac6.32g (5
,00+gnol) in 60ml of 90% acetic acid aqueous solution and activated zinc powder 19.6g (300mmol)
was added and stirred vigorously at room temperature overnight. After filtering the zinc dust, acetic acid was distilled off under reduced pressure, and a 10% aqueous citric acid solution was added to the residue to precipitate it, which was washed with water. After drying under reduced pressure, D? lF
-Re-precipitate with ether to obtain the desired B.

c-Leu-Gin-Lys(Cl-Z)-Tyr(B
r-Z)-Leu-0! I 4.36g (76,1
%) was obtained.

Melting point: 190-92°C TLCiO, 10 (C1:A, 9o:to: 1)
(α) ”=-17,6” (C=1.0. DMF
) Elemental analysis value C5Jt+N, 0+4・Cl-Br・
2/3H! CHN as O Theoretical value (%) 54,99 6.30 8.47 Actual value (
%) 54,69 6.26 8.64 Amino acid analysis; Gin 1.02 (1), Leu 2.08 (2
) Tyr O, 93 (1), Lys O, 96 (1
) Reference Example 18 ``Literature description of Boc-Leu-Ala-OPac'' (G
Boc-Ala-OPac 20. Og
(65,1sa+ol) was dissolved in ditrifluoroacetic acid at 0° C. and stirred for 4 hours. After the reaction, it was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in DMF 25d-TI (F 550
17 and diluted with N-methylmorpholine under cooling at 0°C.
Adjusted to H7. On the other hand, Boc-Leu-OH-Hto
17.9g (71.6mmol) of ethyl acetate15
0- and dry with sodium sulfate. Concentrate under reduced pressure,
Residue and HOBt 11. Dissolve Og (71,6v++ol) in a mixture of DMF 20aj -THF 40-, add 30- of T)IF solution of 6.3 g DCCl (78,8 open o1) under cooling at 0°C, and cool for 30 minutes at 0°C. Stirred. This solution was added to the above solution and stirred for 2 hours at 0°C and overnight in a cold room (+4°C). After the reaction, the precipitate precipitated was collected by filtration, and the reduced diameter residue after reduction with iJI was reduced to 300% in ethyl acetate. −
The solution was washed with a 10% aqueous citric acid solution, a 5% aqueous sodium bicarbonate solution, and water in this order, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, ether-n-hexane (1:1) was added to the residue,
The resulting precipitate was collected by filtration, and the desired Boc-Leu-Ala-
21.9 g (80.3%) of OPac were obtained.

Melting point: 131-32°C TLC: 0.60 (C:M+ 9: 1) [α)
"=-73,5" (c -1,0,MeOH) Elemental analysis value CHN as C2□H:12N206・1/2+1□O Theoretical value (%) 61.52 7.74 6.52 Actual value (% ) 61.75 7.56 6.55 Example 1
9 Boc-L 5(Cl-Z)-Leu-Ala-OPa
Boc-Leu-Ala-OPac manufactured by c. 10.1g
(24, Ommol) was dissolved in a 40% trifluoroacetic acid-methylene chloride solution under cooling at 0°C and stirred for 5 hours.

After the reaction, the mixture was concentrated under reduced pressure, ether-0-hexane was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in THF.
60- and adjusted to p)17 with N-methylmorpholine while cooling at 0°C. On the other hand, Boc-Lys(Cl-Z
) -0R11,0g (26,5mmol) and HOB
t 4.01g (26.5 open o1) in DMF 20t
n! -THP 30mZ was dissolved in a mixed solution of 30mZ, and a TFIF solution of 5.95 g (28.8 mmol) of DCC was added to the mixture under cooling at 0°C, followed by stirring for 50 minutes under cooling at 0°C. This solution was added to the previous solution and kept at 0°C for 2 hours in a cold room (+4°C).
> overnight. After the reaction, the precipitate was collected by filtration,
After concentration under reduced pressure, a 10% aqueous citric acid solution was added to the residue, and the resulting precipitate was collected by filtration and washed with 5% aqueous sodium bicarbonate and water in that order to obtain the desired Boc-Lys(Cl-Z)-Leu-^1a- OPa
17.2 g (100%) of c was obtained.

Melting point; 145-46°C TLC; 0.53 (C:?I:A, 9o:to:
1) [α) ”=-57,4° (c -1,0+
MeOH) Elemental analysis value CHN as C36H49N409Cl Theoretical value (%) 60.28 6.89 7.81 Actual value (%) 60.28 6.99 7.87 Example 2
0 Boc-Ala-L s C1-Z -Leu-Ala
-OPac Boc-Lys(C1-Z)-Leu-A
la-OPac 10. Og (14,0m+5ol)
was dissolved in 15 m7 of trifluoroacetic acid under cooling at 0°C, stirred for 1 hour, and then concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration.

The obtained precipitate was dissolved in TI 30d-DMF 70-, and while cooling at 0°C, the pl was adjusted to 7 with N-methylmorpholine.
,7mmol) and ll0Bt 2.25g (14,7
mmol) was dissolved in a mixed solution of DMF 5d-TIIF 20-, and 3.47 g (16 mmol) of DCC was added under cooling to 0°C.
, 8 mmol) was added, and the mixture was cooled to 0°C and stirred in Shimonoseki.

This solution was added to the previous solution and stirred for 1 hour at O'C and overnight in a cold room (+4°C). After the reaction, the deposited precipitate was collected by filtration, and after concentration under reduced pressure, a 10% aqueous citric acid solution was added to the residue, the resulting precipitate was collected by filtration, and washed with 5% aqueous sodium bicarbonate and water in this order. After drying under reduced pressure, precipitation is performed with methanol-ether to obtain the desired BocAla-Lys(C1-Z)-Leu-
9.15 g (82.6%) of Ala-OPac was obtained.

Melting point: 140-41"C TLC: 0.55 (COMMA, 90:10:1
)(α)”=-31,3° (c=1.0. DM
F) Elemental analysis value C3gHsJsO+.・C as Cl
ON Theoretical value (%) 59.12 6,96 9.00 Actual value (%) 59.42 6.90 8.88 Reference example 2
1 Boc-Leu-Ala-L 5(Cl-Z)-Le
Boc-^1a-Lys(Cl
-Z)-Leu-Ala-OPac 6.30g (8,
00vwol) to 10a of trifluoroacetic acid under cooling at O''C.
It was dissolved in Z and stirred for 1 hour. After reaction, reduce pressure 1Ji1L
Ether was added to the residue, and the resulting precipitate was collected by filtration.

The obtained precipitate was dissolved in THF 10m-DMF 100- and adjusted to pH 7 with N-methylmorpholine while cooling at O''C.Meanwhile, Boc-Leu-OR-Hzo4.
Dissolve OOg (16, Ommol) in ethyl acetate 100- and dry with sodium sulfate. Concentrate under reduced pressure, and dissolve the residue and 2.46 g (16, Ommol) of HOBt in DMF.
20WL! -T) IF Dissolved in 10m7 of mixed solution, 0
A solution of 3.64 g (17.6 mmol) of DCC in TIIF 10- was added under cooling at °C, and the mixture was stirred for 1 hour under cooling at O''C.

This solution was added to the previous solution and stirred for 1 hour at 0°C and overnight in a cold room (+4°C). After the reaction, the precipitate deposited was collected by filtration, concentrated under reduced pressure, 5% aqueous sodium bicarbonate was added, the resulting precipitate was collected by filtration, and washed with a 10% aqueous citric acid solution and water in that order.

After drying under reduced pressure, precipitate with DMF ethyl acetate to obtain the desired B
oc-Leu-^IaLys (Cl -Z) -Le
7.21 g (100%) of u-^1a-OPac was obtained.

Melting point: 180-81"C TLCio, 78 (C:M:A, 90:10:
1)(α)”=-27,9@(c=1.0.
DMF) Elemental analysis value C45HisNaO□・ci・3
CFIN as /2Hgo Theoretical value (%) 58.23 7,34 9.05 Actual value (%) 58,13 7.15 9.29 Reference example 2
2 Boc-Leu-Leu-Ala-L 5(Cl-Z
)-Leu-^1a-OPac's transfer Boc-Leu-^1a-Lys(Cl-Z)-Leu
-Ala-OPac 5.42g (6,00mmol)
was dissolved in 15 m7 of trifluoroacetic acid under O'C cooling, and 3
Stirred for 0 minutes. Depressurize after reaction? After condensation, ether was added to the residue, and the resulting precipitate was collected by filtration. DM the obtained precipitate
The mixture was dissolved in F60- and adjusted to pH 7 with N-methylmorpholine while cooling at 0°C. On the other hand, Boc-Leu-OH
Dissolve 1.79 g (7,18+nol) of -Hzo in ethyl acetate 100- and dry with sodium sulfate. It was concentrated under reduced pressure, and the residue and HOBt 1.11g (7,23w5o
1) was dissolved in a mixed solution of DMF 15aZ-THF 60g 1j, and -5C-HCl 1.52g (
7,95a + mol) and stirred at O″C for 2 hours. Add this solution to the above solution and stir at O″C for 2 hours.
Stir overnight in a cold room (+4°C). After the reaction and concentration under reduced pressure, 5% aqueous sodium bicarbonate was added, and the resulting precipitate was collected by filtration and washed with a 10% aqueous citric acid solution and water in that order. After drying under reduced pressure, DMF
- Perform reprecipitation with methanol to obtain the desired Boc-Leu-
Leu-Ala-Lys(Cl-Z)-Leu-Ala
-OPac 7.28g (83.6%) was obtained.

Melting point; 188-89°C TLC; 0.95 (C: M:^, 90:10:
1) (α) ”= −33,7° (c=1.0.D
MF) Elemental analysis value (: s ll -t h N ?
0 +□・C1・1/2H, Cl as 0
N Theoretical value (%) 59.84 7.58 9.58 Actual value (%) 59,83 7.55 9.54 Reference example 2
3 Boa-Lea-Leu-Ala-Lys (Cl-Z
)-Leu-Ala-OPac3.03g (2,99a
+5ol) was dissolved in trifluoroacetic acid 15- while cooling at O''C and stirred for 1 hour. After the reaction, it was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration.The resulting precipitate was [)MP 30af and adjusted to pH 7 with N-methylmorpholine while cooling at 0°C.
Under cooling, Boc-Lys (Cl-Z)-081,49
g (3,60 open 01) and ll0Bt O,56g (3
, 66 mmol) of DMF 5- was added, and further -SC-HCl 0.83 g (4,33 m5+ol)
was added and stirred overnight in an O''C1 cold room (+4°C).

After the reaction and concentration under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration and washed successively with a 5% aqueous sodium bicarbonate solution and water. After drying under reduced pressure, reprecipitation is performed with DMF-methanol to obtain the desired Boc-LyS(Cl-Z)-Leu-Leu-Al.
a-Lys (Cl-Z) -Leu-^1a-OPa
c3.21g (82.0%) was obtained.

Melting point; 212-14°C TLC; 0.58 (C: M:^, 90:10:
i) (α) ”=-31,4@(c=1.0.DMF
) Elemental analysis value CthsHqJqO+1CL・3/21
1□O 1 Theoretical value (%) Actual value (%) Reference example 24 58.33 7.23 9.42 58.09 7.04 9.45 Boc-Lys (Cl-Z) -Leu-Leu-A
la-Lys (Cl-Z) -Leu-Ala-OP
Dissolve 3.39 g (3,00 mmol) of ac in 95% acetic acid aqueous solution, and add 5.88 g of activated zinc powder.
(89.9 mmol) was added and stirred vigorously at room temperature for 4.5 hours.

After filtering the zinc dust, acetic acid was distilled off under reduced pressure, and 10%
A citric acid aqueous solution was added to precipitate the mixture, which was washed with water. After drying under reduced pressure, reprecipitation was performed with DMF-ether to obtain the desired Boc-
Lys (Cl-Z)-Leu-Leu-Ala-Ly
s(Cl-Z)-Leu-Aia-OR3,08g (8
6.0%) was obtained.

Melting point: 205°C (decomposition) TLC, 0.25 (COMMA, 90:10:1
) [α) ”=-29,1@(c =1.0. DMF
) Elemental analysis value CsJ@vN, 0+ a・t, h・4/
3 ozo and Cl (N Theoretical value (%) 56.24 7.42 10.36 Actual value (%) 56.00 7.22 10.39 Amino acid analysis; ^la 1,92 (2), Leu 3.2
5(3)Lys 1.94(2) Reference Example 25 Boc-Tr Br-Z -5er(Bzl-OPa
c TosOH-1f-Set (Bzl)-0Pac
7.93g (16.3mmol) in DMF 20sd
It was dissolved in -THF 40- and adjusted to pH 7 with N-methylmorpholine while cooling at -15°C. On the other hand, Boc-T
yr(Br-Z)-088,85g (17,9mmol
), HOBL 2.74g (17.9 old o1) D?
Dissolve in a mixture of IP 10mj-Tl 50- and 0°
T of DCC4,01g (19,4ms+ol) under C cooling
HF solution 5- was added and stirred for 1.5 hours under cooling at O''C.

Add this solution to the above solution and hold at -20°C for 1 hour at 0°C.
Stirred for 2 hours at C and overnight in a cold room (+4° C.). After the reaction, the precipitate precipitated was collected by filtration, concentrated under reduced pressure, and the residue was dissolved in 300% ethyl acetate, washed in this order with 10% citric acid aqueous solution, 5% sodium bicarbonate solution, and saturated brine, and dried over anhydrous sodium sulfate. did. After concentration under reduced pressure, ether-petroleum ether (1:l) was added to the residue, the resulting precipitate was collected by filtration, and the desired Boc
-Tyr(Br-Z)-Ser(Bzl)-0Pac
12.9g (100%) was obtained.

Melting point: 95-8°C TLCio, 79 (CAM, 9:1”) Cut
]"-4,9" (c =-1,0+ MeOH) Elemental analysis value C4°Fln+NzO+.・Br as C)I
N Theoretical value (%) 60.84 5.23 3.55 Actual value (%) 61.30 5.28 3.43 Reference example 2
6 Boc-Gln-Tr Br-Z-Ser (Bzl)
-0Pac's Boc-Tyr(Br-Z)-Set(B
zl)-0Pac 6.33g (8.02mmol)
was dissolved in 4N hydrochloric acid-dioxane solution under cooling at O''C and stirred for 1 hour. After the reaction, it was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration.
Dissolved in HF IM-DMF 20- and cooled at 0°C.
The pH was adjusted to 7 with N-methylmorpholine. On the other hand, Bo
c-Gin-OH2, 37g (9,62IIllIlo
l), HOBt 1.63g (10.6mmol)
Dissolved in a mixture of DMF5d-THF20- and heated to 0°C.
2.37 g (11.5 mmol) of TI under cooling DCC
P solution 5+7 was added and stirred for 40 minutes while cooling at 0°C. This solution was added to the above solution and kept at 0°C for 1 hour in a cold room (+
The mixture was stirred overnight at 4°C. After the reaction, the precipitate deposited was collected by filtration, concentrated under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration and washed with 5% aqueous sodium bicarbonate and water in this order.

After drying under reduced pressure, precipitation was performed with methanol-ether to obtain the desired Boc-Gin-Tyr(Br-Z)-5et(B
zl) -0Pac 6.85g (93.1%) was obtained.

Melting point 1164-5°C TLC; 0.39 (C:M:A, 90:10:
1) [α]z5=-3,5@(C-1,0,DhF) Elemental analysis value C! CHN theory as lH3°N206 (%) 62.05 7.44
6.89 Actual value (%) 62.06 7.51 6
．． 76 Reference Example 27 Boc-Gln-Gln-Tr(Br-Z)-Set
(Bzl)-0Pac's 1Boc-Gin-Tyr(B
r-Z)-Set(Bzl)-0Pac 5.51g(
6,00 eamol) was dissolved in 45 mf of 4N hydrochloric acid-dioxane solution under cooling at 0°C, and stirred for 3.5 hours. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in DMF 30mj-THF4
5- and diluted with N-methylmorpholine under cooling at 0°C.
Adjusted to H7. On the other hand, Boc-Gin-0) 11.7
7g (7.19gmol), HOBt 1.21g
(7.94 mmol) was dissolved in a mixed solution of DMF5d-T liver 30-, and 11.52 g of N5C-HC (
7.93 mmol) and stirred for 40 minutes. This solution was added to the previous solution and incubated at 0°C for 30 minutes in a cold room (+4°C).
℃) overnight. After the reaction, the precipitate precipitated was collected by filtration, concentrated under reduced pressure, 10% aqueous citric acid solution was added, and the precipitate formed was collected by filtration, washed in the order of 5% aqueous sodium bicarbonate and water to obtain the desired Boc.
-GlnGln-Tyr(Br-Z)-Ser(Bzl
)-0Pac 5.38g (93.3%) was obtained.

Melting point; 198-201'C TLC; 0.27 (C: M:^, 90:10:
1) [α)” = -5,9° (c = 1.0. DMF
) Elemental analysis value C5ollsJJ□・Br-H2O Theoretical value (%) 56.45 5.59 7.90 Actual value (
%) 56,56 5.48 7.43 Reference Example 28 Boc-Gin-Gln-Tyr(Br-Z)-5er
(Bzl)-0Pac 5.24g (5,OOmmol
) was dissolved in 40@l of 4N hydrochloric acid-dioxane solution under O''C cooling and stirred for 1 hour. After the reaction, it was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The pH was adjusted to 7 with N-methylmorpholine while cooling at O''C. Add Boc to this solution.
-Thr(Bzl)-0H1,86g (6,00nu
wol), FIOBt 0.93g (6,05emr
iol) and further 1.39 g of 5C-HCl (
7,25+nmol) was added and stirred for 4.5 hours at O''C and overnight in a cold room (+4'C). After the reaction, it was concentrated under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration. ,5
% sodium bicarbonate solution and then water. After drying under reduced pressure, precipitation is performed with DMF-ethyl acetate to obtain the desired Boc-Thr (Bzl
)-Gln-Gin-Tyr(Br-Z)-5et(B
zl)-0Pac 5.51 g (89.1%) was obtained.

Melting point; 213-6'C TLCiO,39(CAM, 9:1)(α)''
= -t, o" (c = 1.0. DMF) Elemental analysis value C61■wJyO+1Br-ON as H2O Theoretical value (%) 58,37 5.78 7.81 Actual value (%) 58,25 5.63 7.82 Amino Acid Analysis HThr O, 94(1), Set 0.91 (
1) Gin 2.11 (2), Tyr 1.04 (1
) Reference example 29 Boc-11e-Phe-OPac's notification HBr
・H-Phe-OPac 15.2g (41.7mmo
l) was dissolved in DMF' 100- and adjusted to p) 17 with N-methylmorpholine while cooling at -15°C. on the other hand,
Boc41e-Oll ・1/2HzO12,Og (
50.1+mmol), HOBt 18.3g (11
9 mmol) was dissolved in a mixed solution of DMF 150d-THF 250- and WSC-)1G111. under cooling at 0°C.
3 g (58.9 mmol) was added and stirred for 40 minutes while cooling at 0°C. This solution was added to the above solution and heated to -20℃ for 1 hour.
The mixture was stirred for 1 hour at 0°C and overnight in a cold room (+4°C). After the reaction, the mixture was concentrated under reduced pressure, and the residue was dissolved in 500% ethyl acetate, washed successively with 10% aqueous citric acid, 5% aqueous sodium bicarbonate, and saturated brine, and dried over anhydrous sodium sulfate.

After concentration under reduced pressure, ether was added to the residue, the resulting precipitate was collected by filtration, and the desired Boc-11e-Phe-OPac12.9
g (70.0%) was obtained.

Melting point i 138.5°C TLC; 0.8B (CAM, 9:1) (α)
”= -41,0' (c=1.0. DMF) Elemental analysis value CzsH: +aNzOa as HN Theoretical value (%) 67.72 7.31 5.64 Actual value (%) 67.81 7.36 5 .64 Reference example 3
0 Boc-Ala-11e-Phe-OPac
-11e-Phe-OPac 4.96g (10,0
mmol) was dissolved in 10-trifluoroacetic acid under O''C cooling and stirred for 1.5 hours. After the reaction, it was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The resulting precipitate was diluted with DMF. 25- and adjusted the pH to 7 with N-methylmorpholine while cooling at 0°C.
-0)! 2.27g (12.0mmol), ll
0Bt 1.87g (12.2wmol) in DMF
It was dissolved in a mixed solution of 30-5af-THF, and while cooling at 0°C, 2.768 (14.4 mmol) of -5C-HCl was added, followed by stirring for 30 minutes. This solution was added to the previous solution and stirred for 1 hour at 0°C and overnight in a cold room (+4°C). After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration and washed in the order of 5% aqueous sodium bicarbonate and water. After drying under reduced pressure, precipitation is performed with DMF-petroleum ether to obtain the desired Bo
c-Ala-11e-Phe-OPac5.24g (
92.4%) was obtained.

Melting point; 164-4.5°C TLC: 0.62 (CAM, 9:1) [α'J
"--28,8' (c = 1.0. DMF) Elemental analysis value C311141N30? as Cl (N Theoretical value (%) 65.59 7.28 7.40 Actual value (%") 65.56 7. 36 7.55 Reference Example 31 Boc-As (OcHex)-Ala-11e-Ph
e=OPac's Boc-^1a-11e-Phe-OP
ac 5.24g (9,2411111101) O
The mixture was dissolved in trifluoroacetic acid IQm7 while cooling at °C and stirred for 1 hour. After the reaction, concentrate under reduced pressure, add ether to the residue,
The resulting precipitate was collected by filtration. The obtained precipitate was dissolved in DMF 40a.
It was dissolved in Z-TIIF 5aZ and adjusted to pH 7 with N-methylmorpholine while cooling at 0°C.

On the other hand, Boc-Asp(Octlex)-0H3,50
g (11.1 mmol), HOBt 1.70 g (
11,1mmo+) in DMF 5aZ-THF 20a
Dissolve in the mixture of f and cool at θ°C with -SC-HCl 2.
55 g (13.3 ml) was added and stirred for 1 hour.

This solution was added to the previous solution and stirred overnight in a cold room (+4°C). After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration and washed in the order of 5% aqueous sodium bicarbonate and water. After drying under reduced pressure, precipitation is performed with DMF-ether to obtain the desired Boc-Asp(OcHex)-Ala-11e-
5.89 g (83.4%) of Phe-OPac was obtained.

Melting point, 182-2.5℃ TLC, 0.67 (CAM, 9:1) [α)
”=-23,8” (c=1.0.DMF)
Elemental analysis value C4+HssNJ+HN as o・H2O Theoretical value (%) 63.77 7,35 7.31 Actual value (%>63,71 7.30 7.24 Reference example 32 Boa-Set (Bzl)-As (Octlex) −
Ala-11e-Phe-OPac's ally Boc-Asp(OcHex)-Ala-11e-Ph
e-OPac 4.97g (6.50mmol) at 0℃
The mixture was dissolved in 10 mZ of trifluoroacetic acid under cooling and stirred for 1 hour. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration.

The obtained precipitate was dissolved in DMF 40- and cooled at 0°C.
The pH was adjusted to 7 with N-methylmorpholine. On the other hand, Bo
c-Set (Bzl)-082,30g (7,79m
mol), HOBt 1.19g (7.77mmol
) was dissolved in a mixture of DMF 5aZ-THF 20- and cooled to 0°C. 5C-HCL 1.79g (9,34
mmo+) was added and stirred for 1 hour. This solution was added to the previous solution and stirred overnight in a cold room (+4°C).

After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration and washed successively with a 5% aqueous sodium bicarbonate solution and water. After drying under reduced pressure, precipitation was performed with D liver-ethyl acetate-ether to obtain the desired Boc-Ser(Bzl)-Asp(OcHex).
-^1a-Tie-Phe-OPac 5.90g (
96.3%) was obtained.

Melting point; 189-90°C TLC; 0.55 (CAM, 9:1) [α)”
=-19,2° (c = 1.0. DMF) Elemental analysis value CHN as C51H66NSOI□・H2O Theoretical value (%) 63.87 7.15 7.30 Actual value (%) 63.80 7.04 7 .30 Reference example 3
3 Boc-3et(Bzl)-Asp(OcHex)-8la-11e-Phe-OPac5,OOg (5,3
0 mmol) in 15 mZ of trifluoroacetic acid under cooling at O'c.
and stirred for 1.5 hours. After the reaction, concentrate under reduced pressure,
Ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in DMF 25af-TIIF80@1, and the pH was adjusted to 7 with N-methylmorpholine while cooling at 0°C. 'On the other hand, Boc-His (Tos) -OB
-DCH＾7.50g (12.7mmol) in O″C
After adding to 150% of 10% citric acid aqueous solution under cooling and stirring,
Extract with ethyl acetate 250@1 and dry with sodium sulfate.

Concentrate under reduced pressure, dissolve the residue in 75m7 of methylene chloride, and reduce to 0.
While cooling at °C, 1.33 g (6.45 mmol) of DCC was added, and the mixture was stirred for 30 minutes. After the reaction, the precipitate was collected by filtration, concentrated under reduced pressure, and the residue was dissolved in THP 50-.

This solution was added to the above solution and -
Stirred overnight. After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added, and the resulting precipitate was collected by filtration and washed in the order of 5% aqueous sodium bicarbonate and water. After drying under reduced pressure, precipitation is performed with DMF-ether to obtain the desired Boc-old s(Tos)Set(Bzl)-
Asp(OcHex)-Ala-41e-Phe-OP
5.00 g (76.5%) of ac was obtained.

Melting point, 209-210°C TLC; 0.49 (ethyl acetate: n-hexane, 4
:1) [α) ”=-10,6' (c =1.0.D
MF) Elemental analysis value C1aFItJeO+5S-H2O
HN Theoretical value (%”) 61.47 6.53 8.96 Actual value (%) 60.97 6.62 9.49 Reference example 34 Boc-His(Tos)-Ser(Bzl)-Asp
(OcHex)-Ala-11e-Phe-OPac
4. Dissolve OOg (3,25o+mol) in 95% acetic acid aqueous solution 70fi+7, and add 6.50% activated zinc powder.
g (99.5 mmol) and stirred vigorously at room temperature overnight. After filtering the zinc powder, it was concentrated under reduced pressure, and the residue was
% citric acid aqueous solution was added to precipitate, and the mixture was washed with water.

After drying under reduced pressure, reprecipitation was performed with DMF-ethyl acetate to obtain the desired Boc-His(Tos)-5er(Bzl)-Asp.
(Oc)lex)-^1a-1ie-Phe-OR3
, 16g (87.4%) were obtained.

Melting point, 217-9°C TLC; 0.77 (B:A:W, 4:1:5
)(α) ”=-13,5″’ (c=1.0.0M
5O) Elemental analysis value C5JtJsO□・DMF・3/2+i, o Theoretical value (%) 58,35 6.89 10.38 Actual value (%) 58.34 7.11 11.11 Amino acid analysis; Asp 1. 07(1), Ser 1.05(1)
)Ala 1.00(1), lie O,9B(
1) Phe O, 98 (1), His 0.95 (1)
) Reference Example 35 Boc-Thr(Bzl)-Gln-Gln-Tyr(
Br-Z)-3et(Bzl)-OPac 3.99g
(3.21 mmol) was dissolved in 24 ml of 4N hydrochloric acid-dioxane solution under cooling at 0°C and stirred for 2 hours. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in 0MF27- and O'
The pH was adjusted to 7 with N-methylmorpholine under cooling.

In this solution, Boc-His-Ser(Bzl)-As
p(OcHex)-^1a-Tle-Phe-OH3,
81g (3.42mmol), tlOBt 0.65g
(4,27 mmol) Leaf F 30aZ-DMSO1
Add 5af solution and add WSC/HCl under O″C cooling.
0.98 g (5,12 mmol) of D liver solution 30-
and cooled at 0°C for 1 hour, then in a cold room (+4°C) -
Stirred overnight. After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added to the residue, and the resulting precipitate was washed successively with a 5% aqueous sodium bicarbonate solution and water. After drying under reduced pressure, it is recrystallized with methanol to obtain the desired Boc-His-Ser(Bzl)-Asp (OcH
ex) -Ala-1ie-Phe-Thr (Bz
1)-Gin-Gin-Tyr(Br-Z)-Set(
6.36 g (95.2%) of Bzl)-0Pac were obtained.

Melting point: 248°C (decomposition) TLC: 0.70 (B:A:W, 4:1:5
)(α) ”= −4,0' (c=0.5.0M5
O) Elemental analysis value C+osH+tJ+50g5Br ・
HN as 311tO Theoretical value (%) 59.12 6.28 9.85 Actual value (%) 59.06 5.93 9.61 Amino acid analysis; ^sp 1.06 (1), Thr 1.0H1
) Set 1.94 (2), Gin 2.30 (2)
Ala 0.99(1), lie O,76(1)T
yr 1.15 (1), Phe O, 86 (1) Hi
s O,92(1) Reference Example 36 Boc-His-Ser(Bzl)-Asp(OcH
ex)-^1a-Tie-PheThr(Bzl)-G
in-Gin-Tyr(Br-Z)-3et(Bzl)
-0Pac3.44g (1,75 open o1), N-
5.65 g (86.5 m
mo+) was added and stirred vigorously at room temperature for 7.5 hours. After filtering the zinc dust, acetic acid was distilled off under reduced pressure, and the residue was
A citric acid aqueous solution was added to precipitate the mixture, which was washed with water. After drying under reduced pressure, wash with ether to obtain the desired Boc-His-3e.
r(Bzl)-Asp(OcHex)-Ala-11e
-Phe-Thr(Bzl)-Gin-Gin-Tyr
(Br-Z)-5et(13zl)-081,28g
(41.2%) was obtained.

Melting point: 247°C (decomposition) TLC: 0.21 (C: M:^, 9:2:1)
[α) ”=+3.0” (c =0.1.0M5O
) Elemental analysis value CwJtttN+5OzJr + ci
Theoretical value (%) as tric acid 11H20 Actual value (%) Amino acid analysis; Asp Ser Ala Tyr is CH 52,616,47 52,245,80 1,02 (1), Thr 1.76 (2), Gln 1.06(1), l1e 1.02(1), Phe O,9H1) Reference example 37 8.94 9.03 1.00(1) 2.19(2) 0.99(1) 1.04 (1) Boc-5et (Bzl)-Arg (Mts)-Thr
(Bzl)-5et(Bzl)-Pro-Pro-Pr
o-N41t 4. OOg (3.09 mmol),
The mixture was dissolved in 10 m7 of trifluoroacetic acid under cooling at O°C and stirred for 2 hours. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was heated to D liver 15W.
Boc-^1a-Se was dissolved in Boc-^1a-Se.
t(Bzl)-11e-Leu-Gly-OH2,41
g (3,71ma+ol), HOBL 0.56g
(3,71 sv+ol) of DMF 20-solution was added and further cooled at 0°C -SC-ICl 0.86 g (4,
49 mmol) of DMF solution was added thereto, and the mixture was stirred for 2 hours under cooling at O'C and overnight in a cold room (+4°C). After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added to the residue, and the resulting precipitate was washed successively with a 5% aqueous sodium bicarbonate solution and water. After drying under reduced pressure, the precipitate was applied to a 5ephadex LH-20 column chromatograph (3.2×55 cm) and purified by column chromatography using an eluent of 97% DMF (DMF:water, 97:3).

Fractionate 3g each, collect 24th to 60th fractions, concentrate under reduced pressure,
Add ether and collect the precipitate by filtration to obtain the desired Boc.
-^1a-Ser(Bzl)-11e-Leu-Gly
-3er(Bzl)-Arg(Mts), Thr
(Bz l) -Set (Bz l) -Pro-P
86 g (86.1%) of ro-Pro-NH14 was obtained.

Melting point; 220-3°C TLCio, 75 (BAA:W, 4:1:5
) [α) ” −−27,1@(c =0.1.0M5
O) Elemental analysis value CwsH+sJ+10zoS ・4H
C)l as zO Theoretical value (%) 58.91 7.33 Actual value (%)
59.07 7.13 Amino acid analysis; Thr O,
99(1), 5erGly 1.00(1), T
ie Leu 1.02(1), 8rgPro 2.6
3(3) 11.82 12.01 2.67(3) 0.97(1) 1.08(1) Reference example 38 Boc-Ala-Ser(Bzl)-11e-Leu-
Gly-Ser(Bzl)-Arg(Mts)-Thr
(Bzl)-3et(Bzl) -Pro-Pro-P
ro-Ntlz3.39g (1.86mmol),
The mixture was dissolved in 10 mZ of trifluoroacetic acid while cooling at 0°C and stirred for 2 hours. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in DMF 40
It was dissolved in aZ-0MSO35-, and the pH was adjusted to 7 with N-methylmorpholine while cooling at 0°C. In this solution, Bo
c-LeuGln-Lys(Cl-Z)-Tyr(Br
-Z)-Leu-OH2,55g (2,23mmol
), HOBt O, 35g (2,29ma+ol)
Add a DMF 25-solution and further cool at 0°C-SC
-) DM of ICl O, 51 g (2,66 ml O1)
F solution 10- was added and kept at 0°C for 3 hours in a cold room (
+4°C) overnight.

After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added to the residue, and the resulting precipitate was washed successively with a 5% aqueous sodium bicarbonate solution and water. After drying under reduced pressure, reprecipitation was performed with N-methylpyrrolidone-methanol to obtain the desired Boc-Leu-Gin-Lys (Cl-Z
)-Tyr(Br-Z)-Leu-Ala-Ser (
Bzl)-11e-Leu-Gly-Ser(Bzl)
-Arg(Mts)-Thr(Bzl)-3et(B
zl)-Pro-Pro-Pro-NHx 4.78g
(90.1%) was obtained.

Melting point: 286°C (decomposition) TLC: 0.75 (B:A:W, 4:1:5
) [α) ”=-18,3@(c=1.0.DMS
O) Elemental analysis value CraH+qNzxOs+-Cl-B
r-S ・CHN as 2JO Theoretical value (%') 5B, 65 6.81 11.16
Actual value (%) 58.76 6.91 11.38 Amino acid analysis; Thr O, 99 (1).

Gin 1.04 (1).

Ala 1.02 (1).

Leu　3.30(3).

Lys 1.10(1).

Pro 2.68 (3) Set 2.60 (3) Gly 1.03 (1) 11e 1.04 (1) Tyr 1. IHl, ) Arg 1.08 (1) Reference Example 39 Boc-Leu-Gln-Lys(Cl-Z)-Tyr
(Br-Z)-Leu-Ala-Ser(Bzl)-1
1e-Leu-Gly-Set(Bzl)-Arg(M
ts) -Thr(Bzl)-Set(Bzl)-Pr
o-Pro-Pro-NHz 4.00g (1,4
0 mmol) was added to trifluoroacetic acid 10-
and stirred for 2 hours. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration.

The obtained precipitate was added to DMF 20mj-IIMSO45wL.
I, and diluted with N-methylmorpholine under cooling at 0°C.
Adjusted to H7. Add Boc-Lys (Cl
-Z) -Leu-Leu-^1a-Lys(Cl-Z)
-Leu-Ala-OH2,OOg (1,68mmo
l), HOBt O, 39g (2,55 question 01) D
Add MF Loa & solution and further cool at O″C -5C-
0.50 g (2', 62+++ mol) of HCl was added and stirred in a cold room (+4°C) for 2 days. After the reaction, the mixture was concentrated under reduced pressure, a 10% aqueous citric acid solution was added to the residue, and the resulting precipitate was washed successively with a 5% aqueous sodium bicarbonate solution and water. After drying under reduced pressure,
Reprecipitate with DMSO-methanol to obtain the desired Boc-Ly.
s(C1-Z)-Leu-Leu-Ala-Lys(C
l-Z)-Leu-^1a-Leu-Gin-Lys
(Cl-Z) -Tyr (Br-Z)-Leu-^1
a-3er(Bzl)-11e-Leu-Gly-Se
t(Bzl)-Arg(Mts)-Thr(Bzl)-
Set(Bzl)-Pro-Pro-Pro-NHt
4.14 g (75.3%) were obtained.

Melting point: 295°C (decomposition) TLC: 0.61 CB:A:H, 4: 1: 5)
[α] “= −20,7” (C=1.0.0M5O
) Elemental analysis value C+++5Hz++N+zOnzC1
+BrS ClI N as 6HzO Theoretical value (%) 57.45 6.99 11.11 Actual value (%) 57.21 6.52 11.03 Amino acid analysis; T, hr 1.00 (1), Set 2.
66(3)Gin 1.03(1), Gly 1.
06(1)Ala 2.96(3), lie 1.
06(1)Leu 6.46(6), Tyr 1.
IHl)Lys 2.9H3), Arg 1.09
(1) Pro 2.67 (3)

[Brief explanation of drawings]

Figure 1 is a graph showing the effect of helodermin on rat and guinea pig atrial muscle, Figure 2 is a graph showing the effect of helodermin on guinea pig tracheal muscle, Figure 3 is a graph showing the effect of herodermin on guinea pig ileum, and Figure 4.
is a graph showing the effect of helodermin on the rabbit thoracic aorta.

Claims (1)

[Claims] 1. An asthma treatment whose main ingredient is a peptide (herodermin) represented by the following structural formula [gene sequence is included]. 2 The following structural formula [Gene sequence is available] (In the formula, Boc is a tertiary butyloxycarbonyl group, Bzl is a benzyl group, OcHex is a cyclohexyl ester group, Br-Z is a 2-bromobenzyloxycarbonyl group, Cl-Z is a 2-chlorobenzyloxycarbonyl group, X_1 is a tosyl group or a hydrogen atom, and X_2 is a tosyl group or a mesitylene-2-sulfonyl group. Method. 3. The method for producing herodermine according to claim 2, wherein the protecting group is removed with a strong acid. 4. The method for producing helodermine according to claim 2, wherein the protecting group is removed with hydrogen fluoride. 5. The method for producing herodermine according to claim 2, wherein the protecting group is removed with trifluoromethanesulfonic acid. 6 The following structural formula [There is a gene sequence] [There is a gene sequence] A pentatriacosapeptide amide represented by a carbonyl group, Cl-Z is a 2-chlorobenzyloxycarbonyl group, X_1 is a tosyl group or a hydrogen atom, and X_2 is a tosyl group or a mesitylene-2-sulfonyl group. 7 The following structural formula [Gene sequence is available] (In the formula, Boc is a tert-butyloxycarbonyl group, Bzl is a benzyl group, OcHex is a cyclohexyl ester group, Br-Z is a 2-bromobenzyloxycarbonyl group, and X_1 is a tosyl group. The undecapeptide represented by the following structural formula [gene sequence available] (in which Cl-Z is a 2-chlorobenzyloxycarbonyl group, and A method for producing pentatriacosapeptide amide represented by the following structural formula [gene sequence is available], which is characterized by condensation with tetracosapeptide amide represented by (indicating a sulfonyl group).