JPH06107681A - Peptide derivative - Google Patents

Abstract

PURPOSE:To provide a new derivative useful as a vascularization inhibitor. CONSTITUTION:The objective derivative of formula I (R<1> is H, lower alkoxycarbonyl, etc.; R<2> is OH, amino, etc.; l and m are each 0-8; (l+m)=3-8; amino acid residues are each D-modification, L-modification, or a mixture thereof) or a medically permissible salt thereof, e.g. a compound of formula II (Arg is arginine residue; Boc is t-butoxycarbonyl; phGly is alpha-phenylglycine residue; Asp is aspartic acid residue; Gly is glycine residue). The compound of the formula I can be obtained, for example, by condensation reaction between a compound of formula III (Asp is aspartic acid residue; OBzl is benzyloxy; OBu<t> is t-butoxy) and a compound of formula IV followed by deprotection to produce a compound of formula V, which is then further condensed with a compound of formula VI (OBzI is benzyloxy; TsOH is p-toluenesulfonic acid) to deprotect the N- terminal and C-terminal and reprotect the N-terminal.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a specific novel peptide derivative, and more particularly to a novel peptide derivative having an angiogenesis-inhibiting effect and salts thereof.

[0002]

BACKGROUND OF THE INVENTION Angiogenesis is the development of embryos, inflammatory reactions,
It is widely known that it plays an important role in diabetic retinopathy, wound healing process, cancer and tumor growth, and the like. For example, in the process of growth of cancer or tumor, the growth of cancer or tumor accompanied by angiogenesis in the tissue requires that the new blood vessels supply nutrients and oxygen, excrete waste products, and depend on angiogenesis. I can say. Therefore, it is expected that by suppressing angiogenesis, the growth of cancer or tumor can be suppressed or regressed (eg Denekamp, J., 1984, Acta Ra.
diologica Oncology, 23 , 217-225: Folkman, J., 197
6, Sci. Am., 234 , 58-78, etc.).

In addition to cancers and tumors, diseases caused by abnormal growth of blood vessels or accompanied by angiogenesis, such as angiofibromas, arteriovenous malformations, Osler-Weber syndrome, vascular adhesions, atherosclerotic plaques, Corneal transplant angiogenesis, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity (posterior lens fibroplasia), trachoma, delayed wound healing, burn-associated granulation, hypertrophic scar, purulent granuloma, psoriasis, Treatment of scleroderma, non-union fracture, rheumatoid arthritis, hemophilic arthropathy, etc. is expected by suppressing angiogenesis (Moses, MA and Langer, R., 1991, Bi
oTechnology, 9 , 630-634).

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel compound having an angiogenesis inhibitory action which is expected to have a therapeutic effect on various diseases as described above.

As a result of intensive studies conducted by the present inventors, the novel peptide derivative having a specific structure represented by the following general formula (1) and a salt thereof have been identified as pharmaceuticals which meet the above objectives. The inventors have found that they have a desired pharmacological action and have completed the present invention.

[0006]

That is, the present invention relates to a peptide represented by the following general formula (1) and a salt thereof.

[0007]

[Chemical 2]

[Wherein R ¹ represents a hydrogen atom, a lower alkoxycarbonyl group or a lower alkanoyl group which may have a group selected from the group consisting of a hydroxyl group and a halogen atom as a substituent, and R ² represents a hydroxyl group, an amino group or Group -Gly-
OH, 1 and m show 0 or an integer of 1-8. However, l
+ M is 3 to 8. In addition, each amino acid residue in the formula is D-
Form, L-form or a mixture of D, L-isomers. In the present specification, when abbreviations for amino acids, peptides, protecting groups, active groups, etc. are used, I
According to the regulations of UPAC and IUB or symbols conventionally used in the field, examples are as follows. When amino acids and the like may have optical isomers, the L-form is shown unless otherwise specified.

Asp ... Aspartic acid residue, Arg ... Arginine residue, Arg (NO ₂ ) ... Nω-nitroarginine residue, Glu ... Glutamic acid residue, Gly ... Glycine residue, PhGly ... α-phenylglycine residue, Ph (OH) Gly ... α- 4- hydroxyphenyl glycine residues, OBzl ... benzyloxy group, OBu ^t ... tert-butoxy group, Boc ... tert-butoxycarbonyl group, TFA ... trifluoroacetic acid, TEA ... triethylamine, TsOH ... p- toluenesulfonyl acid, DMF ... dimethylformamide, WSC-HCl ... N-ethyl -N'- dimethylaminopropyl - carbodiimide hydrochloride, THF ... tetrahydrofuran, HOBT ... 1-hydroxybenzotriazole, CH ₂ Cl ₂ ... methylene chloride , DMAP ... - dimethylaminopyridine.

In the present specification, the above general formula (1)
Examples of the lower alkoxycarbonyl group defined by R ¹ include a methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl group and the like having 1 to 6 carbon atoms. A linear or branched alkoxycarbonyl group can be exemplified, and examples of the lower alkanoyl group include acetyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, butylcarbonyl, pentylcarbonyl and hexylcarbonyl groups having 1 to 6 carbon atoms. A straight chain or branched chain alkyl carbonyl group can be illustrated.

The peptide derivative of the present invention represented by the above general formula (1) and a salt thereof have an angiogenesis-inhibiting action based on their specific structure and are effective as an angiogenesis inhibitor. That is, they are
As described above, various diseases that can suppress the growth of cancer and tumor, and are accompanied by angiogenesis, such as angiofibromas, arteriovenous malformations, Osler-Weber syndrome, vascular adhesions, atherosclerotic plaque, and corneal transplantability. Angiogenesis, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity (posterior lens fibroplasia), trachoma, delayed wound healing, burn-associated granulation, hypertrophic scar, purulent granuloma, psoriasis, scleroderma In addition, it is possible to exert a therapeutic effect by inhibiting angiogenesis on fractures with non-adhesion, rheumatoid arthritis, hemophilic arthropathy and the like. In addition, the derivative of the present invention has excellent durability and absorbability, weak side effects such as antigenicity, blood glucose increase, weight loss, intestinal motility suppression, and food intake suppression, low toxicity, and the above-mentioned aspects. It is suitable as a medicine.

The method for producing the derivative of the present invention will be described in detail below.

The peptide derivative represented by the above-mentioned general formula (1) of the present invention is basically according to a general peptide synthesis method and, in accordance with its structure, individual amino acids are sequentially amide-bonded (peptide bond) from the terminal amino acid. It can be produced by a so-called stepwise method in which the above structure is divided, or by a method in which the above structure is divided into several fragments and each fragment is similarly synthesized and then fragment-condensed.

Specific examples of the peptide synthesis method adopted above include "The Peptides", Vol. 1, 1966 [Schroder and Luhke, Academic p.
ress, New York, USA] and "Basics and Experiments of Peptide Synthesis" [Izumiya et al., Maruzen Co., Ltd., 1985], for example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride. Substance method, DCC method, active ester method (p-
(Nitrophenyl ester method, N-hydroxysuccinimide method, cyanomethyl ester method, etc.), method using Woodward reagent K, carbonyldiimidazole method, redox method, DCC / additive (HONB, HOBT,
The HOSu) method or the like can be appropriately adopted. In the above, either the solid phase synthesis method or the liquid phase synthesis method can be applied.
If, for example, a solid phase synthesis method is employed, this is more particularly the C-terminal amino acid (amino group protected) is first bound to its insoluble carrier by its carboxyl group.
Here, the insoluble carrier is not particularly limited as long as it has a bond with a reactive carboxyl group, for example, chloromethyl resin, halogenomethyl resin such as bromomethyl resin or hydroxymethyl resin, phenol resin, tert-alkyloxycarbonyl hydrazide. Resins, benzhydrylamine resins and the like can be used. Then, after removing the amino-protecting group, amino-protected amino acids are sequentially added according to the amino acid sequences represented by the above general formula (1).
The reactive amino group and the reactive carboxyl group are combined by a condensation reaction (a peptide bond forming reaction and an acid amide bond forming reaction, and the following reactions are simply referred to as a "condensation reaction"), and they are synthesized one step at a time The desired peptide can be obtained by extending the chain length up to the sequence and removing the resulting peptide from the insoluble carrier. The peptide of the present invention can also be synthesized by a liquid phase synthesis method according to a conventional method.

In the above, each amino acid having a side chain functional group not involved in the reaction, such as Arg, Glu, Asp.
Etc., it is desirable to protect the side chain functional group, which can be protected by an ordinary protecting group, and the protecting group can be eliminated after the completion of the reaction. In addition, the functional groups involved in the reaction are usually activated. Each reaction method is known,
Reagents and the like used therein can be appropriately selected from known ones.

For example, as a protecting group for an amino group, benzyloxycarbonyl, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoro. Acetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl oil, 9-
Examples thereof include a fluorenylmethoxycarbonyl group.

Examples of the protective group for the carboxyl group include alkyl esters (chain and cyclic alkyl esters such as methyl, ethyl, propyl, butyl, tert-butyl and cyclohexyl), benzyl esters, p-nitrobenzyl esters, p-methoxy. Examples thereof include groups capable of forming benzyl ester, p-chlorobenzyl ester, benzhydryl ester, benzyloxycarbonyl hydrazide, tert-butyloxycarbonyl hydrazide, trityl hydrazide and the like. The carboxyl groups of Asp and Glu can also be protected by esterification with, for example, benzyl alcohol, methanol, ethanol, tert-butyl alcohol, cyclohexyl alcohol and the like.

Examples of activated carboxyl groups include corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, 1). -Hydroxybenztriazole, N-hydroxy-5-norbornene-2,3-dicarboximide and the like) and the like.

In the above method, the condensation reaction between the reactive amino group and the reactive carboxyl group can be carried out in the presence of a basic compound in a suitable solvent. Here, as the basic compound, for example, triethylamine, trimethylamine, N, N-diisopropylethylamine, pyridine, dimethylaniline, N-methylmorpholine, 1,5
-Diazabicyclo (4,3,0) -5-nonene [DB
N], 1,5-diazabicyclo (5,4,0) -5-undecene [DBU], 1,4-diazobicyclo (2,5)
2,2) Organic bases such as octane [DABCO] and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate and sodium hydrogen carbonate can be used. In addition, various solvents known to be usable in this kind of condensation reaction, for example, anhydrous or hydrous DMF, dimethyl sulfoxide (DMSO), pyridine, chloroform, dioxane, methylene chloride, THF, ethyl acetate, N-methyl are used. Pyrrolidone, hexamethylphosphoric triamide (HMP
A) or the like and a mixed solvent thereof or the like may be used. The ratio of the raw material compounds to be used is not particularly limited, but it is usually preferable that the other raw material compound is in an equimolar amount to about 5 times the molar amount, and preferably in the equimolar amount to about 1.5 times the molar amount. . The reaction temperature is in the usual range used for this kind of condensation reaction, generally about -40 ° C to about 60 ° C, preferably about -20 ° C to about 4 ° C.
It is appropriately selected from the range of 0 ° C., and the reaction time is generally several minutes to
A range of about 120 hours is recommended.

Of the above-mentioned various condensation reactions, for example, the mixed acid anhydride method is described in more detail in the presence of a basic compound in a suitable solvent, methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate. , An alkylhalocarboxylic acid such as isobutyl chloroformate. Here, as the basic compound, for example, triethylamine,
Organic bases such as trimethylamine, N, N-diisopropylethylamine, pyridine, dimethylaniline, N-methylmorpholine, DBN, DBU, DABCO, and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogencarbonate, and sodium hydrogencarbonate can be used. As the solvent, various solvents commonly used in the mixed acid anhydride method, for example, methylene chloride, chloroform, halogenated hydrocarbons such as dichloroethane, benzene, toluene, aromatic hydrocarbons such as xylene, diethyl ether, THF, Ethers such as dimethoxyethane, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as DMF, DMSO and HMPA can be used. Reaction is usually -20 to 100 ° C
Temperature, preferably about -20 to 50 ° C, and usually about several minutes to 10 hours, preferably several minutes to 2
It will finish in about time.

The azidation method is carried out by first reacting an activated carboxyl group, for example, a carboxyl group activated with an alcohol such as methyl alcohol, ethyl alcohol and benzyl alcohol with hydrazine hydrate in a suitable solvent. Be implemented. Here, as the solvent, for example, a mixed solvent of dioxane, DMF, DMSO, HMPA, alcohols or the like can be used. The amount of the hydrazine hydrate used is usually about 5 to 20 times, preferably about 5 to 10 times the molar amount of the activated carboxyl group. The reaction is usually carried out at 50 ° C or lower, preferably at about -20 to 30 ° C, and a compound (hydrazine derivative) in which the carboxyl group moiety is substituted with hydrazine can be produced by the reaction.

The above-mentioned various condensation reactions can also be carried out in the presence of a suitable condensing agent, for example, a carbodiimide reagent such as DCC, WSC, WSC.HCl, carbonyldiimidazole, tetraethylpyrophosphine, or the like.
This condensing agent is usually in an equimolar amount to about 4 with respect to the raw material compound.
It is used in the range of about double molar amount. The reaction using the condensing agent is more specifically, for example, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and tetrachloroethane, ethers such as dioxane, THF and dimethoxyethane, ketones such as acetone and methyl ethyl ketone, and acetonitrile. , Ethyl acetate, DMF, dimethylacetamide, DMSO and the like in a suitable solvent, preferably the above-mentioned anhydrous solvent, at a reaction temperature of generally about -10 to 60 ° C, preferably 0 ° C to room temperature, and a few tens of minutes to It may take as long as 120 hours.

When elimination of the protecting group is required in each of the above reaction steps and final reaction step, the elimination reaction can be carried out according to a usual method. Examples of the method include hydrogenation using a catalyst such as palladium-carbon and palladium black, reductive methods such as reduction with metallic sodium in liquid ammonia, elimination under basic conditions using piperidine, trifluoro, and the like. Examples thereof include acidolysis with a strong acid such as acetic acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid and hydrobromic acid. Hydrogenation using the above catalyst is carried out, for example, at a hydrogen pressure of 1
It can be carried out under the conditions of about 10 atm and about 0 to 40 ° C.
The amount of the catalyst used is usually in the range of about 100 mg to 1 g, and the reaction is generally completed in about 1 hour to several days. The above acidolysis can be carried out in the presence or absence of a solvent, usually at about -40 to 60 ° C, preferably at about -20 to 20 ° C, and for about several minutes to several hours. The amount of the acid used is usually a large excess amount with respect to the raw material compound.
In the acidolysis, when only the amino-protecting group is eliminated, trifluoroacetic acid or hydrochloric acid is preferably used as the acid. Further, the reduction with sodium metal in the liquid ammonia is carried out usually at about -40 ° C to -70 ° C using an amount of sodium metal such that the reaction liquid is colored in permanent blue for about 30 seconds to 10 minutes. Can be done.

Specific examples of the method for producing the peptide derivative of the present invention described in detail above are shown in Reference Examples and Examples below, and by taking typical compounds as examples, the above condensation reaction, peptide chain extension reaction, N-terminal The substitution product synthesis reaction and the N-terminal deprotection group reaction are described in detail in the respective reaction process formulas below.

[0025]

[Chemical 3]

[0026]

[Chemical 4]

[0027]

[Chemical 5]

[0028]

[Chemical 6]

[0029]

[Chemical 7]

[0030]

[Chemical 8]

[0031] In the above reaction schemes 3 and 5, R ^{2 'is} a protected hydroxyl group Gly (e.g. Gly-OBz
l), NH ₂ or a protected hydroxyl group (eg OBzl)
Indicates. The R ¹ group and R ² group in Reaction Scheme 5 are the same as above.

According to the above reaction scheme 1, the compound (A)
To obtain a compound (C) by a condensation reaction of the compound (B) with
Then, the compound (D) is obtained by the deprotection reaction of the compound, and the compound (F) is obtained by the condensation reaction of the compound (E) with the compound (E). By the reprotection reaction of
Is obtained. By the same reaction, PhGly becomes Ph (O
H) The desired compound (G ′) which is Gly can be obtained.

According to the reaction process formula 2, the compound (I) can be obtained by the condensation reaction between the compound (H) and the compound (B), and the compound (K) can be obtained by the reaction between the compound (B) and the compound (J). ) Can be obtained.

According to the reaction process formula 3, the compound (L) is obtained by the condensation reaction of the compound (1K) and the compound (G), and the compound (G) is condensed again with the compound (M). By repeating this reaction operation 1 to 6 times, the desired compound (N) in which m is 3 to 8 is obtained. The compound (N) continues to have Arg
The compound (O) can be derived by the elimination reaction of the hydroxyl group and the protecting group of the R ² group.

According to the reaction scheme 4, the compound (R) in which 1 is 1 can be derived by the condensation reaction between the compound (P) and the compound (G ′), and the compound (G ′) is added to this compound again. A compound (Q) in which 1 is 2 or more can be obtained by conducting a condensation reaction and repeating this reaction. The compound (Q)
Can be converted to the compound (R) by the subsequent deprotection reaction of the Arg hydroxyl group.

According to reaction scheme 5, compound (N) is reacted with compound (T) to give the desired R ^{1 at the} N-terminus.
A compound (U) having a group can be obtained, which can be converted to the compound (V) by a deprotecting group reaction.

Similarly, the compound (R) can also be provided with a desired R ¹ group at its N-terminal.

According to reaction scheme 6, compound (W)
The desired compound (X) can be obtained by the elimination reaction of the N-terminal protecting group of.

The peptides obtained by the above-mentioned various methods can be separated and purified from the reaction system by a usual means for peptide separation, such as extraction method, partition method, column chromatography and the like.

Thus, the desired peptide derivative of the present invention can be obtained.

The obtained peptide derivative of the present invention usually has a free acid form or a salt form thereof, and all have the same biological activity and are useful as various pharmaceuticals. According to the method, it can also be converted into a pharmaceutically acceptable base addition salt form. As the salt,
Examples thereof include alkali metal salts such as sodium salts and potassium salts, as well as alkaline earth metal salts and ammonium salts.

When the peptide derivative of the present invention is used as a medicine, it is usually used as a filler, a bulking agent, a binder, a moisturizing agent, a disintegrating agent, a surface active agent, a diluent or a diluent. It is adjusted to the form of a general pharmaceutical preparation using a dosage form. Various forms of this pharmaceutical preparation can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules,
Suppositories, injections (solutions, suspensions, etc.), ointments, nasal drops, oral mucosa-adhesive preparations (troches, buccal tablets, sublingual tablets,
Chewable tablets, drip tablets, etc.) and the like.

In the case of molding into tablets, as a carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, excipients such as silicic acid, water, ethanol, propanol, etc. Single syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, serac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, poly Oxyethylenesorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, disintegrating agents such as lactose, sucrose, stearin,
Decay inhibitor for cocoa butter, hydrogenated oil, quaternary ammonium base, absorption promoter for sodium lauryl sulfate, humectant for glycerin, starch, adsorption of starch, lactose, kaolin, bentonite, colloidal silicic acid, etc. Agents, purified talc, stearates, boric acid powder, lubricants such as polyethylene glycol, and the like can be used. Further, the tablet is a tablet coated with a usual coating as necessary, for example, a sugar-coated tablet,
It may be a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet.

In the case of molding in the form of pills, as carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, gum arabic powder, tragacanth powder, gelatin, ethanol and the like are bound. Agent,
A disintegrating agent such as laminaran or agar can be used.

In the case of molding in the form of suppositories, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like can be used as carriers.

Capsules are generally prepared by mixing the compound of the present invention with the various carriers exemplified above and filling them into hard gelatin capsules, soft capsules or the like according to a conventional method.

When it is prepared as an injection, the liquid, emulsion and suspension are preferably sterilized and isotonic with blood, and when they are molded into other forms, for example, water or ethyl alcohol is used as a diluent. , Macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. still,
In this case, a sufficient amount of sodium chloride, glucose or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. may be added. Good.

Further, in each form of the pharmaceutical preparation prepared as described above, a colorant, a preservative, a flavoring agent, a flavoring agent, a sweetening agent and the like and other medicinal agents can be contained, if necessary.

In the case of molding into a paste, cream or gel, white petrolatum, paraffin, glycerin, cellulose derivative, polyethylene glycol, silicone, bentonite, etc. can be used as a diluent. Nasal drops and buccal mucoadhesive preparations can be sprayed using appropriate binders, diluents, propellants, etc. according to ordinary methods into powder form, aerosol form, liquid form, etc. suitable for spray administration. Is prepared. For the preparation of the powder form, for example, the use of water-absorbing base materials such as celluloses, starches and polyacrylates is suitable, and for the preparation of the aerosol form preparation, water, glycols, alcohols, A nonionic surfactant or the like is advantageously used. Formulations in spray propellant form also include conventional liquefied petroleum gas,
Propellants (liquefied propellants) such as carbon dioxide and fluorinated lower alkanes are used.

The amount of the peptide derivative of the present invention to be contained in the pharmaceutical preparation is not particularly limited and can be appropriately selected in a wide range, but it is usually about 0.5 mg to 5000 mg in the pharmaceutical preparation. Good.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and it is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are intravenously administered alone or in a mixture with a normal replenishing solution such as glucose and amino acid. Independently, it is intramuscularly, intradermally, subcutaneously or intraperitoneally administered, and the suppository is intrarectally administered. Nasal drops are administered into the nasal cavity by inhalation or the like, and oral mucoadhesives are administered to oral mucosa.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on the usage, age of the patient, sex and other conditions, degree of disease and the like, but the amount of the compound of the present invention as an active ingredient is usually about 1 kg / day of body weight. The amount is preferably about 0.01 mg to 100 mg, and the preparation can be administered in 1 to 4 divided doses per day.

[0053]

EXAMPLES In order to explain the present invention in more detail, production examples of starting compounds for producing the compounds of the present invention will be given as reference examples, and then production examples of the compounds of the present invention will be given as examples.

The amino acid analysis in each example was carried out by the following method.

<Amino acid analysis> 6N hydrochloric acid (phenol added) was added to the test substance, which was hydrolyzed at 110 ° C. for 24 hours or 48 hours and dried under reduced pressure. Then, an amino acid analyzer (Hitachi, model 835-30 high speed) was used. Amino acid analyzer).

In this method, since the recovery rate of Arg (NO ₂ ) is low and the analysis cannot be performed correctly, each compound was identified mainly by the analysis values of Asp and PhGly. In addition, for Ph (OH) Gly, the retention time was almost the same as PhGly, and since it could not be quantified separately, it was shown as the total value of both.

[0057]

[Reference Example 1] Boc-PhGly-Asp (OBzl) manufacturing H-Asp (OBzl) of -OBu ^t -OBu ^t 45.87g, Bo
c-PhGly-OH 41.67 g, HOBT24.3
Dissolve 8 g in 350 ml of CH ₂ Cl _{2 and} add W under ice cooling.
SC.HCl 34.59g was added, TEA 17.04m
The pH was adjusted to 6.0 to 7.0 with 1 and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, dissolved in 500 ml of ethyl acetate, and twice with 200 ml of 1N hydrochloric acid, and saturated sodium bicarbonate water 20 times.
It was washed twice with 0 ml and once with 200 ml of water, and dried over anhydrous magnesium sulfate. Ethyl acetate was concentrated under reduced pressure, and n-hexane was added to the residue for crystallization. This was collected by filtration, washed with n-hexane, and the target product (63.0 g)
(Yield 74.8%) was obtained.

Mp. 103 ~ 104 ℃

[0059]

[Reference Example 2] Boc-PhGly-Asp-OBu ^t of production Boc-PhGly-Asp (OBzl) -OBu t 5
2.60 g of ethanol: THF (1: 1) 200 ml
Was dissolved in water, 5.3 g of 5% palladium carbon was added, and catalytic reduction was carried out for 16 hours under normal temperature and atmospheric pressure hydrogen flow. The reaction solution is filtered and concentrated under reduced pressure to give the above-mentioned amorphous product 43.3.
5 g (yield 100%) was obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
7.52 to 7.10 (5H, m), 6.25 to 5.37
(3H, m), 4.90 to 4.57 (1H, m), 3.
29 to 2.57 (2H, m), 1.43 (9H, s),
1.34 (9H, s)

[0061]

[Reference example 3]

[0062]

[Chemical 9]

[0063] ^{Boc-PhGly-Asp-OBu t} 4
_{3.35g, H-Arg (NO 2} ) -OBzl · TsO
H 54.82 g and HOBT 16.91 g were added to CH ₂ Cl.
₂ Dissolve in 350 ml, and under ice cooling, WSC ・ HCl2
3.98 g was added, and the pH was adjusted to 6.0 with TEA18.9 ml.
It was adjusted to ˜7.0 and stirred overnight at room temperature. The reaction solution was washed in the same manner as in Example 1 and then concentrated under reduced pressure to obtain 73.24 g (yield 100%) of the above-mentioned amorphous target product.

¹ H-NMR (CDCl ₃ ) δ ppm:
7.64 to 6.93 (16H, m), 5.81 (1H,
d, J = 7.0 Hz), 5.26 to 4.99 (3H,
m), 4.84-4.36 (2H, m), 3.41-
3.00 (2H, m), 2.91 to 2.68 (2H,
m), 2.02-0.92 (22H, m)

[0065]

[Reference example 4]

[0066]

[Chemical 10]

[0067]

[Chemical 11]

73.24 g was dissolved in 40 ml of CH ₂ Cl _{2, 200} ml of TFA was added under ice cooling, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, 500 ml of diethyl ether was added to the residue, the precipitate was collected by filtration, and dried under reduced pressure over sodium hydroxide to obtain a powdery substance. Then, this product was dissolved in a mixed solvent of 200 ml of dioxane and 100 ml of DMF, 28.54 g of di-tert-butyl dicarbonate and 32.93 ml of TEA were added under ice cooling, and again,
Stir overnight at room temperature.

The reaction mixture was concentrated under reduced pressure, and the residue was diluted with ethyl acetate (1).
It was dissolved in 1 l, washed successively with 1N hydrochloric acid (450 ml × 4 times), saturated saline (250 ml × 4 times), and concentrated under reduced pressure. Ethyl acetate / n-hexane (1: 1) mixed solvent was added to the residue for crystallization, which was collected by filtration and washed with water to give 63.88 g of the desired product.
Got (Yield 89.1%) mp. 139-143 ° C Amino acid analysis value: Asp 0.96 (1) PhGly 1.04 (1)

[0070]

Reference Example 5 Production of Boc-PhGly-Gly-OBzl 5.64 g of H-Gly-OBzl.TsOH and Boc-
In the same manner as in Reference Example 1 using 4.0 g of PhGly-OH, 6.27 g of the desired product was obtained. (Yield 98.9%) mp. 109-111 ° C

[0071]

[Reference Example 6]

[0072]

[Chemical 12]

Boc-PhGly-Gly-OBzl
4.8 ml of TFA was added to 1.60 g under ice cooling to dissolve it, and the mixture was allowed to stand at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure,
The residue was dried under reduced pressure over sodium hydroxide.

16 ml of dimethylformamide was added to the residue.
Dissolve in water and adjust to pH 6-7 with TEA under ice cooling.

[0075]

[Chemical 13]

2.38 g, HOBT 0.54 g, WSC
-0.77 g of HCl was added, and the pH was adjusted to 6 to 7 again with TEA, and then stirred overnight at room temperature. The reaction solution is concentrated under reduced pressure,
Water was added to the residue, the deposited precipitate was collected by filtration, and 1N hydrochloric acid,
The product was washed with water, saturated sodium bicarbonate water, and water in this order, and further with ethyl acetate / n-hexane (5: 2) five times to obtain 3.20 g of the desired product. (Yield 84.9%) Amino acid analysis value: Asp 0.95 (1) PhGly 2.05 (2) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.82-
7.72 (8H, m), 7.45 to 7.14 (20H,
m), 5.46-4.96 (6H, m), 4.84-
4.55 (1H, m), 4.39 to 4.14 (1H,
m), 4.00-3.77 (2H, m), 3.36-
2.96 (2H, m), 2.76 to 2.36 (2H,
m) 1.82 to 1.23 (13H, m)

[0077]

[Reference Example 7]

[0078]

[Chemical 14]

[0079]

[Chemical 15]

3.10 g

[0081]

[Chemical 16]

4.71 g of the desired product was obtained in the same manner as in Reference Example 6 using 2.14 g. (Yield 97.9%) Amino acid analysis value: Asp 1.91 (2) PhGly 3.09 (3) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.80-
7.70 (14H, m), 7.52 to 7.05 (30
H, m), 5.50 to 4.93 (9H, m), 4.84.
~ 4.54 (2H, m), 4.46-4.13 (2H,
m), 4.03 to 3.75 (2H, m), 3.35
2.96 (4H, m), 2.78 to 2.37 (4H,
m), 1.84 to 1.16 (17H, m)

[0083]

[Reference Example 8]

[0084]

[Chemical 17]

With Boc-PhGly-NH ₂ 1.98 g

[0086]

[Chemical 18]

Using the same amount of 4 g as in Reference Example 6, 4.30 g of the desired product was obtained. (Yield 89.5%) Amino acid analysis value: Asp 0.96 (1) PhGly 2.04 (2) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.47-
7.62 (7H, m), 7.41 to 7.19 (17H,
m), 5.24 to 5.18 (2H, m), 5.12 (2
H, s), 4.73 to 4.67 (1H, m), 4.31.
Up to 4.25 (1H, m), 3.16 to 3.11 (2H,
m), 2.74 to 2.48 (2H, m), 1.79 to
1.43 (4H, m), 1.35 (9H, s)

[0088]

[Reference Example 9]

[0089]

[Chemical 19]

[0090]

[Chemical 20]

4.30 g

[0092]

[Chemical 21]

Using 6.66 g, and in the same manner as in Reference Example 6, 6.75 g of the desired product was obtained. (Yield 93.3%) Amino acid analysis value: Asp 1.93 (2) PhGly 3.02 (3) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.59-
7.60 (13H, m), 7.35 to 7.18 (27
H, m), 5.37 to 5.19 (3H, m), 5.11.
(4H, s), 4.73 to 4.66 (2H, m), 4.
33-4.26 (2H, m), 3.15-3.10 (4
H, m), 2.76-2.50 (4H, m), 1.80
~ 1.45 (8H, m), 1.35 (9H, s)

[0094]

[Reference Example 10] Boc-PhGly-OBzl manufacturing Boc-PhGly-OH6.0g, benzyl alcohol 2.98ml, DMAP0.29g the CH ₂ Cl ₂ 3
Dissolve in 0 ml and salt-ice under ice cooling WSC.HCl 5.
49 g was added, and the mixture was stirred under ice cooling for 4 hours. The reaction solution was washed in the same manner as in Reference Example 1 and then concentrated under reduced pressure. N-Hexane was added to the residue under ice-cooling, and the deposited precipitate was collected by filtration and n-
After washing with hexane, 6.46 g (79.2) of the desired product was obtained.
%) Was obtained. mp. 56-58 ° C

[0095]

[Reference Example 11]

[0096]

[Chemical formula 22]

Boc-PhGly-OBzl4.57 g
When

[0098]

[Chemical formula 23]

Using 8.10 g, a reaction was carried out in the same manner as in Reference Example 6. After washing the reaction solution in the same manner as in Reference Example 1,
After concentrating under reduced pressure, n-hexane was added to the residue for crystallization, which was collected by filtration and washed with n-hexane to give the above-mentioned desired product 10.
37 g were obtained. (Yield 95.5%) Amino acid analysis value: Asp 0.96 (1) PhGly 2.00 (2) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.54 to
7.75 (7H, m), 7.36 to 7.18 (20H,
m), 5.40 to 5.16 (2H, m), 5.11 (4
H, s), 4.78 to 4.72 (1H, m), 4.32
Up to 4.26 (1H, m), 3.19 to 3.11 (2H,
m), 2.65-2.50 (2H, m), 1.82-
1.42 (4H, m), 1.34 (9H, s)

[0100]

[Reference Example 12]

[0101]

[Chemical formula 24]

[0102]

[Chemical 25]

10.30 g,

[0104]

[Chemical formula 26]

15.78 g of the desired product was obtained in the same manner as in Reference Example 6 using 8.10 g. (Yield 94.7%) Amino acid analysis value: Asp 1.95 (2) PhGly 3.05 (3) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.53 ~
7.75 (13H, m), 7.35 to 7.18 (30
H, m), 5.40-5.10 (3H, m), 5.10
(6H, s), 4.79 to 4.63 (2H, m), 4.
38-4.24 (2H, m), 3.20-3.08 (4
H, m), 2.68 to 2.50 (4H, m), 1.80
~ 1.40 (8H, m), 1.34 (9H, s)

[0106]

[Reference Example 13]

[0107]

[Chemical 27]

Boc-Ph (OH) Gly-OH2.4
Using 3 g, the above-mentioned target product 2.
17 g was obtained. (Yield 62.3%) Amino acid analysis value: Asp 1.02 (1) Ph (OH) Gly 0.98 (1) ¹ H-NMR (DMSO-d ₆ ) δppm: 9.35
(1H, s), 8.60 to 7.80 (6H, m), 7.
52 to 7.14 (5H, m), 7.15 (2H, d, J
= 8.6 Hz), 6.67 (2H, d, J = 8.6H)
z), 5.18 to 4.95 (3H, m), 4.52
4.36 (1H, m), 4.35-4.21 (1H,
m), 3.20 to 3.00 (2H, m), 2.70 to
2.50 (2H, m), 1.80 to 1.40 (4H,
m), 1.35 (9H, s)

[0109]

[Example 1]

[0110]

[Chemical 28]

[0111]

[Chemical 29]

4.60 g

[0113]

[Chemical 30]

Using 6.03 g of 2.03 g, the desired product (6.06 g) was obtained. (Yield 97.4%) Amino acid analysis value: Asp 2.95 (3) PhGly 4.05 (4) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.93-
7.57 (20H, m), 7.47 to 7.00 (40
H, m), 5.50 to 4.93 (12H, m), 4.8.
6 to 4.52 (3H, m), 4.46 to 4.12 (3
H, m), 4.02 to 3.74 (2H, m), 3.34.
-2.94 (6H, m), 2.77-2.34 (6H,
m), 1.90 to 1.07 (21H, m)

[0115]

[Chemical 31]

[0116]

[Chemical 32]

0.70 g of dimethylformamide 14 m
1 and methanol 2 ml, dissolved in a mixed solvent of 5% palladium carbon 0.14 g, and at room temperature under normal pressure and hydrogen flow,
The catalytic reduction was carried out for 3 hours. The reaction solution was filtered and then concentrated under reduced pressure, water and 1N hydrochloric acid were added to the residue, and the precipitate was collected by filtration to obtain 0.53 g of the desired product. (Yield 97.4%) Amino acid analysis value: Asp 2.86 (3) PhGly 4.14 (4) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.91-
7.54 (20H, m), 7.48 to 6.99 (20
H, m), 5.60 to 5.06 (4H, m), 4.90
~ 4.50 (3H, m), 4.36-4.02 (3H,
m), 3.86 to 3.62 (2H, m), 3.32 to
2.98 (6H, m), 2.80 to 2.34 (6H,
m), 1.92 to 1.15 (21H, m)

[0118]

Example 2

[0119]

[Chemical 33]

[0120]

[Chemical 34]

5.0 g

[0122]

[Chemical 35]

Using 1.62 and in the same manner as in Reference Example 6, 5.89 g of the desired product was obtained. (Yield 93.6%) Amino acid analysis value: Asp 4.00 (4) PhGly 5.00 (5) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.86-
7.56 (26H, m), 7.52 to 7.01 (50
H, m), 5.57 to 4.97 (15H, m), 4.8.
6 to 4.54 (4H, m), 4.47 to 4.13 (4
H, m), 4.00-3.73 (2H, m), 3.33
-2.93 (8H, m), 2.78-2.41 (8H,
m), 1.93 to 1.08 (25H, m)

[0124]

[Chemical 36]

[0125]

[Chemical 37]

Using 0.40 g of the desired product, 0.68 g was obtained in the same manner as in Example 1- (2). (Yield 93.1
%) Amino acid analysis value: Asp 3.77 (4) PhGly 5.23 (5) ¹ H-NMR (DMSO-d ₆ ) δppm: 9.01 to
7.68 (26H, m), 7.46 to 6.88 (25
H, m), 5.58 to 5.08 (5H, m), 4.86
~ 4.51 (4H, m), 4.39 to 4.03 (4H,
m), 3.88 to 3.65 (2H, m), 3.36 to
2.90 (8H, m), 2.81 to 2.31 (8H,
m), 1.93 to 1.18 (25H, m)

[0127]

Example 3

[0128]

[Chemical 38]

[0129]

[Chemical Formula 39]

3.80 g

[0131]

[Chemical 40]

Using 1.02 g and in the same manner as in Reference Example 6, 4.41 g of the desired product was obtained. (Yield 91.5%) Amino acid analysis value: Asp 4.89 (5) PhGly 6.11 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.85
7.58 (32H, m), 7.52 to 7.00 (60
H, m), 5.56 to 4.97 (18H, m), 4.8
8 to 4.53 (5H, m), 4.48 to 4.13 (5
H, m), 4.02 to 3.78 (2H, m), 3.42.
-2.94 (10H, m), 2.81-2.29 (10
H, m), 1.94 to 1.05 (29H, m)

[0133]

[Chemical 41]

[0134]

[Chemical 42]

Using 0.70 g, 0.57 g of the desired product was obtained in the same manner as in Example 1- (2). (Yield 98.7
%) Amino acid analysis value: Asp 4.85 (5) PhGly 6.15 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.85
7.68 (32H, m), 7.54 to 6.95 (30
H, m), 5.60-5.10 (6H, m), 4.90.
Up to 4.48 (5H, m), 4.38 to 4.03 (5H,
m), 3.87 to 3.66 (2H, m), 3.34 to
2.96 (10H, m), 2.80 to 2.34 (10
H, m), 1.97 to 1.18 (29H, m)

[0136]

Example 4

[0137]

[Chemical 43]

[0138]

[Chemical 44]

With 6.65 g

[0140]

[Chemical formula 45]

Using 8.27 g, and in the same manner as in Reference Example 6, 8.96 g of the desired product was obtained. (Yield 95.9%) Amino acid analysis value: Asp 2.86 (3) PhGly 4.14 (4) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.59-
7.61 (19H, m), 7.35 to 7.19 (37
H, m), 5.38 to 5.18 (4H, m), 5.10.
(6H, s), 4.73 to 4.66 (3H, m), 4.
32-4.25 (3H, m), 3.20-3.02 (6
H, m), 2.80-2.50 (6H, m), 1.80
~ 1.40 (12H, m), 1.34 (9H, s)

[0142]

[Chemical formula 46]

[0143]

[Chemical 47]

0.48 g of the desired product was obtained in the same manner as in Example 1- (2) using 0.60 g. (Yield 93.5
%) Amino acid analysis value: Asp 2.80 (3) PhGly 4.20 (4) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.92-
7.49 (21H, m), 7.46 to 6.89 (20
H, m), 5.59 to 5.02 (4H, m), 4.87
Up to 4.46 (3H, m), 4.37 to 3.94 (3H,
m), 3.36-2.92 (6H, m), 2.82-
2.24 (6H, m), 1.93 to 1.10 (21H,
m)

[0145]

Example 5

[0146]

[Chemical 48]

[0147]

[Chemical 49]

4 g

[0149]

[Chemical 50]

In the same manner as in Reference Example 6 using 1.84 g, 4.84 g of the desired product was obtained. (Yield 93.9%) Amino acid analysis value: Asp 3.86 (4) PhGly 5.14 (5) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.71
7.42 (27H, m), 7.42 to 7.09 (45
H, m), 5.41 to 5.11 (13H, m), 4.8
0 to 4.60 (4H, m), 4.40 to 4.25 (4
H, m), 3.20 to 3.00 (8H, m), 2.80.
~ 2.40 (8H, m), 1.85 ~ 1.40 (16
H, m), 1.34 (9H, s)

[0151]

[Chemical 51]

[0152]

[Chemical 52]

0.38 g of the desired product was obtained in the same manner as in Example 1- (2) using 0.40 g. (Yield 89.4
%) Amino acid analysis value: Asp 3.83 (4) PhGly 5.17 (5) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.73-
7.49 (27H, m), 7.35 to 6.89 (25
H, m), 5.49 to 5.05 (5H, m), 4.87
Up to 4.43 (4H, m), 4.36 to 3.87 (4H,
m), 3.32 to 2.92 (8H, m), 2.77 to
2.33 (8H, m), 1.92 to 1.12 (25H,
m)

[0154]

Example 6

[0155]

[Chemical 53]

[0156]

[Chemical 54]

Using 0.240 g and 0.008 g of AcOH, the above target compound 0.1 was prepared in the same manner as in Examples 1- (1) and (2).
g (59.1%) was obtained.

Amino acid analysis value: Asp 4.73 (5) PhGly 6.27 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.56 to
7.32 (27H, m), 7.32 to 7.11 (25
H, m), 5.58 to 5.18 (5H, m), 4.78
Up to 4.60 (4H, m), 4.30 to 4.12 (4H,
m), 3.20 to 3.00 (8H, m), 2.80 to
2.40 (8H, m), 1.88 (3H, s), 1.8
0 to 1.40 (16H, m)

[0159]

Example 7

[0160]

[Chemical 55]

[0161]

[Chemical 56]

3.40 g

[0163]

[Chemical 57]

Using 1.21 g and in the same manner as in Reference Example 6, 4.08 g (97.0%) of the desired product was obtained.

Amino acid analysis value: Asp 4.87 (5) PhGly 6.13 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.70-
7.50 (33 H, m), 7.40 to 7.10 (50
H, m), 5.45 to 5.00 (16H, m), 4.7
5 to 4.65 (5H, m), 4.40 to 4.20 (5
H, m), 3.20 to 3.00 (10H, m), 2.8.
0-2.40 (10H, m), 1.80-1.40 (2
0H, m) 1.34 (9H, s)

[0166]

[Chemical 58]

[0167]

[Chemical 59]

Using 0.1 g of the desired product, 0.2 g was obtained in the same manner as in Example 1- (2). (Yield 82.6
%) Amino acid analysis value: Asp 4.93 (5) PhGly 6.07 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.60-
7.40 (33H, m), 7.40 to 7.10 (30
H, m), 5.40-5.15 (6H, m), 4.75
~ 4.60 (5H, m), 4.30-4.10 (5H, m)
m), 3.20 to 3.00 (10H, m), 2.80 to
2.40 (10H, m), 1.80-1.40 (20
H, m), 1.35 (9H, s)

[0169]

Example 8

[0170]

[Chemical 60]

[0171]

[Chemical formula 61]

1 g

[0173]

[Chemical formula 62]

Using 0.286 g, and in the same manner as in Reference Example 6, 1.03 g of the desired product was obtained. (Yield 87.1%) Amino acid analysis value: Asp 5.82 (6) PhGly 7.18 (7) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.60-
7.40 (39H, m), 7.40 to 7.10 (65
H, m), 5.45-5.10 (19H, m), 4.8
0 to 4.65 (6H, m), 4.35 to 4.20 (6
H, m), 3.20 to 3.00 (12H, m), 2.8
0-2.40 (12H, m), 1.80-1.40 (2
4H, m), 1.34 (9H, s)

[0175]

[Chemical formula 63]

[0176]

[Chemical 64]

Using 0.2 g, 0.14 g (83.5%) of the desired product was obtained in the same manner as in Example 1- (2).

Amino acid analysis value: Asp 6.21 (6) PhGly 6.79 (7) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.70-
7.40 (39H, m), 7.40 to 7.10 (35
H, m), 5. ． 60-5.15 (7H, m), 4.7
4 to 4.60 (6H, m), 4.40 to 4.10 (6
H, m), 3.20 to 3.00 (12H, m), 2.8
0-2.40 (12H, m), 1.80-1.40 (2
4H, m), 1.36 (9H, s)

[0179]

[Example 9]

[0180]

[Chemical 65]

[0181]

[Chemical formula 66]

0.3 g and

[0183]

[Chemical formula 67]

0.28 g of the desired product was obtained in the same manner as in Reference Example 6 using 0.07 g. (Yield 80.8%) Amino acid analysis value: Asp 6.76 (7) PhGly 8.24 (8) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.70-
7.45 (45H, m), 7.40 to 7.10 (75
H, m), 5.40-5.00 (22H, m), 4.8
0 to 4.60 (7H, m), 4.35 to 4.25 (7
H, m), 3.20 to 3.00 (14H, m), 2.8
0-2.40 (14H, m), 1.80-1.40 (2
8H, m), 1.34 (9H, s)

[0185]

[Chemical 68]

[0186]

[Chemical 69]

Using 0.2 g of the above compound, 0.1 g of the desired product was obtained in the same manner as in Example 1- (2). (Yield 59.3%) Amino acid analysis value: Asp 6.82 (7) PhGly 8.18 (8) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.70-
7.20 (45H, m), 7.28 to 7.12 (40
H, m), 5.41 to 5.13 (8H, m), 4.80
Up to 4.60 (7H, m), 4.30 to 4.12 (7H,
m), 3.20 to 3.00 (14H, m), 2.80 to
2.40 (14H, m), 1.80-1.40 (28
H, m), 1.35 (9H, s)

[0188]

Example 10

[0189]

[Chemical 70]

[0190]

[Chemical 71]

Using 0.03 g of 0.23 g and AcOH, 0.09 g of the desired product was obtained in the same manner as in Examples 1- (1) and (2). (Yield 53.3%) Amino acid analysis value: Asp 5.04 (5) PhGly 5.96 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.64-
7.36 (33H, m), 7.36 to 7.05 (30
H, m), 5.55-5.28 (6H, m), 4.78
~ 4.60 (5H, m), 4.25 ~ 4.10 (5H,
m), 3.20 to 3.00 (10H, m), 2.80 to
2.40 (10H, m), 1.88 (3H, s), 1.
80-1.40 (20H, m)

[0192]

[Embodiment 11]

[0193]

[Chemical 72]

[0194]

[Chemical formula 73]

0.23 g and FCH ₂ COONa 0.00
Using 9 g, 0.15 g of the desired product was obtained in the same manner as in Examples 1- (1) and (2). (Yield 89.3%) Amino acid analysis value: Asp 5.04 (5) PhGly 5.96 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.69-
7.30 (33H, m), 7.30 to 7.06 (30
H, m), 5.60-5.20 (6H, m), 4.98.
Up to 4.60 (7H, m), 4.30 to 4.10 (5H,
m), 3.20 to 3.00 (10H, m), 2.80 to
2.40 (10H, m), 1.80-1.40 (20
H, m)

[0196]

[Example 12]

[0197]

[Chemical 74]

[0198]

[Chemical 75]

With 15.70 g

[0200]

[Chemical 76]

Using 7.70 g, and in the same manner as in Reference Example 6, 21.0 g of the desired product was obtained. (Yield 95.7%) Amino acid analysis value: Asp 2.89 (3) PhGly 4.11 (4) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.51-
7.65 (19H, m), 7.34 to 7.21 (40
H, m), 5.39 to 5.18 (4H, m), 5.10.
(8H, s), 4.78 to 4.64 (3H, m), 4.
35 to 4.25 (3H, m), 3.18 to 3.02 (6
H, m), 2.75 to 2.58 (6H, m), 1.75
~ 1.45 (12H, m), 1.34 (9H, s)

[0202]

[Chemical 77]

[0203]

[Chemical 78]

Using 0.30 g of the desired product, 0.50 g was obtained in the same manner as in Example 1- (2). (Yield 80.9
%) Amino acid analysis value: Asp 2.88 (3) PhGly 4.12 (4) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.54 to
7.60 (19H, m), 7.36 to 7.23 (20
H, m), 5.37 to 5.19 (4H, m), 4.75
~ 4.65 (3H, m), 4.25 ~ 4.15 (3H,
m), 3.20 to 3.02 (6H, m), 2.78 to
2.50 (6H, m), 1.80 to 1.40 (12H,
m), 1.35 (9H, s)

[0205]

Example 13

[0206]

[Chemical 79]

[0207]

[Chemical 80]

With 16.0 g

[0209]

[Chemical 81]

20.35 g of the above-mentioned desired product was obtained in the same manner as in Reference Example 6 using 5.64 g. (Yield 99.8%) Amino acid analysis value: Asp 3.91 (4) PhGly 5.12 (5) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.53〜
7.65 (25H, m), 7.33 to 7.20 (50
H, m), 5.39 to 5.18 (5H, m), 5.09
(10H, s), 4.78 to 4.62 (4H, m),
4.35 to 4.25 (4H, m), 3.18 to 3.02
(8H, m), 2.74-2.54 (8H, m), 1.
78 to 1.45 (16H, m), 1.34 (9H, s)

[0211]

[Chemical formula 82]

[0212]

[Chemical 83]

Using 0.10 g of the desired product, 0.38 g was obtained in the same manner as in Example 1- (2). (Yield 74.
4%) Amino acid analysis value: Asp 3.84 (4) PhGly 5.16 (5) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.54 to
7.60 (25H, m), 7.34 to 7.22 (25
H, m), 5.37-5.19 (5H, m), 4.78
~ 4.62 (4H, m), 4.26 ~ 4.14 (4H,
m), 3.12 to 3.00 (8H, m), 2.78 to
2.54 (8H, m), 1.80 to 1.40 (16H,
m), 1.35 (9H, s)

[0214]

Example 14

[0215]

[Chemical 84]

[0216]

[Chemical 85]

With 19.0 g

[0218]

[Chemical 86]

21.92 g of the desired product was obtained in the same manner as in Reference Example 6 using 5.22 g. (Yield 95.4%) Amino acid analysis value: Asp 4.76 (5) PhGly 6.24 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.53 ~
7.60 (31H, m), 7.34 to 7.21 (30
H, m), 5.39 to 5.18 (6H, m), 5.10.
(12H, s), 4.79 to 4.64 (5H, m),
4.37 to 4.25 (5H, m), 3.23 to 3.02
(10H, m), 2.67 to 2.50 (10H, m),
1.80-1.40 (20H, m), 1.34 (9H,
s)

[0220]

[Chemical 87]

[0221]

[Chemical 88]

Using 2.2 g of the desired product, 2.24 g was obtained in the same manner as in Example 1- (2). (Yield 90.8
%) Amino acid analysis value: Asp 4.90 (5) PhGly 6.10 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.51
7.60 (31H, m), 7.34 to 7.22 (30
H, m), 5.37-5.19 (6H, m), 4.76
Up to 4.64 (5H, m), 4.25 to 4.15 (5H,
m), 3.20 to 3.00 (10H, m), 2.78 to
2.54 (10H, m), 1.80 to 1.42 (20
H, m), 1.35 (9H, s)

[0223]

Example 15

[0224]

[Chemical 89]

[0225]

[Chemical 90]

Using 0.45 g and 0.013 ml of AcOH, 0.31 g of the desired product was obtained in the same manner as in Examples 1- (1) and (2). (Yield 90.2%) Amino acid analysis value: Asp 4.96 (5) PhGly 6.04 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.58 to
7.60 (31H, m), 7.35 to 7.21 (30
H, m), 5.52 to 5.19 (6H, m), 4.76.
~ 4.62 (5H, m), 4.26 to 4.16 (5H,
m), 3.12-3.00 (10H, m), 1.88
(3H, s), 1.78 to 1.44 (20H, m)

[0227]

Example 16

[0228]

[Chemical Formula 91]

[0229]

[Chemical Formula 92]

0.45 g and 0.022 g of glycolic acid
Was used in the same manner as in Examples 1- (1) and (2) to obtain 0.31 g of the desired product. (Yield 89.8%) Amino acid analysis value: Asp 4.90 (5) PhGly 6.10 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.74-
7.60 (31H, m), 7.38 to 7.21 (30
H, m), 5.55-5.19 (6H, m), 4.76
~ 4.64 (5H, m), 4.26 ~ 4.14 (5H,
m), 3.87 (2H, s), 3.12-3.00 (1
0H, m), 2.78-2.24 (10H, m), 1.
78-1.24 (20H, m)

[0231]

Example 17

[0232]

[Chemical formula 93]

[0233]

[Chemical 94]

100 mg was dissolved in 1 ml TFA and left at room temperature for 1.5 hours. The reaction solution was evaporated under reduced pressure, Et ₂ O was added to the residue for crystallization, and the residue was collected by filtration. This was dried under reduced pressure on sodium hydroxide to obtain 90 mg of the target product. (Yield 90.0%) Amino acid analysis value: Asp 5.03 (5) PhGly 5.97 (6) ¹ H-NMR (DMSO-d ₆ ) δppm: 8.80-
7.22 (34H, m), 7.28 to 7.08 (30
H, m), 5.44 to 5.12 (6H, m), 4.92
Up to 4.60 (5H, m), 4.28 to 4.12 (5H, m
m), 3.20 to 3.00 (10H, m), 2.80 to
2.40 (10H, m), 1.80-1.40 (20
H, m)

[0235]

Example 18

[0236]

[Chemical 95]

[0237]

[Chemical 96]

3.0 g

[0239]

[Chemical 97]

Using the same amount of 1.08 g as in Reference Example 6, 3.47 g of the desired product was obtained. (Yield 89.2%) Amino acid analysis value: Asp 4.00 (4) Ph (OH) Gly + PhGly 5.11 (1 + 4) ¹ H-NMR (DMSO-d ₆ ) δppm: 9.34
(1H, s), 8.75 to 7.40 (27H, m),
7.40-6.60 (44H, m), 5.42-5.0
0 (13H, m), 4.89 to 4.61 (4H, m),
4.38 to 4.22 (4H, m), 3.20 to 3.00
(8H, m), 2.80-2.40 (8H, m), 1.
80-1.40 (16H, m), 1.34 (9H, s)

[0241]

[Chemical 98]

[0242]

[Chemical 99]

0.33 g (yield 77.5%) of the desired product was obtained in the same manner as in Example 1- (2) using 0.5 g.

Amino acid analysis value: Asp 4.01 (4) Ph (OH) Gly + PhGly 5.07 (1 + 4) ¹ H-NMR (DMSO-d ₆ ) δppm: 9.45 to
9.25 (1H, m), 8.65 to 7.40 (27H,
m), 7.40-6.60 (24H, m), 5.55-
5.00 (5H, m), 4.80-4.60 (4H,
m), 4.30 to 4.10 (4H, m), 3.20 to
3.00 (8H, m), 2.80-2.40 (8H,
m), 1.80 to 1.20 (16H, m), 1.34
(9H, s)

[0245]

Example 19

[0246]

[Chemical 100]

[0247]

[Chemical 101]

2.0 g

[0249]

[Chemical 102]

Using the same amount of 0.50 g as in Reference Example 6, 2.40 g of the desired product was obtained. (Yield 97.6%) Amino acid analysis value: Asp 4.95 (5) Ph (OH) Gly + PhGly 6.12 (2 + 4) ¹ H-NMR (DMSO-d ₆ ) δppm: 9.35
(1H, s), 9.34 (1H, s), 8.63 to 7.
40 (33H, m), 7.40 to 6.58 (53H,
m), 5.40 to 5.00 (16H, m), 4.80 to
4.60 (5H, m), 4.40 to 4.20 (5H,
m), 3.20 to 3.00 (10H, m), 2.80 to
2.40 (10H, m), 1.88-1.40 (20
H, m), 1.33 (9H, s)

[0251]

[Chemical 103]

[0252]

[Chemical 104]

0.24 g (96.4%) of the desired product was obtained in the same manner as in Example 1- (2) using 0.3 g.

Amino acid analysis value: Asp 5.07 (5) Ph (OH) Gly + PhGly 6.04 (2 + 4) ¹ H-NMR (DMSO-d ₆ ) δppm: 9.45 to
9.22 (2H, m), 8.65 to 7.24 (33H,
m), 7.22 to 6.58 (28H, m), 5.58 to
4.97 (6H, m), 4.80 to 4.52 (5H,
m), 4.30 to 4.00 (5H, m), 3.20 to
3.00 (10H, m), 2.80 to 2.40 (10
H, m), 1.82-1.40 (20H, m), 1.3
4 (9H, s) Hereinafter, examples of pharmacological tests conducted on the peptide derivative of the present invention will be described.

[0255]

[Pharmacological test example 1] Angiogenesis inhibition test (1) Cultured endothelial cells (Fetal bovine heart ebdothelia)
Preparation of FBHE (FBHE) FBHE (ATCC CRL1395) is a 10% fetal bovine serum (FBS, Fetal) obtained from the American Type Culture Collection (ATCC).
Dulbecco's Modified Eagle medium (DME) modified with bovine serum, HEZLTON
Medium, manufactured by Nissui Pharmaceutical Co., Ltd., and bovine brain-derived basic fibroblast growth factor (bFGF, Bovine Basic Fibroblast Grow).
th Factor, manufactured by R & D) was subcultured and maintained in a culture medium containing 0.5 ng / ml of the factor.

FBHE cells were treated with bFG one day before the start of the experiment.
The cells were transferred to a 10% FBS-added DME culture solution without F (hereinafter, simply referred to as a culture solution). 0.1% at the start of the experiment
After removing the cells with EDTA-0.2% trypsin solution and centrifuging and washing with the culture solution, the number of viable cells was counted with a hemocytometer, and the culture solution was adjusted so that the cell concentration was 3 × 10 ⁴ cells / ml. Was prepared using. Thus, a cell suspension was prepared.

(2) Preparation of test compound The test compound was a sodium salt in physiological saline or phosphate buffered saline (PBS ^(-) , Phosphate Buffer Salin ⁾ .
e, manufactured by Nissui Pharmaceutical Co., Ltd.), and then diluted with a culture solution so that the final concentration becomes a maximum concentration of 10 μg / ml to 100 μg / ml, and then sterilized by filtration (0.2 μm, It was used as a Millex GV filter manufactured by Millipore).

(3) Experimental procedure The following procedure was carried out. That is, first, 50 μl each of the culture solution was dispensed into a 96-well microplate (manufactured by Corning), and then 50 μl of the test compound solution diluted with the culture solution to the desired final maximum concentration A dilution series was prepared by placing the cells in wells and performing 2-fold serial dilution on the plates. In addition, 50 μl of each well,
A culture solution containing bFGF (0.4 ng / ml) or a culture solution containing no bFGF was added, and finally 1 cell suspension was added to each well.
00 μl was added to each. Final volume of each well is 200μ
l, bFGF final concentration 0.1 ng / ml, FBHE
The number of cells was 3 × 10 ³ / well.

The plate prepared above was incubated at 37 ° C. in a 5% carbon dioxide gas incubator for 3 days.

On the 3rd day of culture, 50 μl of 0.05% neutral red solution was added to each well, and the mixture was further incubated at 37 ° C. for 2 hours.
Then, the cells were placed in a 5% carbon dioxide gas incubator and the dye was incorporated into the cells. Thereafter, 50 μl of 1% glutaraldehyde was added to each well, and the cells were fixed at room temperature for 15 minutes. The liquid in each well was discarded, washed twice with physiological saline, drained, and 100 μl of acid alcohol (1: 1 mixture of 0.1 molar monosodium phosphate aqueous solution and ethanol) was added to each well. , The dye incorporated into the cells was eluted, and the absorbance of each well was measured with an absorptiometer (wavelength 540 nm).

(4) Judgment of effect and calculation of IC ₅₀ value Absorbance values measured according to the above (3) are graphed for each of bFGF addition (FGF ⁺ , proliferation state) and no bFGF addition (FGF ⁻ , normal state). And the growth of cultured endothelial cells (FBHE) was plotted from the graph.
The concentration of each test compound, which suppresses 0%, was calculated and used as an IC _50.
The value (μg / ml) was used.

Table 1 shows the results obtained by using the compounds of the present invention obtained in each of the above Examples as test compounds.

[0263]

[Table 1]

From Table 1, all the compounds of the present invention are bFG.
It was found to have a highly active and selective inhibitory effect on the proliferation of endothelial cells induced in the presence of F.

From the above, the compound of the present invention is used for cancer or tumor associated with angiogenesis, or a disease associated with angiogenesis caused by abnormal growth of blood vessels, such as angiofibroma, arteriovenous malformation, Osler-Weber syndrome, Vascular adhesion, atherosclerotic plaque, corneal transplant angiogenesis, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity (posterior lens fibroplasia), trachoma, delayed wound healing, granulation associated with burns, hypertrophy For scars, pyogenic granulomas, psoriasis, sclerosis, non-union fractures, rheumatoid arthritis, hemophilic arthropathy, etc., the preventive and therapeutic effects of angiogenesis can be exerted, and side effects It turns out that there are few.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical display location A61K 37/02 ADU // C07K 99:00 (72) Inventor Yasuo Yanagihara 10 Hasuike-cho, Otsu City, Shiga Prefecture No. 45 (72) Inventor Tadashi Okada 22-20 Kayanoura, Otsu, Shiga Prefecture (72) Inventor Takeshi Imaoka 2-7-303 Misoracho, Otsu City, Shiga Prefecture (72) Midori Tanaka Mibu, Nakagyo Ward, Kyoto City 9 No. 9 Palm-no-Miyamachi (72) Inventor Mitsuru Hirohashi 2-2 Hanazonocho, Otsu City, Shiga Prefecture

Claims (3)

[Claims] 1. A general formula: [Wherein R ¹ represents a hydrogen atom, a lower alkoxycarbonyl group or a lower alkanoyl group which may have a group selected from the group consisting of a hydroxyl group and a halogen atom as a substituent,
R ² is a hydroxyl group, an amino group or a group -Gly-OH, l and m is an integer of 0 or 1-8. However, l + m is 3 to 8
And Further, each amino acid residue in the formula represents a D-form, an L-form or a D, L-isomer mixture. ] The peptide derivative represented by these, or its pharmaceutically acceptable salt. 2. A peptide derivative represented by the general formula according to claim 1, wherein each amino acid residue represents an L-form, or a pharmaceutically acceptable salt thereof. 3. At least one selected from the peptide derivative according to claim 1 or 2 and a pharmaceutically acceptable salt thereof.
An angiogenesis inhibitor containing a seed as an active ingredient.