JP2778831B2 - Endothelin antagonist peptide - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel peptide having endothelin antagonism and a synthetic intermediate thereof. This peptide has excellent endothelin antagonism, high blood pressure, asthma, stroke,
It is useful as a therapeutic agent for angina pectoris, acute renal failure, myocardial infarction, cerebral vasospasm and the like.

BACKGROUND ART Endothelin is a cyclic peptide having a strong and sustained vasoconstrictor action, and is considered to be one of the substances involved in hypertension, asthma, acute renal failure, myocardial infarction, stroke, angina pectoris and cerebral vasospasm . Therefore, substances that antagonize endothelin and inhibit this action are expected to be useful in the treatment and prevention of these diseases.

It is known that the cyclic peptide represented by the formula (A) exhibits an endothelin antagonism (Japanese Patent Laid-Open No. 3-13 / 1990).
No. 0299): (Where U is D-Val or D-allo-Ile, R ¹ is hydrogen or amino protecting group, R ² is hydrogen or carboxyl protecting group).

DISCLOSURE OF THE INVENTION The inventors of the present invention have conducted repeated studies on products of many microorganisms for the purpose of finding a physiologically active substance exhibiting endothelin antagonism from the natural world. Endothelin antagonism, inhibition of intracellular calcium elevation by endothelin, and intracellular guanosine-
A substance having a 3 ′, 5′-cyclic monophosphate concentration inhibitory action and the like was isolated, and RES-701-1, RES-701-2 and RES-701 were isolated.
-3. As a result of further studies, the structures of these substances were determined, and novel derivatives of these compounds were also synthesized, thereby completing the present invention.

According to the present invention, there is provided a peptide compound represented by the following formula (I) or a pharmacologically acceptable salt thereof: XA-Trp-B-Gly-Thr-EGY (I ) ｛Wherein A represents Asn or Asp, B represents His or Lys, E represents Ala or Ser, G represents Ala or Pro, X represents X ¹ -Gly or Y represents hydroxy, lower alkoxy, amino, Wherein X ¹ and X ³ represent hydrogen, benzyloxycarbonyl, t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl or a carbonyl-substituted or unsubstituted lower alkanoyl, and X ² and Y ² represents hydrogen, Y ¹ represents hydroxy, lower alkoxy or amino, or X ¹ , Y ¹ and X ² , Y ² together represent X ¹ −
Y ¹ and X ² —Y ² represent a single bond, Z is hydroxy, lower alkoxy, benzyloxy, benzhydryloxy, Gly-Z ¹ (wherein Z ¹ is hydroxy, lower alkoxy, benzyloxy or benzhi Stands for drilloxy,
X ¹ and together represent X ¹ -Z ¹ is a single bond), in Ala-Z ¹ (wherein, Z ¹ is as defined above), in Val-Z ¹ (wherein, Z ¹ is and wherein Trp-Z ¹ (wherein, Z ¹ is as defined above), Trp-Gly-Z ¹ (where Z ¹ is as defined above), Trp-Asn-Tyr-Tyr -Trp-Z ¹ (wherein Z ¹ is as defined above), Trp-Phe-Phe-Asn-Tyr-Tyr-7Hyt-Z ¹ , wherein Z ¹
Is as defined above, 7Hyt represents 7-hydroxytryptophan), Trp-Ile-Ile-Trp-Z ¹ (wherein Z ¹ is as defined above), Trp-Val-Tyr-Phe-W -His-Leu-Asp-Ile-Ile-T
rp-Z ¹ (wherein, Z ¹ has the same meaning as described above, and W represents Ala, Ser or Cys), Trp-W-His-Leu-Asp-Ile-Ile-Trp-Z ¹ , wherein , Z ¹ and W are as defined above), Trp-Val-Tyr- Tyr-W-His-Leu-Asp-Ile-Ile-T
rp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Leu-Tyr-Phe-W-His-Gln-Asp-Val-Ile-T
rp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Val-Tyr-Phe-W-Phe-Phe-Asn-Tyr-Tyr-T
rp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Phe-Phe-Asn-Tyr-Tyr-W-His-Leu-Asp-I
le-Ile-Trp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Phe-Phe-Asn-Tyr-Tyr-Asn-Ile-Ile-Trp-
Z ¹ (wherein, Z ¹ is as defined above), or J-Phe-MQ-Tyr-R-T-Z ¹ (where J is Trp or a single bond, M is Phe or a single bond , Q is Asn or a single bond, R is Tyr or a single bond, T is Trp, Ala, Phe, Tyr, Trp-Trp, Asn-Tyr-Tyr-Trp, Trp-Asn-Tyr-Tyr-Trp, Trp-Val -Tyr-Phe-W-His-Leu-Asp-Ile-Ile-Trp (wherein W is as defined above) or a single bond, and at least two of J, M, Q, R and T not represent a single bond or at the same time, Z ¹ is as defined above)]}.

The present invention also provides an intermediate represented by the following formula (II), which is useful for synthesizing the peptide compound represented by the formula (I): X ⁴ -J-Phe-MQ-Tyr- R-T-Z ² (II) (wherein X ⁴ is hydrogen, benzyloxycarbonyl, t-
Represents butyloxycarbonyl or 9-fluorenylmethyloxycarbonyl, Z ² is hydroxy, lower alkoxy, represents a benzyloxy or benzhydryloxy, J, M, Q, R and T are as defined above).

Hereinafter, the peptide compound represented by the above formula (I) is referred to as compound (I), and the same applies to compounds having other formula numbers.

In the definitions of the above formulas (I) and (II), the lower alkyl and the alkyl moiety of the lower alkoxy are linear or branched having 1 to 6 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, se
c-butyl, tert-butyl, pentyl, neopentyl,
Hexyl, isohexyl and the like, and the lower alkanoyl is a linear or branched C1-6, for example,
Examples include formyl, acetyl, propionyl, butyryl, valeryl, pivaloyl, pentanoyl and the like.

The pharmacologically acceptable salts of compound (I) include acid addition salts, metal salts, and organic base addition salts. Specifically, pharmacologically acceptable acid addition salts include hydrochlorides, sulfates, inorganic salts such as phosphates, acetates, maleates,
Organic salts such as fumarate, tartrate, and citrate are mentioned, and pharmacologically acceptable metal salts include sodium salts,
Examples thereof include alkali metal salts such as potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, and zinc salts. Examples of pharmacologically acceptable organic bases include primary amines such as methylamine, ethylamine and aniline, secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine and piperazine, trimethylamine, triethylamine, N, N-dimethylaniline and pyridine. And tertiary amines and ammonia.

Next, the method for producing the compounds (I) and (II) will be described.

In compound (I), RES-701-1, RES having the following structure:
For -701-2 and RES-701-3, microorganisms belonging to the genus Streptomyces and having RES-701-1, RES-701-2, and RES-701-3 producing ability are used. After culturing in a medium, RES-701-1,
RES-701-2 and RES-701-3 are produced and accumulated, and RES-701-1, RES-701-2 and RES-701- are produced from the culture.
3 is obtained.

As a specific preferred example, an actinomycetes Streptomyces sp. RE-70 belonging to Streptomyces newly collected from soil near Kitashitara-gun, Aichi Prefecture by the present inventors.
1 strain and R newly collected from soil near Tono-gun, Yamaguchi Prefecture
E-629 strain.

The bacteriological properties of the Streptomyces sp. Strain RE-701 are as follows.

I. Morphology The strain RE-701 is a commonly used agar medium having a partition wall and forming branched aerial hyphae and basal hyphae. In addition, the characteristic division | segmentation of a base hypha is not seen. Also, no sporangia and sclerotium formation is found.

The spores are formed in a spore stalk that is simply branched from the aerial hyphae in the form of 10 or more long chains, and the shape is bent or spiral. Mature spores are 0.4-0.5 μm × 0.6-
It is 0.8 μm elliptical and its surface is smooth and has no flagella.

II. Growth on Various Media The RE-701 strain shows normal or vigorous growth on commonly used synthetic and natural media, and forms gray aerial hyphae. On the other hand, the basic mycelium shows a grayish white to brown. Little soluble dye is produced.

The characteristics of growth and color when cultured on various media at 28 ° C. for 10 days are shown below. The color display is Color Horm
The color classification according to the ony Manual (Container Corporation of America) was followed.

1. Glucose-asparagine agar medium Growth, back color: normal, patty (1 1 / 2ec) Aerial hypha: normal, white (a) Soluble pigment: none 2. Glycerol-asparagine agar growth, back color: poor , Oyster White (b) Aerial hyphae: slightly poor, white (a) Soluble pigment: none 3. Sucrose / nitrate agar medium Growth, back color: normal, patty (1 1 / 2ec) Aerial hyphae: normal, Gray (f) Soluble pigment: None 4. Starch / inorganic salt agar medium Growth, back color: Normal, light antique gold (1 1/2 ic) Aerial hyphae: Abundant, gray (g) Soluble pigment: None 5. Tyrosine Agar medium Growth, back color: poor, light tan (3gc) Aerial hyphae: poor, ride beige (3ec) Soluble pigment: none 6. Nutrient agar medium Growth, back color: normal, honey gold (2ic) Medium hyphae: slightly poor, white (a) soluble pigment: none 7. Malt extract / yeast extract agar medium Growth, back color: good, master gold (2ne) Aerial hyphae: normal, white (a) soluble pigment: none 8. Oatmeal agar medium Growth, back color: slightly good, white (1 1/2 lg) Aerial hyphae: poor, charcoal gray (o) Soluble pigment: none III. Physiological properties Physiological properties of strain RE-701 It is shown below. The observation results are as follows: growth temperature range: 6 days, others: 28 ° C, 2 weeks later.

(1) Utilization of carbon source; Pridham-Gottlieb inorganic medium (ISP9) was used as a basal medium.

RE-701 strain is D-glucose, D-fructose,
Sucrose, inositol, raffinose and D-mannitol are assimilated, but D-arabinose and L-rhamnose are not assimilated. The assimilation of D-xylose is unknown.

(2) Action on milk: coagulation, no liquefaction (3) Starch hydrolysis action: yes (4) Growth temperature range: 7 to 41 ° C (5) Melanoid pigment formation: no (6) Gelatin liquefaction: None IV. Cell wall composition In the analysis of diaminopimelic acid by hydrolysis of whole cells, only LL-diaminopimelic acid was detected.

Based on the spore chain formed on the aerial mycelium and the stereotype of diaminopimelic acid, this strain is classified into Streptomyces among actinomycetes.

Therefore, this strain was transformed into Streptomyces sp.
701 (Streptomyces sp. RE-701). This strain has been deposited on October 29, 1991 with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology under the name of Microtechnical Research Institute Deposit No. 3624 (FERM BP-3624).

The bacteriological properties of Streptomyces sp. Strain RE-629 are as follows.

I. Morphology The strain RE-629 is a commonly used agar medium, having a septum and forming branched aerial hyphae and basal hyphae. In addition, the characteristic division | segmentation of a base hypha is not seen. Also, no sporangia and sclerotium formation is found.

The spores are formed in a spore stalk that is simply branched from the aerial hyphae in the form of 10 or more long chains, and the shape is bent or spiral. Mature spores are 0.7-0.8 μm x 0.7-
It has an elliptical shape of 1.0 μm, and its surface is smooth and has no flagella.

II. Growth on Various Media The strain RE-629 shows normal or vigorous growth on commonly used synthetic and natural media, and forms aerial hyphae of a gray strain. On the other hand, the basic mycelium shows a grayish white to brown. The medium may produce a brown colored soluble pigment.

The characteristics of growth and color when cultured on various media at 28 ° C. for 14 days are shown below. The color display is Color Horm
The color classification according to the ony Manual (Container Corporation of America) was followed.

1. Glycerol / Asparagine agar medium Growth, back color: good, bamboo (2gc) Aerial hyphae: good, natural (2dc) Soluble pigment: none 2. Starch / inorganic salt agar medium Growth, back color: good, dull Gold (2ng) Aerial hyphae: Abundant, silver gray (3fe) Soluble pigment: Yes 3. Malt extract / Yeast extract agar medium Growth, back color: good, oak brown (4pi) Aerial hyphae: good, silver gray ( 3fe) Soluble dye: Yes 4. Oatmeal agar medium Growth, back color: Normal, olive (1 1 / 2pl) Aerial hyphae: Normal, lamp black (o) Soluble dye: Yes III. Physiological properties RE-629 strain The physiological properties of are shown below. The observation results are as follows: the growth temperature range is 5 days, the others are 28 ° C, and 2 weeks later.

(1) Utilization of carbon source; Pridham-Gottlieb inorganic medium (ISP9) was used as a basal medium.

The RE-629 strain is D-glucose, D-fructose,
It assimilates sucrose, inositol, raffinose, D-mannitol and D-xylose, but not L-rhamnose. The utilization of L-arabinose is unknown.

(2) Growth temperature range: 13-43 ° C (3) Production of melanoid pigment: (a) Peptone yeast / iron agar medium: None (b) Tyrosine agar medium: None IV. Cell wall composition Diaminopimelin by whole cell hydrolysis Analysis of the acid detected only LL-diaminopimelic acid.

Based on the spore chain formed on the aerial mycelium and the stereotype of diaminopimelic acid, this strain is classified into Streptomyces among actinomycetes.

Therefore, this strain was transformed into Streptomyces sp.
629 (Streptomyces sp. RE-629). This strain was deposited on December 17, 1992 with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology, Japan, as No. 4126 (FERM BP-4126).

In culturing the strains RE-701 and RE-629, a usual culture method used for culturing actinomycetes is applied. As the medium to be used, any natural medium or synthetic medium may be used as long as the medium contains a carbon source, a nitrogen source, an inorganic substance, and the like which can be assimilated by bacteria.

As the carbon source, glucose, fructose, sucrose, stabilose, starch, carbohydrates such as textulin, mannose, maltose, molasses, citric acid, malic acid, acetic acid, organic acids such as fumaric acid, alcohols such as methanol and ethanol, Methane, ethane, propane, n
-Hydrocarbons such as paraffin, amino acids such as glutamic acid, and glycerol are used.

As a nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, amino acids such as aspartic acid, glutamine, cystine and alanine, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, Soybean flour, cottonseed meal, soybean casein, casamino acid, Pharmamedia (procter and gambling), and the like are used.

As inorganic substances, monopotassium phosphate, dipotassium phosphate, disodium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate,
Calcium pantothenate, ammonium molybdate,
Potassium aluminum sulfate, barium carbonate, calcium carbonate, cobalt chloride, salt and the like are used.

In addition, if necessary, a substance which promotes the growth of bacterial cells such as vitamins such as thiamine or the production of RES-701-1, RES-701-2 and RES-701-3 can be added to the medium.

If the microorganism used requires a specific substance, it is of course necessary to add what is needed for growth.

The culture is performed by shaking culture, aeration stirring culture, etc.
The reaction is carried out at a temperature around 0 ° C. and near neutral pH. RES-701-1, RES-701-2 and RES-701-3 after 3-7 days of culture.
Has reached the maximum, and the culture is completed.

When RES-701-1, RES-701-2 and RES-701-3 accumulated in the culture were isolated and collected from the culture,
A method of collecting a physiologically active substance from a normal culture solution is applied. When RES-701-1, RES-701-2 and RES-701-3 accumulated in the cells are isolated and collected from the cells, the usual method of collecting physiologically active substances from the cells is applied. .
For example, cells are obtained from the culture by filtration, centrifugation, etc., and extracted using an organic solvent such as methanol or acetone. Then, the extract was distributed, the adsorption resin, silica gel,
Chemically modified silica gel, reverse phase silica gel, alumina, cellulose, diatomaceous earth, magnesium silicate, ion exchange resin,
Alternatively, it is purified by column chromatography such as gel filtration or thin-layer chromatography to obtain RES-701-
1, RES-701-2 and RES-701-3 can be obtained.

RES-701-1, RES-701-2 and RES in the above process
The detection of -701-3 can be performed by developing on a silica gel thin layer chromatography and then spraying with an iodine reaction or 50% sulfuric acid and coloring by heating, or a high-performance liquid using a C-18 reverse phase silica gel column. The measurement is performed by measuring the absorption at a wavelength of 253.7 nm by chromatography (hereinafter referred to as HPLC).

Compounds (I) and (II) are produced by culturing the above microorganisms.
And RES-701-3, and can also be produced by synthetic means.

That is, the compounds (I) and (II) of the present invention
On a peptide synthesizer manufactured by ied Biosystems, Inc., USA (ABI) or a peptide synthesizer manufactured by Shimadzu Corporation, use Nα-t-butyloxycarbonyl-amino acid or the like appropriately protected with a side chain, or use an appropriate side chain. It is obtained by using a chain-protected Nα-9-fluorenylmethyloxycarbonyl-amino acid or the like according to the company's synthesis program.

The cyclic peptide in the compound (I) can be obtained by subjecting a partial peptide appropriately protected in a side chain to the above-mentioned synthesizer or a general liquid-phase peptide synthesis method (“Basic and Experimental Peptide Synthesis” by Nobuo Izumiya et al., Maruzen). The cyclized partial peptide was synthesized using a condensing agent such as benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) as described below. Using the liquid phase synthesis method,
Alternatively, it can be obtained by condensing a C-terminal peptide obtained by appropriately combining both methods. As the C-terminal peptide, for example, compound (II) can be effectively used.

ABI, Shimadzu, Kokusan Chemical Co., Ltd., Nova bioche
It can be obtained from m and Peptide Research Institute.

Compounds (I) and (II) thus obtained are
It can be purified by HPLC using a reversed-phase column or the above-described various chromatography.

When obtaining a pharmacologically acceptable salt of compound (I), a conventional method is used. That is, the acid addition salt and the organic base addition salt of the compound (I) can be obtained by dissolving the compound (I) in an aqueous solution of the corresponding acid or organic base, and freeze-drying. The metal salt of compound (I) can be obtained by dissolving compound (I) in an aqueous solution containing the corresponding metal ion and purifying the solution by gel filtration or HPLC.

Next, specific examples of the compounds (I) and (II) are shown in Table 1.

Next, the pharmacological action of the representative compound (I) will be described in Test Examples.

Test Example 1. Inhibitory effect of compound (I) on endothelin-induced increase in intracellular calcium concentration Cell line A1O (ATCC CRL) derived from rat thoracic aortic smooth muscle
1476) was adhered to a dish with a cover glass on the bottom using a Dulbecco's modified Eagle's medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum (manufactured by HiClone) and used for experiments. The cultured cells were placed in a physiological test solution (NaCl 130 mM, KCl 2.5 m
M, CaCl ₂ 1 mM, MgSO ₄ 1 mM, NaH ₂ PO ₄ 1 mM, N- (2-hydroxyethyl) -piperazine-N′-2-ethanesulfonic acid (HEPES) 10 mM, glucose 15 mM, bovine serum albumin BSA
(Manufactured by Sigma) 2 mg / ml, pH 7.4] and then fura-2 / AM
10 μM (calcium fluorescent indicator, manufactured by Dojindo Laboratories) was added and incubated for 1 hour.

After washing with a physiological test solution, 1 ml of a test solution containing the compound (I-1) (final concentration 1 μg / ml) or the compound (I-1)
The dish was filled with 1 ml of an experimental solution containing no, and endothelin (final concentration: 0.3 nM) was added. The intracellular calcium concentration of each cell and its change were measured by measuring the fluorescence intensity at 510 nm by ultraviolet light with excitation wavelengths of 340 nm and 380 nm using an ARGUS200 system (Hamamatsu Photonics).

Results Compound (I-1) -free group (201 cells) and test compound (I-1) -added group (final concentration 1 μg / ml, cell number 171)
The increase in the intracellular calcium concentration due to the addition of endothelin (final concentration 0.3 nM) was measured in the presence of compound (I-1). The increase in intracellular calcium concentration when not performed was 46%.

Test Example 2. Endothelin receptor antagonism At 4 ° C., bovine lung tissue was treated with buffer A (1 mM MaHCO ₃ , 5 mM ethylenediaminetetraacetic acid, 5 μg / ml leupeptin, 5 μm).
g / ml pepstatin A, 40 μM phenylmethylsulfonyl fluoride, pH 8.3) polytron (type PT10 /
35, manufactured by Kinematica GmbH).

The obtained suspension was centrifuged at 8,000 g for 10 minutes at 4 ° C., and the resulting supernatant was centrifuged at 40,000 g for 60 minutes at 4 ° C. to obtain a solid. The obtained solid was suspended in buffer A and centrifuged again at 4 ° C. at 40,000 g for 60 minutes. The obtained solid was prepared as a suspension solution so that the protein content was 2 mg / ml, and was used as a membrane fraction. 7 μl of the membrane fraction solution was added to 1 ml of buffer B (50 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 0.2% bovine serum albumin, pH 7.6) to prepare a membrane fraction solution.

¹²⁵ I-endothelin-1 (about 30,000 cpm) was added to the membrane fraction solution to which unlabeled endothelin-1 (final concentration: 100 nM) was added, compound (I-1) was added, or compound (I-2) was added, or none was added. added. After allowing these mixtures to stand at 25 ° C. for 2 hours, a glass filter GF / B (Whatman
(Trade name). After washing the filter with buffer C (50 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, pH 7.6), the radioactivity on the glass filter was measured, and the amount of ¹²⁵ I-endothelin bound to the receptor and non-specifically was determined. It measured and calculated the endothelin receptor binding inhibition rate according to the following formula.

Inhibition rate = (CA / CB) × 100 (%) A: Radioactivity in the presence of RES-701-1 or RES-701-2 B: Radioactivity in the presence of unlabeled endothelin-1 C: RES -701-1, RES-701-2, unlabeled endothelin-
1. Radioactivity without any addition Result RES-701-1 that inhibits endothelin-1 binding by 50%
Concentration (IC ₅₀₎ is 25 nM, the concentration (IC ₅₀₎ of RES-701-2 to inhibit the binding of endothelin-1 of 50% was 55 nM.

Test Example 3. Intracellular guanosine-3 'by endothelin,
Inhibitory effect of compound (I) on increase in 5'-cyclic-phosphate concentration Ishii et al. [The Journal of Pharmacology and Experim
ental Therapeutics 259 , 3, 1102-1108 (1991)].

Endothelin-1 (final concentration 1 nM) was added to a test solution containing compound (I-1) (final concentration 3 μg / ml) or compound (I-2) (final concentration 3 μg / ml) or a test solution without any of them. Was added. The change was measured by measuring the intracellular guanosine-3 ', 5'-cyclic monophosphate concentration using a Cyclic GMP Assay Kit (manufactured by Yamasa Shoyu).

Results Endothelin 1n in the presence of 3 μg / ml of compound (I-1)
The increase in intracellular guanosine-3 ', 5'-cyclic monophosphate concentration by M was 0% of the increase in intracellular guanosine-3', 5'-cyclic monophosphate concentration by the addition of 1 nM endothelin alone.
Met. The increase in intracellular guanosine-3 ', 5'-cyclic monophosphate concentration by 1 nM endothelin in the presence of compound (I-2) at 3 µg / ml was determined by the addition of 1 nM endothelin alone to guanosine-3', 5'-intracellular. The increase in cyclic monophosphate concentration was 17%. Administration of 3 μg / ml of compound (I-1) and 3 μg / ml of compound (I-2) resulted in endothelin 1n
The increase in intracellular guanosine-3 ', 5'-cyclic monophosphate concentration by M was suppressed.

Test Example 4 Endothelin receptor antagonism Inhibition of endothelin receptor binding by the same method as in Test Example 2 except that bovine lung tissue in Test Example 2 was changed to bovine cerebellar tissue and the compounds shown in Table 2 were used as test compounds. The rate was determined.

 The results are shown in Table 2.

The bronchoconstriction inhibitory action in in vivo inhibitory activity present invention compounds of compounds against bronchoconstriction (I) according to Test Example 5. Endothelin
C. Touvay et.al., European Journal of
Pharmacology, 176 23-33 (1990)]. That is, propranolol (3 mg / kg) was intravenously administered in advance to be in a β-blocked state.
Using a male Hartley guinea pig, after anesthesia, a tracheal cannula was inserted and fixed and connected to a ventilator. Then Gallamin
After stopping spontaneous respiration with e triethiodide (manufactured by Sigma), artificial respiration was performed at a constant flow rate. A respiratory dose measuring device was connected to the bypass of the tracheal cannula, and the amount of air outflow that changed due to airway constriction was measured and used as an index of airway constriction. Endothelin-3 (1.5 μmol / l; manufactured by Peptide Laboratories) was injected intravenously at 0.1 ml per 100 g body weight. Immediately after the injection, airway constriction (increased air outflow) was observed, and 30 seconds and 3
An early and late biphasic airway constriction response was elicited with a peak in minutes. The compound (I-
Dissolution solution of 1) (dimethyl sulfoxide: saline = 1:
When 1) was intravenously administered before injecting endothelin-3, the airway constriction reaction was suppressed as compared with the control to which only the lysate was administered.

The evaluation of the airway contraction inhibitory effect of the compound (I-1) was performed at two points: 30 seconds (initial phase) and 3 minutes (late phase) after endothelin-3 injection, and the inhibition rate was calculated according to the following formula.

Inhibition rate (%) = (ab) / b × 100 a: airway constriction of compound (I-1) non-administration group (control) b: airway constriction of compound (I-1) administration group From these results, it was confirmed that compound (I-1) has an antagonistic effect on endothelin-3 even in a living body and suppresses airway constriction.

Test Example 6. Acute toxicity Compound (I-1) was intraperitoneally administered to an administration group consisting of three ddy mice weighing 20 ± 1 g. Seven days after administration, death cases were observed and the minimum lethal dose (MLD) was determined. The result was more than 300mg / kg.

BEST MODE FOR CARRYING OUT THE INVENTION In the examples below, the abbreviations for the amino acids and their protecting groups used are in the IUPAC-IUB Commission on Biochemical Nomenclatur.
The recommendation of e) [Biochemistry, 11 , 1726 (1972)] was followed.

Amino acids and protecting groups have the following meanings.

Gly: glycine Val: L-valine Ile: L-isoleucine Leu: L-leucine Glx: L-glutamine or L-glutamine Gln: L-glutamine Ser: L-serine Thr: L-threonine Asp: L-aspartic acid Asn: L-asparagine Lys: L-lysine Tyr: L-tyrosine Cys: L-cysteine Phe: L-phenylalanine Trp: L-tryptophan His: L-histidine Pro: L-proline Asx: L-aspartic acid or L-asparagine t- Boc: t-butyloxycarbonyl Me: methyl Bzl: benzyl Bzl (NO ₂ ): 4-nitrobenzyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl 4-CH ₃ Bzl: 4 - methylbenzyl t-Bu: tert-butyl Bom: benzyloxymethyl Trt: trityl Fmoc: 9--fluorenylmethyloxycarbonyl Z; benzyloxycarbonyl CHO: formyl Ac: acetyl CHPh _2: Nzuhidoriru side chain protected amino acids are expressed as follows.

Trp (CHO): N ^ln -formyl-L-tryptophan t-Boc-Tyr (Br-Z): Nα-t-butyloxycarbonyl-O-2-bromobenzyloxycarbonyl-L-
Tyrosine t-Boc-His (Bom): Nα-t-butyloxycarbonyl-N ^lm -benzyloxymethyl-L-histidine Asp (Ot-Bu): aspartic acid-β-t-butyl ester t-Boc-Thr ( Bzl): Nα-t-butyloxycarbonyl-O-benzyl-L-threonine t-Boc-Asp (OBzl): Nα-t-butyloxycarbonyl-L-aspartic acid-β-benzyl ester t-Boc-Cys ( _{4-CH 3 Bzl): Nα} -t- butyloxycarbonyl -S-4-methylbenzyl -L- cysteine Fmoc-Asp (Ot-Bu) -OH: Nα-9- -fluorenylmethyloxycarbonyl -L- aspartic Acid-β-t-butyl ester Fmoc-Tyr (t-Bu) -OH: Nα-9-Fluorenylmethyloxysicarbonyl-Ot-butyl-L-tyrosine Fmoc-Asn (Trt) -OH: Nα -9-Fluorenylmethylcarbonyl- γ- trityl -L- aspartic Fmoc-His (Trt) -OH: Nα-9- -fluorenylmethyl carbonyl -N ^lm - trityl -L- histidine Fmoc-Thr (t-Bu) -OH: Nα-9- full Oleynylmethyloxycarbonyl-Ot-butyl-L-threonine Fmoc-Gln (Trt) -OH: Nα-9-Fluorenylmethylcarbonyl-Nδ-trityl-L-glutamine Fmoc-Lys (Z) -OH: Nα-9-Fluorenylmethyloxycarbonyl-Nε-carbobenzoxy-L-lysine Fmoc-Lys (t-Boc) -OH: Nα-9-Fluorenylmethylcarbonyl-Nε-t-butyloxycarbonyl-L
-Lysine The abbreviations for the reaction solvent and the reaction reagent have the following meanings.

DCC: dicyclohexylcarbodiimide DEPC: diethylphosphorocyanidate HONSu: N-hydroxysuccinimide PyBOP: benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate HOBt: N-hydroxybenzotriazole NMM: N -Methylmorpholine DMF: N, N-dimethylformamide TFA: Trifluoroacetic acid TosOH: p-Toluenesulfonic acid Pd / C: Palladium-carbon catalyst TEA: Triethylamine The following Examples 1, 3, 6 to 10 and Reference Example 1
In Nos. 2 and 3, peptides were synthesized using an ABI peptide synthesizer model 430A and operating a synthesizer using ABI reagents and solvents according to an ABI synthesis program. The amino acid condensation reaction was carried out under standard conditions as a symmetric acid anhydride.

In Examples 16, 17, 21 to 40, and Reference Examples 3 and 4, a peptide synthesizer was operated by Shimadzu's synthesis program using Shimadzu's reagents and solvents, using Shimadzu's peptide synthesizer PSSM8. Was synthesized. Amino acid condensation reactions were performed under standard conditions by the Fmoc method [Basic and Experimental Peptide Synthesis Nobuo Izumiya et al. (Maruzen)]. .

BEST MODE FOR CARRYING OUT THE INVENTION Example 1. Compound (I-1) (RES-701-1) As the inoculum, Streptomyces sp. RE-701
Using the strain as a first-class medium, glucose 10 g / l, beef extract (manufactured by Far East Pharmaceutical Industries) 3 g / l, powdered yeast extract S (manufactured by Nippon Pharmaceutical) 5 g / l, bactotripton (manufactured by Difco) 5 g / l A medium containing 1 g / l of monopotassium phosphate, 0.5 g / l of magnesium sulfate heptahydrate, 2 g / l of calcium carbonate, and pH 7.2 is used. One platinum loop of the inoculum was inoculated into 10 ml of the above medium placed in a 50 ml thick test tube, and cultured with shaking at 28 ° C for 5 days.

9 ml of this first-type culture was inoculated into 300 ml of a second-type medium in an Erlenmeyer flask with a 2 l baffle. The composition of the second type medium was the same as the composition of the first type medium. The second seed culture was performed at 28 ° C. for 2 days. 300 ml of the second seed culture was inoculated into 9.7 l of the third seed medium in a 30-liter stainless steel jar fermenter. The composition of the third medium was the same as the composition of the first medium. The third seed culture was performed at 28 ° C. for 3 days by aeration and agitation (rotational speed 250 rpm, aeration 10 l / min). 10 l of this third seed culture was inoculated into 90 l of the main fermentation medium in a 200 l stainless steel jar fermenter. The main fermentation medium is glucose 40g / l, soluble starch 40g
/ l, soy flour 10g / l, corn steep liquor 5g / l, dry yeast 5g / l, monopotassium phosphate 0.5g / l, cobalt chloride 1
A medium of μg / l, nickel sulfate 1 μg / l, zinc sulfate 10 μg / l, magnesium phosphate 0.5 g / l, pH 7.0 was used. The main fermentation culture is aerated at 28 ° C for 4 days (rotation speed 200rpm,
The air flow rate was 100 l / min).

100 l of the obtained culture was continuously centrifuged to separate the cells, 70 l of acetone was added to the obtained cells, and the mixture was stirred and filtered. The obtained filtrate was concentrated under reduced pressure to remove acetone, and then passed through a Diaion HP-20 column (5 l) for adsorption. The column was washed with 25 l of 50% methanol and eluted with methanol. Fractions containing RES-701-1 were collected, diluted with water, and then filled with Diaion HP-
The mixture was passed through a 20SS column (2.5 L) and adsorbed. After washing with 7.5 l of 70% methanol, elution was performed with 80% methanol.
The fractions containing -701-1 were collected and concentrated to dryness under reduced pressure to obtain 2.2 g of a light brown solid. The obtained solid was dissolved in methanol, passed through a HP-20 column (2 l) filled with methanol, and eluted with methanol. RES-701-1
Is collected, concentrated under reduced pressure to dryness to 350 m
g of white solid were obtained. After the obtained solid was dissolved in methanol, preparative purification was performed using preparative HPLC (LC-8 system, manufactured by Shimadzu Corporation) under the following conditions. Obtained RES-7
The fraction containing 01-1 was treated with Seppak C-18 (manufactured by Waters) to remove TFA, and concentrated to dryness under reduced pressure to obtain 20 mg of a dry powder of RES-701-1.

HPLC preparative conditions Column: SH-363-10 (C-18 reverse phase silica gel, manufactured by YMC) Eluent: 0.1% TFA solution-acetonitrile (30% -65%)
Linear concentration gradient Separation time: 20 minutes Flow rate: 25 ml / min The physicochemical properties of RES-701-1 are shown below.

The physicochemical properties were measured by the following instruments.

Mass spectrum: JEOL JMS-SX102A (measured by FAB method) Amino acid analysis was performed by Bidlingmeyer.BA etc. [J. Chromatogr., 3
36 , 93 (1984)]. The hydrolysis was carried out in hydrochloric acid vapor at 110 ° C. for 22 hours. The amino acid composition of the hydrolyzate was analyzed using a Waters Pico Tag amino acid analyzer. For tryptophan only, see Matsubara et al. [Biochem.Biophys.Res.Co.
mmun., 35 , 175 (1969)]
Analysis was performed using a JLC-300 amino acid analyzer. The measured value is Al
The value of a was expressed as 1.00.

Mass spectrometry [FABMS]: Found 2042.8722, calcd ^{_{2042.8614 (12 C 103 H 115 N}} 23 O 23
Amino acid analysis: Actual value (theoretical value): Asx 2.54 (3), Gly
2.13 (2), His 0.93 (1), Thr 1.00 (1), Ala
1.00 (1), Pro 1.15 (1), Try 1.95 (2), Phe 2.
06 (2), Trp 2.54 (3) The configuration of the amino acid is (+)-1- (9
-Fluorenyl) ethyl chloroformate was derivatized and analyzed by reverse phase HPLC to find that it was all L-form.

The primary structure of the amino acid was partially hydrolyzed in 0.05N hydrochloric acid at 108 ° C. for 2 hours and then subjected to automated Edman degradation of a partially hydrolyzed peptide obtained by separation by HPLC (470A protein sequencer,
120A Online PTH Amino Acid Analyzer, ABI) and FA
Determined by B-MS spectral analysis. In addition, tryptophan at the C-terminal can be obtained by a hydrazinolysis method [Akahori et al.
m. Soc. Jap., 25 , 214 (1967)].

Further, the Rf value of RES-701-1 was measured by developing it with thin layer chromatography using various developing agents. Detection was performed by an iodine reaction, sulfuric acid coloring, or ultraviolet irradiation at 253.7 nm.

Expand Conditions 1 thin layer; Kiserugeru 60F ₂₅₄ (Merck, Art.5629) developing solvent; chloroform: methanol: ethanol: water = 10: 4: 4: 2 deployment method; room temperature rise process, 15 to 60 minutes Rf value : 0.4 Developing condition 2 Thin layer and developing method are the same as developing condition 1. Developing solvent; 100% methanol Rf value; 0.6 Developing condition 3 Developing method is the same as developing condition 1. Thin layer; RP-18 (Merck, Art. 13724) Developing solvent; 80% methanol Rf value; 0.3 Developing condition 4 Developing method is the same as developing condition 1. Thin layer; RP-18 (Merck, Art. 13724) Developing solvent; 90% acetonitrile Rf value; 0.4 Example 2. Compound (I-2) (RES-701-2) As the inoculum, Streptomyces sp. RE-701
Using a strain, one loopful of the inoculum was placed in four 250 ml Erlenmeyer flasks in a first-class medium [glucose 10 g /
l, soluble starch 10g / l, beef extract (Kyokuto Pharmaceutical) 3g / l, powdered yeast extract S (Nippon Pharmaceutical) 5g / l, bactotripton (Difco) 5g / l,
Monopotassium phosphate 1g / l, magnesium sulfate heptahydrate 0.
5g / l, calcium carbonate 2g / l, LG-109 (Shin-Etsu Chemical)
0.5 g / l (pH 7.2)], and cultured with shaking at 28 ° C for 3 days.

10 ml of this first-type culture was inoculated into 300 ml of a second-type medium placed in six Erlenmeyer flasks each having a 2 L baffle. The composition of the second type medium was the same as the composition of the first type medium. The second seed culture was performed at 28 ° C. for 2 days. 1800 ml of the second seed culture was inoculated into 100 liter of the third seed medium in a 200-liter stainless steel jar fermenter. The composition of the third medium was the same as the composition of the first medium. The third seed culture is aerated and agitated at 27 ° C for 27 hours (rotation speed: 220 rpm, aeration volume)
60 l / min). Add 100l of this third seed culture to 2,000
Main fermentation medium in l-volume stainless steel jar fermenter
Inoculate 1,000 l. Soluble starch as the main fermentation medium
50g / l, dried yeast 30g / l, monopotassium phosphate 0.5g /
l, magnesium phosphate 0.5 g / l, LG-109 (Shin-Etsu Chemical) 0.5 g / l, pH 7.0 medium was used. This main fermentation culture is 2
Ventilation stirring method at 8 ° C for 3 days (rotation speed 140rpm, ventilation volume 400l /
min).

400 l of n-propyl alcohol was added to 1,000 l of the obtained culture solution.
Then, a filter aid was added, the mixture was sufficiently stirred, and filtered by a filter press. Water was added to the filtrate to make the concentration of n-propyl alcohol 20%, and the mixture was passed through a 50-liter column filled with Diaion HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). R
ES-701-2 was adsorbed, washed with 200 l of 60% methanol and eluted with 250 l of 80% methanol. Water and ammonium acetate were added to 150 l of the fraction containing RES-701-2, and the pH was adjusted to 7.0 with 30% methaneol containing 50 mM ammonium acetate.
% Methanol and 30% methanol (pH 7.0) containing 50 mM ammonium acetate, and passed through a column (15 l) filled with Sepabeads FP-DA13 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). pH 7.0) 15 l, then
45L of 30% methanol (pH 7.0) containing 1M ammonium acetate
And elute with 60 liters of 50% methanol containing 0.6M acetic acid. The fractions containing RES-701-2 were collected, passed through an HP-20SS column (10 l) packed with 50% methanol, washed with 50% methanol, then with 70% methanol, and eluted with 75% methanol. The fractions containing RES-701-2 were collected and dried under reduced pressure to obtain a brown solid. This was dissolved in 25% acetonitrile, and fractionation was repeatedly performed using preparative HPLC (manufactured by Soken Chemical) under the following conditions.

Carrier: ODS AQ S-50 (manufactured by YMC) Eluent: acetonitrile (25% -50%) linear gradient Separation time: 250 minutes Sample amount: 500 mg / time Flow rate: 250 ml / min Retention time of 3.5 to 4.5 hours Fractions were collected and dried under reduced pressure to obtain 800 mg of a dry powder of RES-701-2.

The physicochemical properties of RES-701-2 are shown below.

The physicochemical properties were measured by the following instruments.

Mass spectrum: JEOL JMS-HX110A (measured by FAB method) Amino acid analysis was performed by Bidlingmeyer.BA [J. Chromatogr., 3
36 , 93 (1984)]. The hydrolysis was carried out in hydrochloric acid vapor at 110 ° C. for 22 hours. The amino acid composition of the hydrolyzate was analyzed using a Waters Pico Tag amino acid analyzer. For tryptophan only, use RJ Simpson et al. (J. Biol. Chem., 25
1, 1936 (1976)] and analyzed by the amino acid analyzer described above. In addition, the actual measurement value represented the value of Ala as 1.00.

Mass spectrometry: measured value 2058.8496 Amino acid analysis: actual measured value (theoretical value): Asx 2.61 (3), Gly
2.25 (2), His 0.92 (1), Thr 1.02 (1), Ala
1.00 (1), Pro 1.14 (1), Tyr 1.74 (2), Phe 1.
90 (2), Trp 2.15 (2) The configuration of the amino acid is (+)-1- (9
-Fluorenyl) ethyl chloroformate was derivatized and analyzed by reverse phase HPLC to find that it was all L-form.

The primary structure other than the C-terminal amino acid is 108 ° C in 0.1N hydrochloric acid.
Automated Edman degradation of partially hydrolyzed peptides obtained by partial hydrolysis with HPLC for 2 hours and separation by reverse phase HPLC (470A protein sequencer, 120A online PTH amino acid analyzer, ABI
And FAB-MS spectrum analysis.

The C-terminal amino acid is determined by a hydrazinolysis method [Akahori et al., Bull.
m. Soc. Jap., 25 , 214 (1964)] and carboxypeptidase A (manufactured by Sigma), and the C-terminal amino acid was separated by reversed-phase HPLC. The FAB-MS spectrum analysis and NMR spectrum analysis (Bruker) Ltd., was identified as aM500, ¹ H-500MHz) by 7-hydroxy-tryptophan. The amino acid configuration was determined to be L-type by a polarimeter (JASCO DIP-370).

Example 3. Compound (I-3) (RES-701-3) Streptomyces sp. RE-629 as the inoculum
Using a strain, one loopful of the inoculum was placed in four 250 ml Erlenmeyer flasks in a first-class medium [glucose 10 g / l,
10g / l soluble starch, 3g / l beef extract (Kokuto Pharmaceutical), 5g / powder yeast extract S (Nippon Pharmaceutical)
l, bactotripton (Difco) 5g / l, monopotassium phosphate 1g / l, magnesium sulfate heptahydrate 0.5g / l, calcium carbonate 2g / l, LG-109 (Shin-Etsu Chemical) 0.5g / l (pH 7.
2)] 40 ml of each was inoculated, and cultured with shaking at 28 ° C. for 3 days. 10 ml of this first-type culture was inoculated into 300 ml of a second-type medium placed in six Erlenmeyer flasks each having a 2 L baffle. The composition of the second type medium was the same as the composition of the first type medium. The second seed culture was performed at 28 ° C. for 2 days.
1800 ml of this second seed culture was inoculated into 100 l of the main fermentation medium in a 200 l stainless steel jar fermenter. The main fermentation medium is soluble starch 50 g / l, dry yeast 30 g / l, monopotassium phosphate 0.5 g / l, magnesium phosphate 0.5 g / l,
A medium of LG-109 (Shin-Etsu Chemical Co., Ltd.) 0.5 g / l, pH 7.0 was used.
The main fermentation medium was aerated and agitated at 28 ° C. for 3 days (at 14 rpm).
0 rpm, aeration 400 l / min). Obtained culture solution
To 100 l, n-propyl alcohol (40 l) and a filter aid were added, sufficiently stirred, and filtered by a filter press. Water was added to the filtrate and the n-propyl alcohol concentration was 20
%, Diaion HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.)
And passed through a 5 l column. After adsorbing RES-701-3, it was washed with 20 l of 60% methanol,
Eluted in 5 l. Water and ammonium acetate are added to 15 l of the fraction containing RES-701-3, containing 50 mM ammonium acetate
Sepabeads FP, pH 7.0 with 30% methanol, filled with 30% methanol (pH 7.0) containing 50 mM ammonium acetate
-Passed through a column (1.5 l) of DA13 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) and passed through 30% methanol (pH 7.
0) 1.5 l, then washed with 4.5 l of 30% methanol (pH 7.0) containing 1 M ammonium acetate and eluted with 6 l of 50% methanol containing 0.6 M acetic acid. Fractions containing RES-701-3 were collected and HP-20SS column packed with 50% methanol (1)
And washed with 50% methanol, then with 70% methanol and eluted with 75% methanol. The fractions containing RES-701-3 were collected and dried under reduced pressure to obtain a brown solid. This was dissolved in 25% acetonitrile, and fractionation was repeated using preparative HPLC (manufactured by Shimadzu Corporation) under the following conditions.

Column: ODS (diameter 5 cm, length 50 cm) (manufactured by YMC) Eluent: acetonitrile (25% -50%) linear gradient Separation time: 250 minutes Sample amount: 10 mg / time Flow rate: 30 ml / min 180 from retention time From 210 minutes, and dried under reduced pressure to obtain 7 mg of a dry powder of RES-701-3.
Obtained.

 The physicochemical properties of RES-701-3 are shown below.

 The physicochemical properties were measured by the following instruments.

Mass spectrum: JEOL JMS-HX110A Amino acid analysis was performed in the same manner as in Example 2. The measured value is
The value of Phe was expressed as 2.00.

a) Mass spectrometry: measured value 2059.4626 b) amino acid analysis Actual value (theoretical value): Asx 3.16
(3), Ser 0.90 (1), Gly 1.97 (2), His 0.96
(1), Thr 0.89 (1), Pro 1.12 (1), Tyr 1.92
(2), Phe 2.00 (2), Trp 2.91 (3) The amino acid configuration was reversed after hydrolysis by derivatization with (+)-1- (9-fluorenyl) ethyl chloroformate.
All were separated into the L form by HPLC.

The primary structure was determined by automated Edman degradation (470A protein sequencer, 120A online PT) of the peptide obtained by partial hydrolysis in 0.1N hydrochloric acid at 108 ° C for 2 hours and separation by reverse phase HPLC.
H amino acid analyzer, manufactured by ABI) and FABMS spectrum analysis (manufactured by JEOL Ltd., JMS-HX110A).

The C-terminal amino acid is determined by a hydrazinolysis method [Akahori et al., Bull.
em.Soc.Jap., 25 , 214 (1964)]
It was identified as tryptophan.

Example 4. Compound (I-4): H-Gly-Asn-Trp-His-Gly-Thr
-Ala-Pro-Asp-Trp-Phe-Phe-AsN-Tyr-Tyr-Trp
Synthesis of -OH According to the synthesis program of ABI, t-Boc-Trp (CHO)
0.73 g of the carrier resin to which 0.5 mmol was bound was put into a reactor of an automatic synthesizer, and the following operation was performed.

(A) A methylene chloride solution containing 33% TFA was added, and the mixture was stirred for 1 minute and 20 seconds, and the solution was discharged.

(B) A methylene chloride solution containing 50% TFA was added, and the mixture was stirred for 18 minutes and 30 seconds, and the solution was discharged.

(C) The carrier resin was washed three times with methylene chloride.

(D) A methylene chloride solution containing 10% diisopropylethylamine was added and the mixture was stirred for 1 minute, the solution was drained and this operation was repeated once more.

(E) The carrier resin was washed five times with DMF.

 Thus, a carrier resin to which Trp (CHO) was bound was obtained.

(F) To this carrier resin was added 4 ml of a DMF solution containing 2.0 mmol of t-Boc-Tyr (Br-Z) symmetrical anhydride, and the mixture was stirred for 18 minutes and the solution was discharged.

(G) The carrier resin was washed five times with methylene chloride.

Thus, t-Boc-Tyr (Br-Z) -Trp (CH
O) was synthesized on the support. Next, the above (a) to (e)
After performing the deprotection step of t-Boc-Tyr in step (f)
A condensation reaction is carried out by adding a symmetric acid anhydride of (Br-Z) -OH, and then, through a washing step (g), t-Boc-Tyr (Br
-Z) -Tyr (Br-Z) -Trp (CHO) was synthesized on a carrier resin. Hereinafter, in step (f), t-Boc-Asn-OH, t
-Boc-Phe-OH, t-Boc-Phe-OH, t-Boc-Trp (CHO)
-OH, t-Boc-Asp (OBzl) -OH, t-Boc-Pro-OH, t-Bo
c-Ala-OH, t-Boc-Thr (Bzl) -OH, t-Boc-Gly-OH,
t-Boc-His (Bom) -OH, t-Boc-Trp (CHO) -OH, t-B
Using oc-Asn-OH and t-Boc-Gly-OH, the steps (a) to (g) were sequentially repeated to obtain 2.0 g of a carrier resin having a protected peptide bonded thereto.

Using 0.8 g of the obtained carrier resin, 0.8 ml of 1,2-ethanedithiol, 0.8 ml of dimethyl sulfide and 0.2 ml of anisole were added thereto, and the mixture was left for 3 hours. 18 ml of hydrogen fluoride was added, and the mixture was cooled on ice for 70 minutes. After stirring, hydrogen fluoride was removed under reduced pressure, 100 ml of ethyl acetate was added to the carrier resin, and the mixture was stirred for 0.5 hour. 100 ml of DMF was added to the carrier resin obtained by filtration, and the mixture was stirred for 1 hour. Fully automatic high-speed cooling centrifuge (RS-20)
(Tomy Seiko), and centrifuged at 10,000 rpm for 10 minutes to obtain a supernatant. The DMF of this supernatant is concentrated in a concentrator (ROTARY VA
Remove with CUUM EVAPORATOR N-2 type Tokyo Rikaki
Redissolved in M acetic acid to obtain a crude product. This crude product was purified by HPLC using a reversed-phase column (CAPCELL PACK C18 SG-120 30 × 250 mm manufactured by Shiseido). 0.1% TFA and 0-90%
The fraction was eluted with a linear concentration gradient using acetonitrile and detected at 220 nm to obtain a fraction containing compound (I-4). This fraction was freeze-dried to obtain 18.2 mg of compound (I-4).

Mass spectrometry of compound (I-4) was performed by JEOL JMS-HX110A.
The amino acid analysis was performed in the same manner as in Example 2.

In the following Examples, mass spectrometry and amino acid analysis were performed in the same manner as in Example 4.

Mass spectrometry [FABMS]: 2062 (M + H) Amino acid analysis: Asx2.5 (3), Gly2.2 (2), His1.1
(1), Thr1.1 (1) Ala1.1 (1), Pro1.1 (1), Tyr2.
1 (2), Phe1.9 (2), (Trp was not analyzed) Compound (I-5) cyclo (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-A
Synthesis of sp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp) TFA salt of compound (I-4) synthesized according to Example 4.
1.84 mg was dissolved in 0.35 ml of methanol, then 1.7 μl of 5% hydrochloric acid-methanol solution was added, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, a gel filtration column [Sephadex G-15]
(Manufactured by Pharmacia), 44 × 9 mm ID] to elute with methanol to obtain a fraction containing the compound (I-4). next,
The solvent was distilled off under reduced pressure to obtain 1.25 mg of compound (I-4) hydrochloride. 0.2 mg of the hydrochloride of compound (I-4) in 0.3 ml of dry DMF
0.4 mg / ml HOBt prepared in dry DMF, 0.61 mg /
0.1 ml of each DCC was added in order under ice cooling. The reaction solution was left under ice cooling for 2 hours, returned to room temperature, and left overnight.
Insoluble matter was removed by centrifugation (15000 rpm × 5 minutes, 0 ° C.), and the supernatant was purified by reverse phase HPLC. Reversed phase column is YMC
YMC Pack ODS-AM312 (150 × 6 mm I.D.) manufactured by the company was used.
Elution was performed with a linear concentration gradient using 0.1% TFA and 0 to 90% acetonitrile, detection was performed at 220 nm, and a fraction containing 5% of compound (I-1) and 95% of compound (I-5) was obtained. Was. The solvent of this fraction was distilled off under reduced pressure to obtain 0.01 mg of a residue. This was dissolved in 50 μl of ethanol, and reversed phase using an ion pair agent.
Purified by HPLC. The reversed-phase column is YMC Pac manufactured by YMC.
Using k ODS-AM312 (150 × 6 mm ID), elution was carried out with 1 mM tetraethylammonium hydroxide containing 36% acetonitrile and 0.1 M aqueous monosodium phosphate (pH = 3.3). The fraction containing the compound (I-1) is evaporated under reduced pressure, and the residue is purified again by reversed-phase HPLC using a linear concentration gradient using 0.1% TFA and 0 to 90% acetonitrile. 0.5 μg of compound (I-1) was obtained.

 Similarly, 25 μg of the compound (I-5) was obtained.

Compound (I-1) Mass spectrometry [FABMS]: 2044 (M + H) Amino acid analysis: Asx2.5 (3), Gly2.2 (2), His1.1
(1), Thr1.1 (1), Ala1.1 (1), Pro1.1 (1), Tyr
2.1 (2), Phe1.9 (2), (Trp is not analyzed) Compound (I-5) Mass spectrometry [FABMS]: 2044 (M + H) Amino acid analysis: Asx2.5 (3), Gly2.2 (2 ), His1.1
(1), Thr1.1 (1), Ala1.1 (1), Pro1.1 (1), Tyr
2.1 (2), Phe1.9 (2), (Trp was not analyzed) Example 6. Compound (I-6): H-Cys-Asn-Trp-His-Gly-Thr
-Ala-Pro-Cys-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp
Synthesis of -OH t-Boc-Tyr (Br-Z) -OH, t-Boc as a carrier resin to which t-Boc-Trp (CHO) is bound and a protected amino acid
-Tyr (Br-Z) -OH, t-Boc-Asn-OH, t-Boc-Phe-O
H, t-Boc-Phe-OH, t-Boc-Trp (CHO) -OH, t-Boc-C
_{ys (4-CH 3 Bzl)} -OH, t-Boc-Pro-OH, t-Boc-Ala-
OH, t-Boc-Thr (Bzl) -OH, t-Boc-Gly-OH, t-Boc-
His (Bom) -OH, t-Boc-Trp (CHO) -OH, t-Boc-Asn
-OH and t-Boc-Cys (4- CH 3 Bzl) -OH, using,
Carrier resin bonded with protected peptide in the same manner as in Example 4.
2.0 g were obtained. 0.8 g of the obtained carrier resin was subjected to treatment from hydrogen fluoride treatment to purification by HPLC in the same manner as in Example 4 to obtain 298.4 mg of compound (I-6).

Mass spectrometry [FABMS]: 2096 (M + H) Amino acid analysis: Asx1.7 (2), Gly1.0 (1), His1.0
(1), Thr1.0 (1), Ala1.0 (1), Pro1.0 (1), Tyr
2.0 (2), Phe2.0 (2), Cys1.9 (2), (Trp not analyzed) 3 mg of the compound (I-6) obtained in Example 6 was dissolved in 10 ml of anhydrous methanol, and 50 mM Tris-HCl buffer (pH 7.5) was dissolved.
0.2 ml was added under ice cooling. Next, 1 ml of a hydrated methanol solution (V / V: 5/1) containing 1.6 mM glutathione oxidized form was added, and the mixture was stirred at room temperature for 16 hours. Then, the reaction solution was adjusted to pH with hydrochloric acid.
The mixture was adjusted to 4 and purified by reverse phase HPLC in the same manner as in Example 5 to obtain 1.5 mg of compound (I-7).

Mass spectrometry [FABMS]: 2094 (M + H) Amino acid analysis: Asx1.8 (2), Gly1.0 (1), His1.0
(1), Thr0.9 (1), Ala1.0 (1), Pro1.0 (1), Tyr
2.0 (2), Phe1.9 (2), (Cys, Trp were not analyzed. 0.2 mg of the compound (I-7) was dissolved in 0.2 ml of DMF, and 40 μMED
100 μl of 20 mM sodium phosphate buffer (pH 7.2) containing TA was added, 50 μg of N-acetylsuccinimide was added under ice cooling, and the mixture was stirred at 10 ° C. for 16 hours. PH of the resulting reaction solution with hydrochloric acid
The mixture was adjusted to 4 and purified by reverse phase HPLC in the same manner as in Example 5 to obtain 110 μg of the compound (I-8).

Mass spectrometry [FABMS]: 2136 (M + H) Amino acid analysis: Asx1.8 (2), Gly1.0 (1), His1.0
(1), Thr0.9 (1), Ala1.0 (1), Pro1.0 (1), Tyr
2.0 (2), Phe1.9 (2), (Cys, Trp not analyzed) Example 9. Compound (II-9): H-Trp-Phe-Phe-Asn-Tyr-Tyr-
Synthesis of Trp-OH Carrier resin to which t-Boc-Trp (CHO) is bound and N-
T-Boc-Tyr (Br-Z) -OH, t
-Boc-Tyr (Br-Z) -OH, t-Boc-Asn-OH, t-Boc-P
he-OH, t-Boc-Phe-OH, and t-Boc-Trp (CHO)-
1.35 g of a carrier resin having a protected peptide bonded thereto was obtained in the same manner as in Example 4 using OH. Of these, 0.8 g of the synthetic peptide was treated with hydrogen fluoride in the same manner as in Example 4, cut out of the resin, dissolved in 2M acetic acid, and lyophilized to obtain 46.2 mg of a crude product. Of these, 4.0 mg was purified by HPLC in the same manner as in Example 4 to obtain 2.4 mg of compound (II-9).

Mass spectrometry [FABMS]: 1126 (M + H) Amino acid analysis: Asx1.0 (1), Phe2.0 (2), Tyr2.0
(2), (Trp was not analyzed) Example 10. Compound (II-10): H-Trp-Phe-Asn-Tyr-Tyr-Trp-
Synthesis of OH t-Boc-Trp (CHO) bound carrier resin and N-
T-Boc-Tyr (Br-Z) -OH, t
-Boc-Tyr (Br-Z) -OH, t-Boc-Asn-OH, t-Boc-P
Using he-OH and t-Boc-Trp (CHO) -OH, a carrier resin bound with a protected peptide in the same manner as in Example 4.
1.3 g was obtained. Of these, 0.8 g was treated with hydrogen fluoride in the same manner as in Example 4, cut out from the resin, dissolved in 2M acetic acid, and lyophilized to obtain 215.2 mg of a crude product. 4.0 mg thereof was subjected to HPLC by the same method as in Example 4.
Then, 2.0 mg of compound (II-10) was obtained.

Mass spectrometry [FABMS]: 979 (M + H) Amino acid analysis: Asx0.9 (1), Tyr2.1 (2), Phe1.0
(1), (Trp is not analyzed) Example 11. Compound (II-11): H-Trp-Phe-Phe-Asn-Tyr-Trp-
Synthesis of OH t-Boc-Trp (CHO) bound carrier resin and N-
T-Boc-Tyr (Br-Z) -OH, t
-Boc-Asn-OH, t-Boc-Phe-OH, t-Boc-Phe-OH and t-Boc-Trp (CHO) -OH, and a carrier to which a protected peptide is bound in the same manner as in Example 4. 1.2 g of resin was obtained. Of these, 0.8 g was cut out of the resin by hydrogen fluoride treatment in the same manner as in Example 4, dissolved in 2 M acetic acid, and lyophilized to obtain 264.7 mg of a crude product. 8.0 mg of this was subjected to HPLC by the same method as in Example 4.
And 4.7 mg of the compound (II-11) was obtained.

Mass spectrometry [FABMS]: 963 (M + H) Amino acid analysis: Asx0.8 (1), Tyr1.1 (1), Phe2.2
(2), (Trp is not analyzed) Example 12. Compound (II-12): H-Trp-Phe-Phe-Tyr-Tyr-Tyr-
Synthesis of OH t-Boc-Trp (CHO) bound carrier resin and N-
T-Boc-Tyr (Br-Z) -OH, t
-Boc-Tyr (Br-Z) -OH, t-Boc-Phe-OH, t-Boc-P
Using he-OH and t-Boc-Trp (CHO) -OH, a carrier resin bound with a protected peptide in the same manner as in Example 4.
1.3 g was obtained. Of these, 0.8 g was treated with hydrogen fluoride in the same manner as in Example 4, cut out from the resin, dissolved in 2M acetic acid, and freeze-dried to obtain 243.4 mg of a crude product. 78.0 mg of this was subjected to HPLC in the same manner as in Example 4.
And 12.2 mg of compound (II-12) was obtained.

Mass spectrometry [FABMS]: 1012 (M + H) Amino acid analysis: Tyr2.0 (2), Phe2.0 (2), (Trp was not analyzed) Example 13. Compound (II-13): H-Phe-Phe -Asn-Tyr-Tyr-Trp-
Synthesis of OH t-Boc-Trp (CHO) bound carrier resin and N-
T-Boc-Tyr (Br-Z) -OH, t
-Boc-Tyr (Br-Z) -OH, t-Boc-Asn-OH, t-Boc-P
Using he-OH and t-Boc-Phe-OH, 1.2 g of a carrier resin having a protected peptide bonded thereto was obtained in the same manner as in Example 4. Of these, 0.8 g was treated with hydrogen fluoride in the same manner as in Example 4, cut out from the resin, dissolved in 2M acetic acid, and freeze-dried to obtain 285.5 mg of a crude product. Of these, 150.0 mg was purified by HPLC in the same manner as in Example 4 to obtain 39.1 mg of compound (II-13).

Mass spectrometry [FABMS]: 940 (M + H) Amino acid analysis: Asx0.8 (1), Tyr2.0 (2), Phe2.2
(2), (Trp is not analyzed) 11.3 mg of the compound (a) obtained in Reference Example 1 was dissolved in 12.3 ml of DMF, 5.2 mg of DEPC and 6.4 mg of TEA were added while cooling at -10 ° C, and the mixture was stirred for 30 minutes. 4.0 mg of aspartic acid α-benzyl β-diphenylmethyl ester obtained by reacting diphenyldiazomethane with benzyl ester of aspartic acid in the same manner as in Example 27 was added to the obtained reaction solution, and
Stirred at C for an additional 7 days. The obtained compound was subjected to reverse-phase HPLC equipped with a YMC ODS-AQ column (250 × 30 mm ID).
And purified by a linear concentration gradient of 0.1% TFA and 0 to 90% acetonitrile, and lyophilized to give 5 mg of a condensate.
I got

To the condensate were added methylene chloride (500 µl), anisole (10 µl), ethanedithiol (1 µl) and TFA (500 µl) sequentially under ice cooling, and the mixture was stirred and left for 30 minutes. The solvent is distilled off under reduced pressure,
Purification by reverse-phase HPLC and freeze-drying in the same manner as described above gave 2 mg of the dediphenylmethyl ester of the condensate.

The obtained ester compound was dissolved in DMF (800 μl), piperidine (200 μl) was added, the mixture was stirred, and left at room temperature for 10 minutes. 300 μl of acetic acid and 450 μl of methanol were added to neutralize, purified by reverse phase HPLC as above, and lyophilized to remove Fm.
1.5 mg of the ocylated ester was obtained.

The obtained de-Fmoc ester was dissolved in 1.9 ml of DMF,
While cooling at 10 ° C, add 0.47mg of DEPC and 0.59mg of TFA to add 30
The mixture was allowed to stand for 4 minutes, returned to 4 ° C., and stirred for 3 days. Purification by reverse-phase HPLC and freeze-drying in the same manner as described above gave 1 mg of the benzyl ester of compound (I-14).

The obtained benzyl ester compound was dissolved in 150 μl of methanol and 50 μl of acetic acid, hydrogen gas was added in the presence of about 1 mg of 10% Pd / C, and catalytic reduction was performed with stirring at room temperature for 2 hours. Purification by reverse phase HPLC and freeze-drying as described above gave 1 mg of compound (I-14).

Mass spectrometry [FABMS]: 936 (M + H) Amino acid analysis: Asx1.9 (2), Gly2.3 (2), His1.0
(1), Thr0.7 (1), Ala0.9 (1), Pro1.0 (1), Trp
0.4 (1). Example 15. Step 1: H-Asp (Ot-Bu) -Trp-OBzl (a) 41 mg of Fmoc-Asp (Ot-Bu) -OH was mixed with 1 ml of methylene chloride.
, Cooled to 0 ° C, added with 12 mg of HONSu and 21 mg of DCC, and stirred at 0 ° C for 30 minutes. Here, H-Trp-OBzlHC133
Add 1 ml of methylene chloride solution containing 14 mg of TEA and 14 μl of TEA, and add
For 3 hours. The insoluble material was separated by filtration, washed with cold methylene chloride, and the filtrate was collected. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (Wako Pure Chemical Industries, Ltd., Wakogel C-200, 50 g, eluted with chloroform / methanol = 25/1) to obtain Fmoc-Asp (Ot-Bu ) -Trp
-167 mg of OBz were obtained as a white powder. Mass spectrometry (FABMS);
688 (M + H) (b) 10 mg of the dipeptide obtained in (a) was dissolved in 3 ml of DMF, 0.75 ml of piperidine was added, and the mixture was allowed to stand at room temperature for 10 minutes. Ether and hexane were added to the reaction mixture, and the precipitated white crystals were collected by filtration, dried under reduced pressure, and dried under H-Asp (Ot-Bu) -Trp.
2 mg of -OBzl were obtained.

Mass spectrometry [FABMS]; 466 (M + H) Step 2: H-Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-A
sp-Trp-OBzl (a) To 4.4 mg of the compound (a) obtained in Reference Example 1, a 5.5 ml DMF solution containing 1.7 mg of the dipeptide obtained in Step 1 was added, and the mixture was cooled to 0 ° C. Here, 0.5μl of DEPC and 1.0μl of TEA
Were added successively, and the mixture was stirred at 0 ° C. for 5 days. The solvent was distilled off under reduced pressure, the residue was dissolved again in 1 ml of DMF, and HPL using a reversed-phase column (YMC-PackODS-AM312 150 × 6 mm ID) was used.
Purified with C, Fmoc-Gly-Asn-Trp-His-Gly-Thr-Ala
-Pro-Asp (Ot-Bu) -Trp-OBzl 320 µg was obtained as a white powder.

(B) TFA90 was added to 250 μg of the protected peptide obtained in (a).
0 μl, 1,2-ethanedithiol 50 μl, anisole 50 μl
l, 50% of the mixture consisting of 5 mg of 2-methylindole
μl was added and left at room temperature for 1.5 hours. The white precipitate formed by adding ether was collected by filtration, dried, added with 100 μl of DMF containing 20% piperidine, and allowed to stand at room temperature for 15 minutes. The white precipitate formed by adding ether again was collected by filtration and dried, and H-Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-As
200 μg of p-Trp-OBzl was obtained.

Step 3: Compound (I-15) (a) 66 μg of the peptide obtained in Step 2 was dissolved in 60 μl of DMF, and a DMF solution containing 0.1 M of PyBOP was added at room temperature to 1.6 μm.
μl, 1.6 μl of DMF solution containing 0.1 M of HOBt, 1% of NMM
3 μl of the DMF solution was added, and the mixture was stirred at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and the product was subjected to a reverse phase column (YMC, YMC
HPLC using —Pack ODS-AM312 150 × 6 mm ID) gave 20 μg of the benzyl ester of compound (I-15).

(B) Dissolve 250 μg of the benzyl ester obtained in (a) in 80 μl of a mixed solution of methanol / acetic acid = 3/1, add about 0.5 mg of 10% Pd / C under a nitrogen atmosphere, and place the system under a hydrogen atmosphere. And stirred at room temperature for 1 hour. Pd / C was separated by filtration, ether was added to the filtrate, and a white precipitate formed was collected by filtration and dried to obtain 100 μg of compound (I-15).

Mass spectrometry [FABMS]: 1123 (M + H) amino acid analysis; Gly2.3 (2), Asx1.6 (2), His0.9
(1), Thr1.0 (1), Ala1.0 (1), Pro1.1 (1) (Trp
Is not analyzed) Example 16. 10 μg of the compound (I-14) obtained in Example 14 was added to DMF10
dissolved in ice, HOBt 4.3 μg, PyBOP 16.7 μg, NM
M4.9 μg was sequentially added and left for 30 minutes. Add H-Gly to the reaction solution
-Add 14.0 µg of -OMe · HC and leave it at 4 ° C overnight to prepare Example 1.
Purification was performed by reverse phase HPLC equipped with a YMC Pack ODS-AM 312 column (150 × 6 mm I.D.) manufactured by YMC in the same manner as in 5, and lyophilized to obtain 6 μg of the compound (I-16).

Mass spectrometry [FABMS]: 1008 (M + H) Amino acid analysis: Asx1.8 (2), Gly3.2 (3), His1.0
(1), Thr1.0 (1), Ala0.9 (1), Pro1.0 (1) (Trp
Is not analyzed) Example 17. 7.1 mg of compound (I-1) is dissolved in 2.84 ml of methanol, 25.56 ml of 0.1 M Tris-HCl buffer (pH 8.0) is added, and about 0.3 mg of carboxypeptidase A (Sigma C-9762). Was added and stirred at 37 ° C. overnight. The reaction solution was acidified by adding an appropriate amount of hydrochloric acid, and NUCLEOSIL 5C18 (Chemco
Using 250 × 20 mm I.D.)
With a linear concentration gradient that increases the acetonitrile concentration in a 0.1% TFA solution from 0% to 50% in 30 minutes at a flow rate of 10 ml per minute,
3.5 mg of compound (I-17) was obtained. Mass spectrometry (FABMS): 1
858 (M + H) amino acid analysis; Asx2.6 (3), Gly2.2 (2), His0.9
(1), Thr1.0 (1), Ala1.0 (1), Pro1.1 (1), Tyr
1.8 (2), Phe1.9 (2), (Trp was not analyzed) PyBOP was added to a 270 μl DMF solution containing 0.49 mg of compound (I-1).
375 μg, HOBt 97 μg, and NMM121 μg were added, and the mixture was stirred at room temperature for 1 hour. 80 μl of a DMF solution containing 0.16 mg of H-Trp-OBzl was added, and the mixture was stirred at 4 ° C. for 4 days. The solvent is distilled off under reduced pressure,
The residue was again dissolved in 80 μl of DMF solution, and H was equipped with a reverse phase column (YMC, YMC-Pack ODS-AM312 150 × 6 mm ID).
The concentration of acetonitrile in a 0.1% TFA solution was increased from 0% to 90% in 60 minutes by a PLC at a flow rate of 1 ml / min.
g was obtained. Mass spectrometry [FABMS]: 2320 (M + H) Then, 70 μg of the above benzyl ester compound was added to 35 μl of DMF.
And a saturated methanol solution of ammonium formate in 35
μl and about 0.1 mg of 10% Pd / C were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was centrifuged to collect the supernatant, and the mixture was subjected to preparative purification using a reversed-phase column (YMC, YMC-Pack ODS-AM312 150 × 6 mm ID) in the same manner as described above to obtain the compound (I-18) )twenty five
μg was obtained.

Mass spectrometry [FABMS]: 2230 (M + H) amino acid analysis; Gly2.2 (2), Asx2.8 (3), His0.9
(1), Thr0.9 (1), Ala1.0 (1), Pro1.3 (1), Phe
1.9 (2), Tyr1.7 (2) (Trp was not analyzed) Step 1: H-Asn-Tyr-Tyr-Trp-OBzl Fmoc-Tyr (t-Bu) 66 μmol bound carrier resin 110m
g and Fmoc-Tyr (t
-Bu) -OH, Fmoc-Asn (Trt) -OH was used to synthesize a peptide in the same manner as in Reference Example 3, and the peptide was cut out from the resin to obtain 89 mg of a crude peptide Fmoc-Asn-Tyr-Tyr-OH. Was.

According to a method similar to that in Example 15, 8.
2 mg and 7.6 mg of H-Trp-OBzl were condensed and de-Fmocized in the same manner as in Example 15 to give H-Asn-Tyr-Tyr-Tyr-Trp-OB.
1 mg of zl were obtained.

Step 2: Compound (I-19) By a method similar to that in Example 18, compound (I-1) 0.29
mg and the above peptide H-Asn-Tyr-Tyr-Trp-OBzl 0.10m
g was condensed to obtain a benzyl ester. Example 18
Debenzylation was carried out in the same manner as described above, and the compound (I)
-19) 19 μg was obtained.

Mass spectrometry [FABMS]: 2671 (M + H) amino acid analysis; Gly2.3 (2), Asx3.6 (4), His0.9
(1), Thr1.0 (1), Ala1.0 (1), Pro1.1 (1), Phe
2.1 (2), Tyr3.7 (4), (Trp was not analyzed) Step 1: H-Val-Tyr-Phe-Ala-His-Leu-Asp (OBzl)
-Ile-Ile-Trp-OBzl Fmoc-Val-Tyr-Phe-Ala-His-Leu-Asp (OBzl)
-Ile-Ile-OH was prepared in the same manner as in Reference Example 3 by using Fmoc-Ile-OH.
Using a carrier resin to which N-protected amino acid Fmoc-Il
e-OH, Fmoc-Asp (OBzl) -OH, Fmoc-Leu-OH, Fmoc-Hi
s (Trt) -OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Tyr
The synthesis was performed by sequentially condensing (t-Bu) -OH and Fmoc-Val-OH. Excision from the resin was performed in the same manner to obtain the above crude peptide.

Dissolve 2.0 mg of the obtained peptide in 200 μl of dry DMF, dry DMF containing 42 mg / ml HOBt prepared in dry DMF under ice-cooling 10 μl of dry DMF containing 162 mg / ml PyBOP, and
1, 10 μl of dry DMF containing 34.3 μl / ml NMM and 10 μl of dry DMF containing 62 mg / ml H-Trp-OBzl hydrochloride were added in order. The mixture was allowed to stand at 4 ° C. overnight, and the insolubles were filtered and purified by reverse phase HPLC. Column is Chemco Pack NUCLEOSIL5C18 250 × 2
Using 0 mm ID (Chemco), elution was performed with a linear concentration gradient using 0.1% TFA and 0 to 90% acetonitrile. The fraction containing the target substance was freeze-dried, and Fmoc-Val-Tyr-Phe-Ala-
2.7 mg of His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl
Obtained.

Then, 100 μl of dry DMF containing 20% piperidine was added, and the mixture was allowed to stand at room temperature for 5 minutes. Diethyl ether was added to allow crystallization, ether washing, drying under reduced pressure, and H-Val-Tyr.
-Phe-Ala-His-Leu-Asp (OBzl) -Ile-Ile-Trp-
467 μg of OBzl was obtained.

(2) Compound (I-20) In the same manner as in Example 18, 0.14 mg of the compound (I-1) and 10 mg of the peptide obtained in the above step 1 were condensed to obtain a benzyl ester, and debenzylation was carried out. The compound (I-
20) 10.5 μg was obtained.

Mass spectrometry [FABMS]: 3303 (M + H) Amino acid analysis; Gly2.3 (2), Asx4.0 (4), His1.8
(2), Thr1.0 (1), Ala2.0 (2), Pro1.2 (1), Phe
2.7 (3), Tyr2.6 (3), Val 0.9 (1), Leu1.0 (1),
Ile1.7 (2), (Trp not analyzed) 0.2 containing 0.22 mg of the compound (I-17) obtained in Example 17.
In 2 ml DMF solution, PyBOP 187 μg, HOBt 49 μg, NMM 61 μg
Was added and stirred at room temperature for 1 hour. Then, H-Ala-OBz
A 35 μl DMF solution containing 0.12 mg l · TosOH and 35 μg NMM was added, and the mixture was stirred at 4 ° C. for 2 days. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in 1 ml of DMF and fractionated and purified by HPLC in the same manner as in Example 15 to obtain 36 μg of the benzyl ester of compound (I-21). .

Mass spectrometry [FABMS]: 2019 (M + H) 36 μg of the obtained benzyl ester compound was dissolved in 20 μl of DMF, and a saturated methanol solution of ammonium formate was added to a solution of 20 μl.
1 and about 0.1 mg of 10% Pd / C were added, followed by stirring at room temperature for 1 hour. The reaction solution was centrifuged to collect a supernatant. The crude product was fractionated and purified by reverse phase HPLC in the same manner as in Example 18 to obtain 27 μg of compound (I-21).

Mass spectrometry [FABMS]: 1929 (M + H) Amino acid analysis: Gly2.2 (2), Asx2.6 (3), His1.0
(1), Thr1.1 (1), Ala2.0 (2), Pro1.1 (1), Phe
2.0 (2), Tyr1.9 (2), (Trp was not analyzed) Compound (I-17) 0.22 was prepared in the same manner as in Example 15.
mg and H-Phe-OBzl.TosOH 0.15 mg were condensed to obtain a benzyl ester, which was debenzylated to give the compound (I-
22) 27 μg were obtained.

Mass spectrometry [FABMS]: 2005 (M + H) Amino acid analysis: Gly2.1 (2), Asx2.6 (3), His1.0
(1), Thr1.0 (1), Ala1.0 (1), Pro1.0 (1), Phe
3.0 (3), Tyr 2.0 (2), (Trp was not analyzed) Compound (I-17) 0.22 was prepared in the same manner as in Example 15.
mg and H-Tyr-OBzl.TosOH 0.16 mg were condensed to give a benzyl ester, debenzylated, and the compound (I
-23) 30 μg was obtained.

Mass spectrometry [FABMS]: 2021 (M + H) Amino acid analysis: Gly2.2 (2), Asx2.6 (3), His1.0
(1), Thr1.0 (1), Ala1.0 (1), Pro1.1 (1), Phe
2.0 (2), Tyr2.9 (3), (Trp was not analyzed) Compound (I-17) 0.22 was prepared in the same manner as in Example 15.
mg and H-Asn-Tyr-Tyr-Trp-OBzl 0.17 mg were condensed to obtain a benzyl ester form, which was debenzylated to obtain 36 μg of the compound (I-24).

Mass spectrometry [FABMS]: 2485 (M + H) Amino acid analysis: Gly2.1 (2), Asx4.0 (4), His1.0
(1), Thr1.0 (1), Ala1.0 (1), Pro1.1 (1), Phe
1.9 (2), Tyr3.9 (4), (Trp was not analyzed) 113 μl of dry DMF was added to 150 μg of the compound (I-15),
0.53 mg / ml HOBt prepared in dry DMF under ice cooling, 2.0 mg / ml
PyBOP and 0.71 μl / ml NMM were added in order of 100 μl. The resulting mixture was mixed with the H-Val-Tyr-Ph obtained in Example 20.
e-Ala-His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl
87 μl of a dry DMF solution containing 2.4 mg / ml was added. The obtained mixture was left under ice cooling for 5 hours, and purified by reverse phase HPLC in the same manner as in Example 15. The fraction containing the condensate is lyophilized to give 140 μg of the benzyl ester of compound (I-25)
I got Then, under a nitrogen atmosphere, 200 μl of a methanol solution containing 25% acetic acid and 50 μl of dry DMF were added, and a 10%
0.5 mg of Pd / C was added, hydrogen gas was introduced, and the mixture was vigorously stirred at room temperature for 1 hour. The catalyst was removed by filtration and purified by reverse phase HPLC in the same manner as in Example 15. The solvent of the fraction containing the target compound was distilled off under reduced pressure to obtain 6.5 μg of the compound (I-25).

Mass spectrometry [FABMS]: 2472 (M + H) Amino acid analysis: Asx2.6 (3), Gly2.0 (2), His1.6
(2), Thr0.9 (1), Ala1.6 (2), Pro0.8 (1), Val
0.7 (1), Tyr0.9 (1), Phe0.8 (1), (Trp was not analyzed) Example 26. Compound (I-26): H-Gly-Asn-Trp-His-Gly-Thr-
Ala-Pro-Asp-Trp-Val-Tyr-Phe-Ala-His-Leu-
Synthesis of Asp-Ile-Ile-Trp-OH In the same manner as in Reference Example 3, using 20 mg of a carrier resin to which 10.4 μmol of Fmoc-Trp was bound, and sequentially as an N-protected amino acid,
Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Asp (Ot-Bu)
-OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-A
la-OH, Fmoc-Phe-OH, Fmoc-Tyr (t-Bu) -OH, Fm
oc-Val-OH, Fmoc-Trp-OH, Fmoc-Asp (Ot-Bu) -O
H, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Thr (t-B
u) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH, Fmoc
-Trp-OH, Fmoc-Asn (Trt) -OH and Fmoc-Gly-OH
The synthesis is performed using. Only the final step was different from that of Reference Example 3, and the piperidine treatment (b) was also performed. The peptide was cut out from the resin in the same manner as in Reference Example 3 to obtain 25 mg of a crude product.
I got 4.0 mg of H was prepared in the same manner as in Example 4.
Purification by PLC yielded 1.8 mg of compound (I-26).

Mass spectrometry [FABMS]: 2400 (M + H) Amino acid analysis: Gly2.4 (2), Ala2.0 (2), Asx2.9
(3), His1.9 (2), Ile1.0 (2), Leu0.9 (1), Phe
0.9 (1), Pro1.1 (1), Thr1.1 (1), Tyr0.9 (1), V
al0.7 (1), (Trp is not analyzed) Step 1: H-Ile-Ile-Trp-OCHPh ₂ Fmoc-Ile-Ile-Trp-OH, in the same manner as in Reference Example 3, using a carrier resin to which Fmoc-Trp is bound, and sequentially as an N-protected amino acid, Fmoc-Ile-OH and Fmoc-Ile-OH
Was synthesized and cleaved from the resin to obtain the above crude peptide. To 126 μg of this peptide, 200 μl methanol and 200 μl methylene chloride were added and dissolved, and 30 μl of methylene chloride containing 7.8 mg / ml diphenyldiazomethane and 30 μl of methanol containing 28.6 μl / ml hydrochloric acid were added under ice cooling. The mixture was allowed to stand under ice cooling for 2 hours, returned to room temperature, and left overnight. The insolubles were filtered and purified by reversed-phase HPLC (column: YMS-Pack ODS-AM-312 manufactured by YMC) as in Example 20. This is freeze-dried and Fmoc-Ile-Ile-Trp-OCHPh
75 g of ₂ was obtained. Then, 100 μl of dry DMF containing a 20% piperidine solution was added, and the mixture was allowed to stand at room temperature for 5 minutes. Then, insoluble materials were removed by filtration and purified by reverse-phase HPLC in the same manner as described above. The solvent of the fraction containing the target substance was distilled off under reduced pressure, and H-Ile-Ile-Tr
the p-OCHPh ₂ was obtained 32μg.

Step 2: Compound (I-27) 40 μl of compound (I-15) 64 μg obtained in Example 15
1.1mg / ml prepared in dry DMF under ice-cooling
HOBt, 4.1 mg / ml PyBOP, 1.5 μl / ml NMM, H-Ile-
Ile-Trp-OCHPh the ₂ 31.7 mg / ml were sequentially added in 20 [mu] l. The mixture was allowed to stand under ice cooling for 2 hours, and the reaction solution was purified by reversed-phase HPLC in the same manner as in Example 15. The solvent of the obtained fraction was distilled off under reduced pressure to obtain 37 μg of a benzhydryl ester of the compound (I-27). Then TFA 900μl, anisole 50μ
l, 50 µl of 1,2-ethanedithiol and 25 µl of a mixed solution of 5 mg of 2-methylindole were added, and the mixture was stirred at room temperature for 1 hour. 0.5 ml of diethyl ether was added to the reaction solution to crystallize the compound (I-27), washed with ether and dried under reduced pressure to obtain 10.1 μg of the compound (I-27).

Mass spectrometry [FABMS]: 1535 (M + H) Amino acid analysis: Asx1.7 (2), Gly2.1 (2), His0.9
(1), Thr1.0 (1), Ala1.0 (1), Pro1.3 (1), Ile
2.0 (2), (Trp is not analyzed) Step 1: H-Leu-Tyr-Phe-Ala-His-Gln-Asp (OBzl)
-Val-Ile-Trp-OBzl 555 μg of the compound (d) obtained in Reference Example 4 was dissolved in 200 μl of dry DMF, and 15.9 mg / ml of HOBt,
61.2 mg / mL of PyBOP and 21.5 μL / mL of NMM were successively added under ice cooling to 10 μM.
7.8 mg / ml of H-Trp-OBzl hydrochloride was further added to the solution.
μl was added. The reaction solution was left overnight at 4 ° C., the insolubles were filtered, and the filtrate was purified by reverse phase HPLC in the same manner as in Reference Example 3. At this time, the column was Chemco-
Lyophilized using Pack NUCLEOSIL 5C18 (250 × 20 mm ID). Then, this freeze-dried product was prepared with dry DMF 20
After adding 100 μl of a 100% piperidine solution and leaving the mixture at room temperature for 5 minutes, the solvent was concentrated under reduced pressure to 50 μl, and precipitated by adding diethyl ether, and further washed with ether and dried under reduced pressure to obtain H-Leu-Tyr-Phe-Ala- His-Gln-Asp (OBzl)
250 μg of -Val-Ile-Trp-OBzl was obtained.

Step 2: Compound (I-28) Compound (I-15) obtained in Example 15 was dissolved in 100 μl of dry DMF, and 0.72 mg / ml of HOBt prepared in dry DMF was prepared.
2.8 mg / ml of PyBOP, 1.0 μl / ml of NMM, and 2.5 mg / ml of the above peptide were added in an order of 50 μl under ice-cooling. The mixture was left overnight at 4 ° C., the insolubles were removed by filtration, and purified by reversed-phase HPLC in the same manner as in Example 15. The obtained fraction was freeze-dried to obtain 66 μg of the benzyl ester of the compound (I-28). Then, 30 μl of dry DMF and 30 μl of a methanol solution saturated with ammonium formate were added, and a small amount of 10% Pd / C was further added, followed by vigorous stirring at room temperature for 1 hour. Thereafter, the catalyst was removed by filtration and purified by reverse phase HPLC in the same manner as in Example 15. The column is
YMC Pack ODS-AM312 (150 × 6 mm I.D.) manufactured by YMC was used. The solvent of the fraction containing the target compound was distilled off under reduced pressure to obtain 1.2 μg of compound (I-28).

Mass spectrometry [FABMS]: 2397 (M + H) Amino acid analysis: Asx2.6 (3), Glx1.0 (1), Gly2.2
(2), His2.0 (2), Thr1.0 (1), Ala2.0 (2), Pro
1.2 (1), Tyr1.1 (1), Val 0.9 (1), Ile0.7 (1),
Leu1.1 (1), Phe1.3 (1), (Trp not analyzed) 10.0 μg of the compound (I-14) obtained in Example 14 was added to DMF1
HOBt 4.3 μg, PyBOP 16.7 μg, N
4.9 μg of MM was added and left for 30 minutes. H-Ala-OB
Add 11.3 μg of zl · TosOH and leave at 15 ° C overnight,
Purification by reverse phase HPLC equipped with a YMC Pack ODS-AM312 column (150 × 6 mm I.D.) manufactured by YMC in the same manner as in 14, and lyophilization gave 5.0 μg of compound (I-29).

Mass spectrometry [FABMS]: 1098 (M + H) Amino acid analysis: Asx1.6 (2), Gly2.2 (2), His1.0
(1), Thr0.9 (1), Ala1.8 (2), Pro.1.2 (1),
(Trp was not analyzed). 10.0 μg of the compound (I-14) obtained in Example 14 was added to DMF1
HOBt 4.3 μg, PyBOP 16.7 μg, N
4.9 μg of MM was added and left for 30 minutes. H-Val-OB
After adding 4.0 μg of zl · TosOH and leaving the mixture at 15 ° C. overnight,
Purified by reverse phase HPLC equipped with a YMC Pack ODS-AM312 column (150 × 6 mm ID) manufactured by YMC in the same manner as described above, and lyophilized to obtain 7.0 μg of compound (I-30).

Mass spectrometry [FABMS]: 1126 (M + H) Amino acid analysis: Asx1.8 (2), Gly2.2 (3), His1.0
(1), Thr1.0 (1), Ala1.1 (1), Pro1.0 (1), Val
1.2 (1), (Trp is not analyzed) Dry DM was added to 30 μg of the compound (I-15) obtained in Example 15.
30 μl of F, 0.53 mg / ml HOBt prepared in dry DMF,
2.0 mg / ml of PyBOP and 0.71 μl / ml of NMM were successively added in an amount of 10 μl under ice cooling.
Was added. Further, 40 μl of dry DMF containing 0.064 mg / ml of H-Gly-OMe was added. The reaction solution was left under ice cooling for 1 hour,
After returning to room temperature, the reaction was further performed for 2 hours. Next, the insolubles were filtered and purified by reverse phase HPLC in the same manner as in Example 15. The solvent of the fraction containing the condensate is distilled off under reduced pressure, and the compound (I)
-31) was obtained in an amount of 8.0 μg.

Mass spectrometry [FABMS]: 1194 (M + H) Amino acid analysis: Asx1.8 (2), Gly2.4 (2), His0.9
(1), Thr1.1 (1), Ala1.0 (1), Pro1.2 (1), (T
rp is not analyzed) Using a carrier resin to which Fmoc-Tyr is bound in the same manner as in Reference Example 3 as a starting material, an N-protected amino acid Fmoc-Tyr is used.
Tyr (t-Bu) -OH and Fmoc-Asn (Trt) -OH were sequentially bonded to synthesize a protected peptide, which was cut out from the carrier resin to obtain a crude peptide Fmoc-Asn-Tyr-Tyr-OH. Then, 8.2 mg of the obtained peptide was dissolved in 5 ml of dry DMF,
15.4 mg / ml HOBt, 59.6 mg / ml PyBO prepared in dry DMF
P, 21 μl / ml of NMM, and 15.2 mg / ml of H-Trp-OBzl hydrochloride were added in order of 250 μl under ice cooling. The reaction was carried out at 4 ° C. for 5 hours, and the insolubles were filtered and purified by reverse phase HPLC. The column is
Chemco Pack NUCLEOSIL 5C18 (250 × 2
0 mm ID) was used. Lyophilize the fraction containing the target substance and
6.75 mg of c-Asn-Tyr-Tyr-Trp-OBzl was obtained.

Next, 2.7 mg of this compound contained 20% piperidine in dry D
After adding 100 μl of MF and leaving the mixture at room temperature for 5 minutes, diethyl ether was added for crystallization, washed with ether and dried under reduced pressure.
2.25 mg of -Asn-Tyr-Tyr-Trp-OBzl was obtained.

Next, 100 μg of the compound (I-15) obtained in Example 15
100 μl of dry DMF was added to 0.72 mg / ml prepared with dry DMF
HOBt, 2.8 mg / ml PyBOP, 1 μl / ml NMM, 0.45 μg / μ
of H-Asn-Tyr-Tyr-Trp-OBzl in an order of 50 μl under ice-cooling.
Was added. After the reaction solution was left under ice cooling for 3 hours, insolubles were removed by filtration and purified by reverse phase HPLC in the same manner as in Example 25. The solvent of the obtained fraction was distilled off under reduced pressure to obtain 80 μg of a benzyl ester of the compound (I-32).

Next, 30 μl of dry DMF was added to 80 μg of the benzyl ester.
l, 30 μl of methanol solution saturated with ammonium formate
Was added, and a small amount of 10% Pd / C was further added, followed by vigorous stirring at room temperature for 1 hour. Thereafter, the catalyst was removed by filtration and purified by reverse-phase HPLC as described above. The column used was ODS-AM312 manufactured by YMC. The solvent of the fraction containing the target compound was distilled off under reduced pressure to obtain 42 μg of the compound (I-32).

Mass spectrometry [FABMS]: 1750 (M + H) Amino acid analysis: Asx2.0 (2), Gly2.4 (2), His1.1
(1), Thr0.9 (1), Ala1.0 (1), Pro1.4 (1), Tyr
2.0 (2), (Trp is not analyzed) Step 1: (1) H-Val-Tyr-Phe-Ala-Phe-Phe-Asn
-Tyr-Tyr-Trp-OBzl In the same manner as in Reference Example 3, an N-protected amino acid Fmoc was obtained using a carrier resin to which Fmoc-Tyr was bound as a starting material.
-Tyr (t-Bu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Phe
-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmo
c-Tyr (t-Bu) -OH and Fmoc-Val-OH are sequentially bonded, cut out from the carrier resin, and the crude peptide Fmoc-Val-Tyr
-Phe-Ala-Phe-Phe-Asn-Tyr-Tyr-OH was obtained.

Then, 4.9 mg of the obtained peptide was dissolved in 2 ml of dry DMF, and 2.8 mg / ml of HOBt and 10.6 mg / ml of prepared in dry DMF.
PyBOP, 4 μl / ml of NMM, and 2.7 mg / ml of H-Trp-OBzl hydrochloride were added in an amount of 0.5 ml in this order under ice cooling. The mixture was allowed to stand at 4 ° C. overnight, and the insolubles were filtered and purified by reverse phase HPLC. The column is Chemco Pack NUCLEOSIL 5C18 (250 x 20m) manufactured by Chemco.
elution with 0.1% TFA and 0-90% acetonitrile. The fraction containing the condensate was lyophilized and Fmoc-Val-Tyr-Phe-Ala-Phe-Phe-Asn
-5.4 mg of -Tyr-Tyr-Trp-OBzl was obtained.

Dry DMF100 containing 20% piperidine in 5.4 mg of this compound
Then, the mixture was left at room temperature for 5 minutes, crystallized by adding diethyl ether, washed with ether, dried under reduced pressure,
-Val-Tyr-Phe-Ala-Phe-Phe-Asn-Tyr-Tyr-Trp
3.0 mg of -OBzl was obtained.

Step 2: Compound (I-33) Dry D was added to 100 μg of the compound (I-15) obtained in Example 15.
100 μl of MF was added, and 0.72 mg / ml HOB prepared in dry DMF was added.
t, 2.8 mg / ml PyBOP, 1 μl / ml NMM in 50 μl
Then, 50 μl of a 3.0 mg / ml dry DMF solution of the partial peptide obtained in the above step 1 was added. The reaction solution was allowed to stand under ice cooling for 3 hours, and insolubles were filtered and purified by reversed-phase HPLC (column used is ODS-AM-312 manufactured by YMC). The solvent of the fraction containing the condensate was distilled off under reduced pressure to give Compound (I-33)
110 μg of the benzyl ester of was obtained. Next, 50 μl of dry DMF and 50 μl of a methanol solution saturated with ammonium formate were added to 110 μg of the benzyl ester, and a small amount of 10% Pd / C was further added, followed by vigorous stirring at room temperature for 2 hours. Thereafter, the catalyst was removed by filtration, and reverse phase HPLC was performed in the same manner as above.
Was purified. The solvent of the fraction containing the target substance is distilled off under reduced pressure,
2.0 μg of compound (I-33) was obtained.

Mass spectrometry [FABMS]: 2525 (M + H) Amino acid analysis: Asx3.0 (3), Gly2.5 (2), His1.1
(1), Thr1.3 (1), Ala2.0 (2), Pro1.1 (1), Phe
3.0 (3), Tyr2.9 (3), Val 1.0 (1), (Trp was not analyzed). Step 1: H-Val-Tyr-Tyr-Ala-His-Leu-Asp (OBzl)
-Ile-Ile-Trp-OBzl In the same manner as in Reference Example 3, an N-protected amino acid Fmoc-Ile-OH, Fmoc
-Asp (OBzl) -OH, Fmoc-Leu-OH, Fmoc-His (Trt)-
OH, Fmoc-Ala-OH, Fmoc-Tyr (t-Bu) -OH, Fmoc-Tyr
(T-Bu) -OH and Fmoc-Val-OH are sequentially bonded to synthesize a protected peptide, which is excised from the carrier resin and the crude peptide Fmoc-Val-Tyr-Tyr-Ala-His-Leu-Asp (OBz
l) -Ile-Ile-OH was obtained. 4.1 mg of the obtained peptide 1 m
lmg / ml HOB dissolved in l DMF and prepared in dry DMF
t, 12 mg / ml PyBOP, 4 μl / ml NMM, 3 mg / ml H-Trp
-0.5 ml of OBzl hydrochloride was added in order under ice cooling. The mixture was allowed to stand at 4 ° C. overnight, and the insolubles were filtered and purified by reverse phase HPLC. The column is Chemco Pack NUCLEOSIL 5C18 (25
0 × 20 mm ID) and eluted with a linear concentration gradient using 0.1% TFA and 0 to 90% acetonitrile. The fraction containing the target substance was freeze-dried, and Fmoc-Val-Tyr-Tyr-Ala-His-L
2.95 mg of eu-Asp (OBzl) -Ile-Ile-Trp-OBzl was obtained.

2.95 mg of the obtained peptide containing 20% piperidine
100 μl of DMF was added, the mixture was allowed to stand at room temperature for 5 minutes, diethyl ether was added to crystallize the desired product, washed with ether, dried under reduced pressure, and H-Val-Tyr-Tyr-Ala-His-Leu-Asp (O
1.87 mg of Bzl) -Ile-Ile-Trp-OBzl were obtained.

Step 2: Compound (I-34) Dry D was added to 100 μg of the compound (I-15) obtained in Example 15.
100 μl of MF was added, and 0.72 mg / ml HOB prepared in dry DMF was added.
t, 2.8 mg / ml PyBOP, 1 μl / ml NMM in 50 μl
of the compound obtained in the above step 1 2.2 mg /
50 μl of dry DMF solution was added. The reaction solution was cooled under ice
After standing for an hour, insolubles were filtered and purified by reverse phase HPLC. The column used was ODS-AM-312 manufactured by YMC. The solvent of the fraction containing the condensate was distilled off under reduced pressure to obtain 74.5 μg of the benzyl ester of the compound (I-34). Then, this compound 74.5μ
30 μl of dry DMF and 30 μl of a methanol solution saturated with ammonium formate, and further a small amount of 10% Pd
/ C was added and stirred vigorously at room temperature for 2 hours. Thereafter, the catalyst was removed by filtration and purified by reverse-phase HPLC as described above. The solvent of the fraction containing the desired compound was distilled off under reduced pressure to give Compound (I-34)
Was obtained in an amount of 0.74 μg.

Mass spectrometry [FABMS]: 2398 (M + H) Amino acid analysis: Asx 2.8 (3), Gly 2.8 (2), His 1.9
(2), Thr 1.1 (1), Ala 2.0 (2), Pro 1.3 (1), Va
l 1.0 (1), Tyr 1.6 (2), Leu 1.0 (1), Ile 2.4
(2), (Trp is not analyzed). Step 1: H-Ala-His-Leu-Asp (OBzl) -Ile-Ile-Trp
-OBzl In the same manner as in Reference Example 3, a carrier resin to which Fmoc-Ile was bound was used as a starting material, and the N-protected amino acid Fmoc
-Ile-OH, Fmoc-Asp (OBzl) -OH, Fmoc-Leu-OH, Fmoc
-His (Trt) -OH and Fmoc-Ala-OH are sequentially bonded,
Crude peptide Fmoc-Ala-His-Le cut from carrier resin
u-Asp (OBzl) -Ile-Ile-OH was obtained. 9.9 mg of the obtained peptide was dissolved in 1 ml of dry DMF, and HOBt 2.7 mg, P
Add 40.4 mg of yBOP and 4.4 μl of NMM, stir for 5 minutes, dry DMF
1 ml of the 3.3 mg / ml H-Trp-OBzl hydrochloride prepared in 1) was added. The mixture was reacted at room temperature for 3 hours, and the insoluble material was filtered.
Purified by reverse phase HPLC in the same manner as described above. The fraction containing the peptide was lyophilized and Fmoc-Ala-His-Leu-Asp (OBz
l) -Ile-Ile-Trp-OBzl was obtained in an amount of 5.7 mg.

DMF150 containing 20% piperidine in 5.7 mg of this compound
μl, left at room temperature for 5 minutes, and crystallized by adding diethyl ether, washed with ether and dried under reduced pressure.
-Ala-His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl
964 μg was obtained.

Step 2: Compound (I-35) Dry D was added to 100 μg of the compound (I-15) obtained in Example 15.
130 μl of MF was added, and 0.72 mg / ml HOB prepared in dry DMF was added.
t, 2.8 mg / ml PyBOP, 1 μl / ml NMM 50 μl
1 mg each, and 6 mg /
19 μl of dry DMF solution was added. The reaction solution was left under ice-cooling for 5 hours, insolubles were filtered, and purified by reverse phase HPLC in the same manner as in Example 15. The solvent of the fraction containing the condensate was distilled off under reduced pressure, and the benzyl ester of compound (I-35) was removed.
172 μg was obtained. Then, 5 ml of dry DMF was added to 172 μg of the compound.
0 μl of a methanol solution saturated with ammonium formate
0 μl was added, a small amount of 10% Pd / C was further added, and the mixture was vigorously stirred at room temperature. Thereafter, the catalyst was removed by filtration and purified by reverse-phase HPLC as described above. The solvent of the fraction containing the desired product was distilled off under reduced pressure to obtain 56 μg of the compound (I-35).

Mass spectrometry [FABMS]: 1973 (M + H) Amino acid analysis: Asx2.7 (3), Gly2.1 (2), His2.1
(2), Thr1.1 (1), Ala2.0 (2), Pro1.0 (1), Phe
1.0 (1), Ile1.9 (2), Leu 1.1 (1), (Trp is not analyzed). Step 1: H-Asp (Ot-Bu) -Trp-Phe-Phe-Asn-Tyr-T
yr-Trp-OBzl (a) In the same manner as in Reference Example 3, Fmoc-Tyr (t-B
Using the carrier resin bound with u) as a starting material, N-protected amino acids were sequentially bound and cut out from the carrier resin to obtain a crude peptide Fmoc-Trp-Phe-Phe-Asn-Tyr-Tyr-OH.
12.75 mg of the obtained peptide and 10.4 mg of PyBOP were dissolved in 0.5 ml of DMF, 0.5 ml of a 0.5 ml DMF solution containing 4.4 μl of NMM, and HOBt3.
A 0.5 ml DMF solution containing 1 mg was added, and the mixture was stirred at room temperature for 5 minutes. To this mixture was added H-1.1 μl of NMM in advance.
A 0.5 ml DMF solution containing 13.3 mg of Trp-OBzl.HC was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated to 0.5 ml, and a white precipitate formed by adding ether was collected by filtration, dried, and dried with Fmoc-Tr.
9 mg of p-Phe-Phe-Asn-Tyr-Tyr-Trp-OBzl were obtained.

(B) 9 mg of the protected peptide obtained in (a) was dissolved in 300 μl of DMF containing 20% piperidine and allowed to stand at room temperature for 15 minutes. A white precipitate formed by adding ether was collected by filtration, dried, and H-Trp-Phe-Phe-Asn-Tyr-Tyr-Tyr-Trp-OBzl6.6.
mg was obtained.

Mass spectrometry [FABMS]: 1216 (M + H) (c) 3.4 mg of Fmoc-Asp (Ot-Bu) -OH and 8.4 mg of PyBOP
Dissolved in 0.5 ml of DMF, 2.7 μl of NMM, 3.1 mg of HOBt in 0.5 ml of DMF
Each solution was added and stirred at room temperature for 5 minutes. here,
A 1 ml DMF solution containing 6.6 mg of the peptide obtained in (b) was added, and the mixture was stirred at room temperature for 2 hours. The product was fractionated and purified by reversed-phase HPLC in the same manner as in Example 17, and Fmoc-Asp (Ot-B
u) -Trp-Phe-Phe-Asn-Tyr-Tyr-Trp-OBzl 3.6 m
g obtained.

Mass spectrometry [FABMS]: 1610 (M + H) (d) 3.6 mg of the protected peptide obtained in (c) was added to (b)
In the same manner as in the above, 2 mg of the compound described in Step 1 was obtained.

Mass spectrometry [FABMS]: 1386 (M + H) Step 2: H-Gly-Asn-Trp-His-Gly-Thr-Ala-Ala-A
sp-Trp-Phe-Phe-Phe-Asn-Tyr-Tyr-Trp-OBzl 4.2 mg of the compound (c) obtained in Reference Example 3 was dissolved in 56 μl of DMF, 8.1 μl of a 0.2 MDMF solution of PyBOP, and 0.2 MDMF of HOBt.
Add 8.1 µl of solution and 15 µl of 2% solution of NMM in DMF.
Stirred for 0 minutes. The resulting mixture is added to the H-Asp (Ot-Bu) -Trp-Phe-Phe-Asn-Tyr-Ty obtained in the above step 1.
Add r-Trp-OBzl 0.27 μmol of 0.1 ml DMF solution and add 4 ° C
For 24 hours and at room temperature for 6 hours. The solvent was distilled off under reduced pressure, the residue was dissolved again in DMF, and the product was fractionated and purified by reversed-phase HPLC in the same manner as in Example 15, and the compound Fmoc-Gly-Asn- Trp-His-Gly-Thr-Ala
-Ala-Asp (Ot-Bu) -Trp-Phe-Phe-Asn-Tyr-Tyr
0.18 mg of -Trp-OBzl was obtained.

(B) To 180 μg of the protected peptide obtained in (a) was added 20 μl of 98% formic acid, and the mixture was left at room temperature for 2 hours. Ether was added, and the precipitated precipitate was collected by filtration, dried and dried with 20 μl of DMF containing 20% piperidine. Was added and left at room temperature for 15 minutes. Ether was added again, and the resulting precipitate was collected by filtration and dried to give 140 μg of the title compound.

Mass spectrometry [FABMS]: 2126 (M + H) Step 3: Compound (I-36) (a) 500 μl DM containing 600 μg of the compound obtained in Step 2
The F solution is cooled to 0 ° C., and 0.2 μl of DEPC and 0.4 μl of TEA
Was added and stirred at 4 ° C. for 3 days. The solvent is distilled off under reduced pressure,
Purification by reverse phase HPLC was performed in the same manner as in Step 2 (a) to obtain 420 μg of the benzyl ester of Compound (I-36).

Mass spectrometry [FABMS]: 2108 (M + H) (b) Dissolve 150 μg of the compound obtained in (a) in 50 μl of DMF, and add 50 μl of a saturated methanol solution of ammonium formate.
About 0.1 mg of 10% Pd / C was added, respectively, and the mixture was stirred at room temperature for 1.5 hours. The reaction product was centrifuged, and the supernatant was collected and purified by reverse-phase HPLC in the same manner as in (a) to obtain Compound (I-36)
52 μg were obtained.

Mass spectrometry [FABMS]: 2016 (M + H) Amino acid analysis: Gly2.1 (2), Asx2.7 (3), His1.0
(1), Thr1.1 (1), Ala2.0 (2), Phe2.3 (2), Tyr
2.4 (2), (Trp is not analyzed) Example 37. Compound (I-37): H-Gly-Asn-Trp-His-Gly-Thr-
Ala-Pro-Asp-Trp-Val-Tyr-Tyr-Ala-His-Leu-
Synthesis of Asp-Ile-Ile-Trp-OH In the same manner as in Reference Example 3, using 30 mg of a carrier resin to which 15.6 μmol of Fmoc-Trp was bound as a starting material, Fmoc-Ile-OH, Fmoc-Ile-OH,
Fmoc-Asp (Ot-Bu) -OH, Fmoc-Leu-OH, Fmoc-His
(Trt) -OH, Fmoc-Ala-OH, Fmoc-Tyr (t-Bu) -O
H, Fmoc-Tyr (t-Bu) -OH, Fmoc-Val-OH, Fmoc-T
rp-OH, Fmoc-Asp (Ot-Bu) -OH, Fmoc-Pro-OH, Fm
oc-Ala-OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Gly-O
H, Fmoc-His (Trt) -OH, Fmoc-Trp-OH, Fmoc-Asn
Synthesis was performed using (Trt) -OH and Fmoc-Gly-OH. Only the final step was different from that of Reference Example 3, and the piperidine treatment (b) was also performed. Excision of peptide from resin
As above, 63.5 mg of crude peptide was obtained. Of these, 1.
7 mg was purified by reverse phase HPLC in the same manner as in Reference Example 3 and freeze-dried to obtain 0.44 mg of a purified product.

Mass spectrometry [FABMS]: 2415 (M + H) Amino acid analysis: Gly2.3 (2), Ala2.0 (2), Asx2.6
(3), His2.0 (2), Ile1.6 (2), Leu0.9 (1), Pro
1.1 (1), Thr1.1 (1), Tyr 1.6 (2), Val 0.7
(1), (Trp was not analyzed) Example 38. Compound (I-38): H-Gly-Asn-Trp-His-Gly-Thr-
Ala-Pro-Asp-Trp-Leu-Tyr-Phe-Ala-His-Gln-
Synthesis of Asp-Val-Ile-Trp-OH In the same manner as in Reference Example 3, using 30 mg of a carrier resin to which 15.6 μmol of Fmoc-Trp was bound as a starting material, Fmoc-Ile-OH, Fmoc-Val-OH,
Fmoc-Asp (Ot-Bu) -OH, Fmoc-Gln (Trt) -OH, Fmo
c-His (Trt) -OH, Fmoc-Ala-OH, Fmoc-Phe-OH, F
moc-Tyr (t-Bu) -OH, Fmoc-Leu-OH, Fmoc-Trp-
OH, Fmoc-Asp (Ot-Bu) -OH, Fmoc-Pro-OH, Fmoc-
Ala-OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Gly-OH, F
moc-His (Trt) -OH, Fmoc-Trp-OH, Fmoc-Asn (Tr
t) Synthesis was performed using -OH and Fmoc-Gly-OH. Only the final step was different from that of Reference Example 3, and the piperidine treatment (b) was also performed. Excision of peptide from resin
As above, 56.0 mg of crude peptide was obtained. Of these, 2.
5 mg was purified by reverse-phase HPLC in the same manner as in Reference Example 3 and freeze-dried to obtain 0.42 mg of a purified product.

Mass spectrometry [FABMS]: 2414 (M + H) Amino acid analysis: Gly2.2 (2), Ala2.0 (2), Asx2.7
(3), His2.0 (2), Ile0.6 (1), Leu1.1 (1), Pro
1.1 (1), Thr1.0 (1), Tyr 1.0 (1), Val0.6 (1),
Glx1.2 (1), Phe1.1 (1), (Trp not analyzed) Step 1: H-Asn-Ile-Ile-Trp-OBzl In the same manner as in Reference Example 3, using a carrier resin to which Fmoc-Ile is bound, N-protected amino acids Fmoc-Ile and Fmo
c-Asn (Trt) -OH is sequentially bonded to synthesize a protected peptide, which is cleaved from the resin and the crude peptide Fmoc-Asn-Ile-Il
e-OH was obtained. The obtained 5.8 mg of the present peptide was dissolved in 1 ml of DMF, 10.4 mg of PyBOP, 2.7 mg of HOBt, and 4.4 μl of NMM were added, and the mixture was allowed to stand at room temperature for 5 minutes. 13.3 mg of H-Trp-OBzlHC in the reaction solution
Was added and stirred at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reverse phase HPLC in the same manner as in Step 1 of Example 36 to obtain the Fmoc compound of the compound described in Step 1
5.4 mg were obtained. This compound was converted to DMF containing 20% piperidine
It was dissolved in 1 ml and left at room temperature for 10 minutes. The precipitate formed upon addition of ether was collected by filtration and purified by reverse phase HPLC to give 5 mg of the compound titled in Step 1.

Step 2: Compound (I-39) 0.19 mg of compound (I-17) and H-
Condensation with 0.1 mg of Asn-Ile-Ile-Trp-OBzl to give a benzyl ester, debenzylation and title compound 10
μg was obtained.

Mass spectrometry [FABMS]: 2385 (M + H) Amino acid analysis; Gly2.2 (2), Asx3.3 (4), His0.9
(1), Thr0.9 (1), Ala1.0 (1), Pro1.0 (1), Phe
1.8 (2), Tyr1.7 (2), Ile1.4 (2), (Trp was not analyzed) By a method similar to that in Example 21, 0.19 mg of compound (I-17) obtained in Example 17 and H obtained in Step 1 of Example 35 were used.
-Ala-His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBz1
By condensing with 0.1 mg, a benzyl ester was obtained. The obtained product was debenzylated in the same manner as in Example 18 to obtain 9.5 μg of the title compound.

Mass spectrometry [FABMS]: 2708 (M + H) Amino acid analysis; Gly2.3 (2), Asx3.4 (4), His1.9
(2), Thr1.1 (1), Ala2.0 (2), Pro1.2 (1), Phe
1.8 (2), Tyr1.8 (2), Ile2.5 (2), Leu1.0 (1),
(Trp was not analyzed) Example 41. Step 1: Fmoc-Gly-Asp (OBzl) -Trp-His-Gly-Thr-
Ala-OH In the same manner as in Reference Example 3, 100 mg of a carrier resin bound with 60 μmol of Fmoc-Ala was used as a starting material, and Fmoc-Thr (t-Bu) -OH and Fmoc were sequentially used as N-protected amino acids.
-Gly-OH, Fmoc-His (Trt) -OH, Fmoc-Trp-OH, Fm
A protected peptide was synthesized using oc-Asp (OBzl) -OH and Fmoc-Gly-OH, cleaved from the resin, and purified by HPLC in the same manner as in Example 15 to obtain 40.5 mg of the title compound.

Mass spectrometry [FABMS]: 1056 (M + H) Amino acid analysis: Asx0.6 (1), Gly1.6 (2), His1.0
(1), Thr0.9 (1), Ala1.0 (1), (Trp is not analyzed) Step 2: H-Gly-Asp (OBzl) -Trp-His-Gly-Thr-AlA
-Pro-Asp-Trp-OBzl ( NO 2) (a) H-Pro-Asp (Ot-Bu) -Trp-OBzl (NO 2) t-
0.33 g of Boc-Trp (CHO) -OH was dissolved in 5 ml of DMF, and 0.17 g of sodium hydrogencarbonate was added and stirred. Where bromide p
1.08 g (5 mmol) of -nitrobenzyl was added, and the mixture was stirred at room temperature for 24 hours. Water and ethyl acetate were added to the reaction solution, and the mixture was shaken. The organic phase was recovered, and the solvent was distilled off. The residue was purified by a silica gel column (Kelselgel 60, 50 g, manufactured by Merck, eluting with hexane / ethyl acetate = 6/4), and t-Boc-Trp
(CHO) -OBzl (NO ₂₎ was obtained 0.2 g. This was dissolved in 5 ml of DMF, 0.5 ml of piperidine was added, and the mixture was stirred at 0 ° C. for 1.5 hours. The solvent was distilled off under reduced pressure, and 0.5 ml of 98% formic acid was added, followed by stirring at room temperature for 4 hours. About 30 ml of ether was added to the reaction solution, and the resulting precipitate was collected by filtration, dried, and treated with H-Trp-OBz.
0.16 g of l (NO ₂ ) was obtained.

41 mg of Fmoc-Asp (Ot-Bu) -OH was dissolved in 2 ml of methylene chloride and cooled to 0 ° C. PyBOP78mg, HOBt20mg, NMM22μ
1 was added in sequence and stirred at 0 ° C. for 5 minutes. A solution of 34 mg of H-Trp-OBzl (NO ₂ ) obtained above in 2 ml of methylene chloride was cooled to 0 ° C. and added to the obtained reaction solution. The mixture was stirred at 4 ° C. for 4 hours at room temperature for 1 hour, the solvent was distilled off under reduced pressure, and the residue was subjected to a silica gel column (Merck Kieselgel 60, 50 g,
Eluted with chloroform / methanol = 25/1)
oc-Asp was obtained (Ot-Bu) -Trp-OBzl (NO 2) 53mg. The obtained product was dissolved in 100 μl of DMF, and 25 μl of piperidine was used.
l, and the mixture was left at room temperature for 10 minutes, purified by a silica gel column (Kieselgel 60, 50 g, eluted with hexane / ethyl acetate = 2/1) and purified by H-Asp (Ot-Bu) -Trp-O.
1 mg of Bzl (NO ₂ ) was obtained.

Dissolve 3.4mg Fmoc-Pro-OH in 0.5ml DMF, PyBOP5.2m
g, HOBt 1.4 mg, and NMM 1.7 μl were sequentially added, followed by stirring at room temperature for 10 minutes. The H-Asp obtained in Step 2 was added to the obtained reaction solution.
(Ot-Bu) -Trp-OBzl (NO 2) by adding 0.5mlDMF solution of 1 mg, and stirred at room temperature for 1.5 hours. 0.2 ml of the obtained reaction solution
Concentrated to a degree. The residue was purified by reverse phase HPLC in the same manner as in Example 17, and Fmoc-Pro-Asp (Ot-Bu) -Trp-O
1.5 mg of Bzl (NO ₂ ) were obtained.

This was dissolved in 160 μl of DMF, 40 μl of piperidine was added, the mixture was allowed to stand at room temperature for 12 minutes, and the solvent was distilled off under reduced pressure.
The residue was purified by HPLC as described above and H-Pro-Asp
It was obtained (Ot-Bu) -Trp-OBzl (NO 2) 1.4mg.

Mass spectrometry [FABMS]: 608 (M + H) Dissolve 2.4 mg of the peptide obtained in Step 1 in 0.5 ml of DMF, add 4.8 mg of PyBOP, 1.2 mg of HOBt, and 1.2 μl of NMM, and stir at room temperature for 15 minutes. Cooled to 0 ° C. Here, obtained above H-Pro-Asp (Ot- Bu) -Trp-OBzl (NO 2) 0.7mg
Was added and the mixture was stirred at 4 ° C. for 18 hours and at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase HPLC in the same manner as described above. The obtained compound was added with 98% formic acid at room temperature for 2 hours, followed by adding 20% piperidine / DMF to room temperature.
After stirring for 15 minutes, H-Gly-Asp (OBzl) -Trp-His-Gly
-Thr-Ala-Pro-Asp- Trp-OBzl (NO 2) to obtain a 400 [mu] g.

In 100 μl of DMF solution of 200 μg of peptide obtained in step 2
468 μg of PyBOP, 122 μg of HOBt, and 152 μg of NMM were added at 0 ° C., and the mixture was stirred at 0 ° C. for 1 hour and at room temperature for 3.5 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase HPLC as described above.
40 μg of 4-nitrobenzyl ester of the title compound were obtained. The obtained product was dissolved in 50 μl of 90% acetic acid, cooled at 0 ° C., added with about 0.1 mg of zinc dust, and stirred at 0 ° C. for 30 minutes. Example of removing the zinc dust by centrifugation and using the supernatant
Purify by reverse phase HPLC in the same manner as 15 to give the title compound
27 μg were obtained.

Step 4: Compound (I-41) 25 μg of the compound obtained in Step 3 was dissolved in 23 μl of DMF, cooled to 0 ° C., and PyBOP 68 μg, HOBt 18 μg, NMM 21 μm
g was added as a 0.2 MDMF solution, and the mixture was allowed to stand at 0 ° C. for 10 minutes. The H-Va obtained in Step 1 of Example 20 was added to the reaction solution.
l-Tyr-Phe-Ala-His-Leu-Asp (OBzl) -Ile-Ile
A solution of 44 μg of -Trp-OBzl in 27 μl of DMF was added, followed by stirring at 4 ° C. for 24 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reversed-phase HPLC in the same manner as in Step 3 to obtain 15 μg of the side chain of compound (I-41), a protected C-terminal compound. 13 μg of this was dissolved in 10 μl of DMF, 10 μl of a saturated methanol solution of ammonium formate and about 0.1 mg of 10% Pd / C were added, and the mixture was stirred at room temperature for 1.5 hours. Pd / C is removed by centrifugation, and the supernatant is reversed phase as above.
Purification by HPLC yielded 2 μg of compound (I-41).

Mass spectrometry [FABMS]: 2383 (M + H) Amino acid analysis: Asx2.1 (3), Gly2.3 (2), His1.4
(2), Thr1.1 (1), Ala2.0 (2), Pro1.0 (1), Tyr
0.9 (1), Val1.3 (1), Ile2.5 (2), Leu1.3 (1), P
he0.9 (1), (Trp is not analyzed) Step 1: Fmoc-Gly-Asn-Trp-Lys (Z) -Gly-Thr-Al
Synthesis of a-OH In the same manner as in Reference Example 3, using 100 mg of a carrier resin bound with 60 μmol of Fmoc-Ala, Fmoc-Thr (t-Bu) -OH, Fmoc-Gly-OH, Fmoc
-Lys (Z) -OH, Fmoc-Trp-OH, Fmoc-Asn (Trt)-
A protected peptide was synthesized using OH and Fmoc-Gly-OH, and the peptide was cut out from the resin and purified by HPLC in the same manner as in Example 17 to obtain 9.9 mg of the title compound.

Mass spectrometry [FABMS]: 1090 (M + H) Amino acid analysis: Asx0.6 (1), Gly1.5 (2), Thr0.8
(1), Ala1.0 (1), Lys0.9 (1), (Trp is not analyzed) Step 2: H-Gly-Asn-Trp-Lys (Z) -Gly-Thr-Ala-
Pro-Asp-Trp-OBzl (NO ₂ ) Dissolve 2.5 mg of the peptide obtained in step 1 in 0.5 ml DMF, add 2.4 mg of PyBOP, 0.6 mg of HOBt, and 0.8 μl of NMM, and stir at room temperature for 15 minutes. Cooled to ° C. H-Pro-Asp (Ot-Bu) -Trp-OBz obtained in Example 41 was added to the obtained reaction solution.
A solution of 0.7 mg of l (NO ₂ ) in 0.5 ml of DMF was added, and the mixture was stirred at 4 ° C. for 6 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase HPLC as described above. Add 70% of 98% formic acid to the obtained compound.
For 2 hours at room temperature, followed by 10 μl / DM of 20% piperidine.
After adding 40 μl of F, the mixture was allowed to stand at room temperature for 15 minutes, crystallized with 3 ml of ether, washed with ether, and dried under reduced pressure to obtain 420 μg of the title compound in Step 2.

In 100 μl of DMF solution of 200 μg of peptide obtained in step 2
468 μg of PyBOP, 122 μg of HOBt, and 152 μg of NMM were added at 0 ° C., and the mixture was stirred at 0 ° C. for 1 hour and at room temperature for 3.5 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase HPLC in the same manner as described above to obtain 43 μg of 4-nitrobenzyl ester of the title compound. The product obtained is dissolved in 50 μl of 90% acetic acid,
After cooling to 0 ° C, about 0.1 mg of zinc dust was added, and the mixture was stirred at 0 ° C for 30 minutes. The zinc dust was removed by centrifugation, and the supernatant was purified by reverse-phase HPLC in the same manner as in Example 15 to obtain 59 μg of the title compound in Step 3.

Step 4: Compound (I-42) 59 μg of the compound obtained in Step 3 was dissolved in 32 μl of DMF and cooled to 0 ° C. The mixture was prepared with 0.2 ml DMF solution
156 μg of PyBOP, 41 μg of HOBt, and 51 μg of NMM were added, respectively, and left at 0 ° C. for 10 minutes. In the obtained reaction solution,
20 H-Val-Tyr-Phe-Ala-His-Leu obtained in Step 1
Asp (OBzl) -Ile-Ile-Trp-OBzl containing 109 μg 68
μl DMF solution was added, and the mixture was stirred at 4 ° C. for 13 hours. The solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase HPLC in the same manner as in Step 3 to obtain a side chain of compound (I-42), 31 μg of the protected C-terminal.
I got 28 μg of the obtained product was dissolved in 15 μl of DMF, and 15 μl of a saturated methanol solution of ammonium formate and
About 0.1 mg of 10% Pd / C was added, and the mixture was stirred at room temperature for 2 hours. Pd / C is removed by centrifugation, and the supernatant is reversed phase as above.
Purification by HPLC yielded 10.6 μg of compound (I-42).

Mass spectrometry [FABMS]: 2373 (M + H) Amino acid analysis: Asx2.8 (3), Gly2.6 (2), His1.0
(1), Thr0.9 (1), Ala2.0 (2), Pro1.2 (1), Tyr
1.0 (1), Val1.1 (1), Ile2.4 (2), Phe1.0 (1), L
ys0.9 (1), (Trp was not analyzed) Example 43. Compound (I-43): H-Gly-Asp-Trp-His-Gly-Thr-
Ala-Pro-Asp-Trp-Val-Tyr-Phe-Ala-His-Leu-
Synthesis of Asp-Ile-Ile-Trp-OH In the same manner as in Reference Example 3, using 20 mg of a carrier resin to which 10.4 μmol of Fmoc-Trp was bound, Fmoc-Ile-OH and Fmoc-Ile were sequentially used as N-protected amino acids. -OH, Fmoc-Asp (Ot
-Bu) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, F
moc-Ala-OH, Fmoc-Phe-OH, Fmoc-Tyr (t-Bu)-
OH, Fmoc-Val-OH, Fmoc-Trp-OH, Fmoc-Asp (Ot-B
u) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Thr
(T-Bu) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -O
H, Fmoc-Trp-OH, Fmoc-Asp (Ot-Bu) -OH, Fmoc-G
A protected peptide was synthesized using ly-OH, and was subjected to piperidine treatment (b), unlike in Reference Example 3, only in the final step.
The peptide was cut out from the resin in the same manner as in Reference Example 3 to obtain 19.2 mg of the crude peptide. 6.2 mg of this product was obtained in Example 1.
Purified by reverse-phase HPLC and lyophilized in the same manner as in 7, and 1.2
4 mg of purified product was obtained.

Mass spectrometry [FABMS]: 2400 (M + H) Amino acid analysis: Gly2.1 (2), Ala2.0 (2), Asx2.1
(3), His1.4 (2), Ile2.5 (2), Leu1.3 (1), Pro
1.0 (1), Thr1.1 (1), Phe0.9 (1), Tyr0.9 (1), V
al1.3 (1), (Trp was not analyzed) Example 44. Compound (I-44): H-Gly-Asn-Trp-Lys-Gly-Thr-
Ala-Pro-Asp-Trp-Val-Try-Phe-Ala-His-Leu-
Synthesis of Asp-Ile-Ile-Trp-OH In the same manner as in Reference Example 3, using 20 mg of a carrier resin to which 10.4 μmol of Fmoc-Trp was bound, Fmoc-Ile-OH and Fmoc-Ile were sequentially used as N-protected amino acids. -OH, Fmoc-Asp (Ot
-Bu) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, F
moc-Ala-OH, Fmoc-Phe-OH, Fmoc-Tyr (t-Bu)-
OH, Fmoc-Val-OH, Fmoc-Trp-OH, Fmoc-Asp (Ot-B
u) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Thr
(T-Bu) -OH, Fmoc-Gly-OH, Fmoc-Lys (t-Bo
c) -OH, Fmoc-Trp-OH, Fmoc-Asn (Trt) -OH, Fmoc
A protected peptide was synthesized using -Gly-OH, and unlike the reference example 3 only in the final step, piperidine treatment (b) was also performed. The peptide was cut out from the resin in the same manner as in Reference Example 3 to obtain 30.0 mg of a crude peptide. 6.9 mg of the product obtained
Was purified by reverse phase HPLC in the same manner as in Example 17 and lyophilized to obtain 1.30 mg of a purified product.

Mass spectrometry [FABMS]: 2390 (M + H) Amino acid analysis: Gly2.6 (2), Ala2.0 (2), Asx2.8
(3), His1.0 (1), Ile2.4 (2), Leu1.1 (1), Pro
1.2 (1), Thr0.9 (1), Tyr1.0 (1), Val1.1 (1), P
he1.0 (1), Lys0.9 (1), (Trp is not analyzed) Reference Example 1. Compound (a): Fmoc-Gly-Asn-Trp-His-Gly-Thr-A
Synthesis of la-Pro-OH In the same manner as in Example 4, t-Boc-Ala- was sequentially used as the carrier resin to which t-Boc-Pro was bound and the protected amino acid.
OH, t-Boc-Thr (Bzl) -OH, t-Boc-Gly-OH, t-Boc-
His (Bom) -OH, t-Boc-Trp (CHO) -OH, t-Boc-Asn
Using -OH and Fmoc-Gly-OH, 1.2 g of a carrier resin having a protected peptide bonded thereto was obtained. 1.2 g of this carrier resin was used in Example 4.
The synthetic peptide was cleaved from the resin with hydrogen fluoride in the same manner as described above, dissolved in 2M acetic acid, and lyophilized to obtain 464.0 mg of a crude product. Purification by HPLC in the same manner as in Example 4 gave 131.9 mg of compound (a).

Mass spectrometry [FABMS]: 1061 (M + H) Amino acid analysis: Asx0.7 (1), Gly2.1 (2), His1.0
(1), Thr1.0 (1), Pro1.1 (1), Ala1.1 (1), (T
(rp is not analyzed) Reference Example 2. Compound (b): H-Val-Tyr-Phe-Ser-His-Leu-Asp
Synthesis of -Ile-Ile-Trp-OH In the same manner as in Example 4, t-Boc-Trp (CHO) was bonded to a carrier resin and an N-protected amino acid in the order of t.
-Boc-Ile-OH, t-Boc-Ile-OH, t-Boc-Asp (OBzl)
-OH, t-Boc-Leu-OH, t-Boc-His (Bom) -OH, t-Boc
-Ser-OH, t-Boc-Phe-OH, t-Boc-Tyr (Br-Z) -O
Using H and t-Boc-Val-OH, 1.5 g of a carrier resin to which the protected peptide was bound was obtained. Of these, 0.8 g was cut out of the resin with hydrogen fluoride, dissolved in 2M acetic acid, and lyophilized to obtain 243.3 mg of a crude product. Of these, 101 mg was purified by HPLC in the same manner as in Example 4 to obtain 7.0 mg of compound (b).

Mass spectrometry [FABMS]: 1293 (M + H) Amino acid analysis: Asx1.1 (1), Ser1.1 (1), His1.2
(1), Tyr1.0 (1), Val1.0 (1), Ile1.5 (2), Leu
1.2 (1), Phe1.1 (1) Reference Example 3. Compound c: Fmoc-Gly-Asn-Trp-His-Gly-Thr-Ala-A
Synthesis of la-OH 50 mg of a carrier resin to which 30 μmol of Fmoc-Ala was bound was placed in a reactor of an automatic synthesizer, and the following operation was performed according to a synthesis program of Shimadzu Corporation.

(A) The carrier resin was washed with DMF for 3 minutes, and the solution was discharged.

(B) DMF solution containing 30% piperidine was added and the mixture was stirred for 4 minutes, the solution was drained and this operation was repeated once more.

(C) The carrier resin was washed with DMF for 1 minute, the solution was discharged, and this operation was repeated five times.

Thus, a carrier resin to which Amo had been removed from which Fmoc had been removed was obtained.

(D) Fmoc-Ala-OH 300 μmol, PyBOP 300 μmol, HOBt30
A solution obtained by adding 1.05 ml of DMF containing 0 μmol and 450 μmol of NMM and stirring for 3 minutes was added to the resin, and the mixture was stirred for 30 minutes and the solution was discharged.

(E) The carrier resin was washed with DMF for 1 minute, and this was repeated 5 times. Thus, Fmoc-Ala-Ala was synthesized on the carrier. Next, after performing the washing and deprotection steps of the above (a) to (c), a condensation reaction is carried out using Fmoc-Thr (t-Bu) -OH in the step (d), and then (e) Get the cleaning process of Fm
oc-Thr (t-Bu) -Ala-Ala was synthesized on a carrier resin. Hereinafter, steps (a) to (e) were sequentially repeated to obtain a carrier resin to which the protected peptide was bound. In step (d), Fmoc-Gly-OH and Fmoc-His (Trt) -OH, Fmoc-Gly-OH
c-Trp-OH, Fmoc-Asn (Trt) -OH and Fmoc-Gly-O
H was used. Step (b) only in the final step of condensing Gly
Was not treated with piperidine. The obtained carrier resin was washed with methanol and butyl ether, and then dried under reduced pressure for 3 hours. To this was added 200 μl of a mixed solution of 90% TFA, 5% thioanisole, and 5% 1,2-ethanedithiol containing 2-methylindole at a rate of 5 mg / ml, and the mixture was left at room temperature for 2 hours to cut out the peptide from the resin. Was. The resin was then filtered off, approximately 10 ml of ether was added to the resulting solution, and the resulting precipitate was filtered off as 23 mg of crude peptide. This crude product is subjected to a reverse phase column (column; ChemcoPac, Chemco
kNUCLEOSIL 5C18 250 × 20 mm I.D.). Elution was performed with a linear concentration gradient using 0.1% TFA and 0 to 90% acetonitrile, and detection was performed at 220 nm to obtain a fraction containing the title compound. This fraction was lyophilized to give compound (c) 16
mg was obtained.

Mass spectrometry [FABMS]: 1036 (M + H) Amino acid analysis: Gly1.9 (2), Asx0.7 (1), His1.0
(1), Thr0.9 (1), Ala2.0 (2), (Trp was not analyzed) Reference Example 4. Compound (d): Fmoc-Leu-Tyr-Phe-Ala-His-Gln-A
Synthesis of sp (OBzl) -Val-Ile-OH In the same manner as in Reference Example 3, Fmoc-Val-OH and Fmoc-Asp (OBzl) were sequentially used as N-protected amino acids using 70 mg of a carrier resin to which 31 μmol of Fmoc-Ile was bound. ) -OH, Fmoc-Gl
n (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Ala-O
H, Fmoc-Phe-OH, Fmoc-Tyr (t-Bu) -OH and Fmo
Synthesis was performed using c-Leu-OH. Cleavage of the peptide from the resin was achieved by 90% TFA, 5% thioanisole, 5% 1,2
The reaction was carried out by leaving 300 μl of a mixed solution of ethanedithiol at room temperature for 2 hours. The obtained crude peptide
25 mg was purified by HPLC in the same manner as in Reference Example 3 to obtain 3.6 mg of compound (d).

Mass spectrometry [FABMS]: 1419 (M + H) Amino acid analysis: Ala1.0 (1), Asx1.0 (1), His0.9
(1), Glx1.0 (1), Ile0.8 (1), Leu0.8 (1), Phe
0.9 (1), Tyr1.0 (1), Val0.7 (1) Industrial Applicability According to the present invention, a peptide having endothelin antagonism is provided.

Sequence Listing SEQ ID NO: 1 Sequence Length: 16 Sequence Type: Amino Acid Number of Chains: Single Chain Topology: Linear Sequence Type: Peptide Sequence H-Gly-Asn-Trp-His-Gly-Thr-Ala -Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp-OH SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid number of chains: Single chain Topology: Cyclic Sequence type: Peptide Sequence cyclo (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp) SEQ ID NO: 3 Sequence length: 16 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence H-Cys-Asn-Trp-His-Gly-Thr-Ala-Pro-Cys-Trp-Phe-Phe-Asn-Tyr-Tyr- Trp-OH SEQ ID NO: 4 Sequence length: 7 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence H-Trp-Phe-Phe-Asn-Tyr-Tyr- Trp-OH SEQ ID NO: 5 Sequence Length: 6 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence H-Trp-Phe-Asn-Tyr-Tyr-Trp-OH SEQ ID NO: 6 Sequence length Length: 6 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence H-Trp-Phe-Phe-Asn-Tyr-Trp-OH SEQ ID NO: 7 Sequence length: 6 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence H-Trp-Phe-Phe-Tyr-Tyr-Trp-OH SEQ ID NO: 8 Sequence length: 6 Sequence Type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence H-Phe-Phe-Asn-Tyr-Tyr-Trp-OH SEQ ID NO: 9 Sequence length: 20 Sequence type : Number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence H-Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Val-Tyr-Phe-Ala- His -Leu-Asp-Ile-Ile- Trp-OH SEQ ID NO: 10 Sequence length: 20 Sequence type: Number of amino acid chains: Single chain Topology: Linear Sequence type: Peptide sequence H-Gly-Asn -Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Val-Tyr-Tyr-Ala-His-Leu-Asp-Ile-Ile-Trp-OH SEQ ID NO: 11 Sequence length: 20 Type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence H-Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Leu-Tyr-Phe-Ala -His-Gln-Asp-Val-Ile- Trp-OH SEQ ID NO: 12 Sequence length: 20 Sequence type: Number of amino acid chains: Single chain Topology: Linear Sequence type: Peptide sequence H-Gly -Asp-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp-OH SEQ ID NO: 13 Sequence length: 20 Sequence type: amino acid Number of chains: single chain Topology: linear Column Type: peptide sequence H-Gly-Asn-Trp-Lys-Gly-Thr -Ala-Pro-Asp-Trp-Val-Tyr- Phe-Ala-His-Leu-Asp-Ile- Ile-Trp-OH

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 1/20 C12N 1/20 A // A61K 38/00 ACD A61K 37/02 ACD ADD ADD AED AED (C12P 21/04 C12R 1 (465) (C12N 1/20 C12R 1: 465) (72) Inventor Chieko Shimazaki 71-1 Okagami, Aso-ku, Kawasaki City, Kanagawa Prefecture (72) Inventor Kazuo Yamaguchi 2121-8 Isobe, Sabehara-shi, Kanagawa Prefecture (72) Inventor Joe Matsuda 1-22-7 Nukii Minamicho, Koganei City, Tokyo (72) Inventor Shigeto Kitamura 1-3-7, Naruse, Machida City, Tokyo (72) Inventor Toshihide Ikemura 4352-1 Wakamatsucho, Mishima City, Shizuoka Prefecture (72) Inventor Tatsuhiro Ogawa 3-6-6 Asahimachi, Machida, Tokyo (72) Inventor Keiichi Yano 1-38-6, Highland, Yokosuka, Kanagawa (72) Inventor Toshiyuki Suzawa Setagaya-ku, Tokyo Yamada 3-29-11 (72) Inventor Takeshi Shibata 3-6-6 Asahimachi, Machida-shi, Tokyo (72) Inventor Yamazaki Sakimoto 3-9-13 Nakamachi, Machida-shi, Tokyo Examiner Kazuyoshi Yoshizumi (58) Field surveyed (Int.Cl. ⁶ , DB name) C12P 21/04 C12N 1/20 C07K 7 / 06,7 / 08,7 / 64 CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A peptide represented by the following formula (I) or a pharmacologically acceptable salt thereof: XA-Trp-B-Gly-Thr-EGY (I) wherein A is Asn Or Asp, B represents His or Lys, E represents Ala or Ser, G represents Ala or Pro, X represents X ¹ -Gly or Y represents hydroxy, lower alkoxy, amino, Wherein X ¹ and X ³ represent hydrogen, benzyloxycarbonyl, t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl or carbonyl-substituted or unsubstituted lower alkanoyl; X ² and Y ² Represents hydrogen, Y ¹ represents hydroxy, lower alkoxy or amino, or X ¹ , Y ¹ and X ² , Y ² together represent X ^1-
Y ¹ and X ² —Y ² represent a single bond, Z is hydroxy, lower alkoxy, benzyloxy, benzhydryloxy, Gly-Z ¹ (wherein Z ¹ is hydroxy, lower alkoxy, benzyloxy or benzhi Stands for drilloxy,
X ¹ and together represent X ¹ -Z ¹ is a single bond), in Ala-Z ¹ (wherein, Z ¹ is as defined above), in Val-Z ¹ (wherein, Z ¹ is and wherein Trp-Z ¹ (wherein, Z ¹ is as defined above), Trp-Gly-Z ¹ (where Z ¹ is as defined above), Trp-Asn-Tyr-Tyr -Trp-Z ¹ (wherein, Z ¹ is as defined above), Trp-Phe-Phe-Asn-Tyr-Tyr-7Hyt-Z ¹ (wherein, Z ¹ is as defined above, and 7Hyt is 7-hydroxytryptophan), Trp-Ile-Ile-Trp-Z ¹ (wherein Z ¹ is as defined above), Trp-Val-Tyr-Phe-W-His-Leu-Asp-Ile- Ile-T
rp-Z ¹ (wherein, Z ¹ has the same meaning as described above, and W represents Ala, Ser or Cys), Trp-W-His-Leu-Asp-Ile-Ile-Trp-Z ¹ (wherein Z
¹ and W are as defined above), Trp-Val-Tyr-Tyr-W-His-Leu-Asp-Ile-Ile-T
rp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Leu-Tyr-Phe-W-His-Gln-Asp-Val-Ile-T
rp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Val-Tyr-Phe-W-Phe-Phe-Asn-Tyr-Tyr-T
rp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Phe-Phe-Asn-Tyr-Tyr-W-His-Leu-Asp-I
le-Ile-Trp-Z ¹ (wherein Z ¹ and W are as defined above), Trp-Phe-Phe-Asn-Tyr-Tyr-Asn-Ile-Ile-Trp-
Z ¹ (wherein, Z ¹ is as defined above), or J-Phe-MQ-Tyr-R-T-Z ¹ (where J is Trp or a single bond, M is Phe or a single bond , Q is Asn or a single bond, R is Tyr or a single bond, T is Trp, Ala, Phe, Tyr, Trp-Trp, Asn-Tyr-Tyr-Trp, Trp-Asn-Tyr-Tyr-Trp, Trp-Val -Tyr-Phe-W-His-Leu-Asp-Ile-Ile-T
rp (wherein W is as defined above), or a single bond, at least two of J, M, Q, R and T do not simultaneously represent a single bond, and Z ¹ is as defined above. )]｝. 2. The peptide according to claim 1, which is represented by the following formula (I-1): 3. A peptide represented by the following formula (II): X ⁴ -J-Phe-MQ-Tyr-RTZ ² (II) wherein X ⁴ is hydrogen, benzyloxycarbonyl, represents t- butyloxycarbonyl or 9-fluorenylmethyloxycarbonyl, Z ² is hydroxy, lower alkoxy, represents a benzyloxy or benzhydryloxy, J, M, Q, R and T are each claim (1 )
Synonymous with J, M, Q, R and T described above). 4. A compound belonging to the genus Streptomyces and having the formula (I-
1) RES-701-1, represented by the formula (I-2) RES-701-2 represented by the formula or formula (I-3) A microorganism having the ability to produce RES-701-3 represented by the formula is cultured in a medium, and RES-701-1, RES-701-2 or RES-701-3 is accumulated in the culture solution. RE
A method for producing RES-701-1, RES-701-2 or RES-701-3, comprising collecting S-701-1, RES-701-2 or RES-701-3. 5. The RES-701-1, RES-7 according to claim (4).
Streptomyces sp. RE-701 (FERM BP-3624) having the ability to produce 01-2 or RES-701-3. 6. The RES-701-1, RES-7 according to claim (4).
Streptomyces sp. RE-629 (FERM BP-4126) capable of producing 01-2 or RES-701-3.