JPS61227554A - Amino acid derivative and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R<1> is H, amino or protected amino; R<2> is H, carboxy or protected carboxy; R<3> is H, carboxy or protected carboxy; R<4> is lower alkyl, lower aminoalkyl, etc.; R<5> is H or lower alkyl; R<6> is H, lower aralkyl, etc.). EXAMPLE:(2S,3R)-2-[[ (2S)-2-Amino-3-carbamoylpropionyl ]amino]-3-[[(1S)-1-car-boxy-2-(4-imidazolyl)ethyl]amino]butyric acid. USE:An agent effective to recover the colony-forming and antibody-forming activity of a host having immunodeficiency and to inhibit the metastasis of tumor. PREPARATION:The compound of formula I can be produced by reacting the compound of formula II or its reactive derivative at the carboxyl group with the compound of formula III or its reactive derivative at the amino group in a solvent under cooling or at room temperature.

Description

[Detailed description of the invention] This invention relates to novel amino acid derivatives and salts thereof.

More specifically, this invention has pharmacological effects such as the effect of restoring the reduced colony forming ability of bone marrow cells of an immunodeficient host, the effect of restoring the reduced antibody formation ability of an immunodeficient host, the effect of inhibiting metastasis of ll+1 tumor, etc. The present invention relates to novel amino acid derivatives, salts thereof, methods for producing the same, and pharmaceutical compositions containing the same.

Therefore, one object of this invention is to provide novel amino acid derivatives and salts thereof.

Another object of the present invention is to provide a method for synthesizing the amino acid derivative and its salt, and a method for producing a specific compound among the compounds by culturing bacteria belonging to the genus Discosia in a medium. including fermentation methods,
An object of the present invention is to provide a method for producing the amino acid derivative and its salt.

The object compound of this invention can be represented by the following formula (I).

[Wherein, R1 is hydrogen, amino or a protected amino group, R2 is hydrogen, carboxy or a unprotected carboxy group, R3 is hydrogen, carboxy or a protected carboxy group, R4 is lower alkyl, amino (lower) alkyl,
Protected amino (lower) alkyl, carbamoyl (lower) alkyl or protected carbamoyl (lower) alkyl, R is hydrogen or lower alkyl, R6 is hydrogen, alk (lower) which may have a suitable substituent Alkyl or heterocyclic (lower) alkyl. ] According to this invention, the novel amino acid derivative (I) can be produced by various production methods.

[Manufacturing by synthesis] 1 yield R-CH-C0OH (I[) or its reactive derivative at the carboxy group or its salt + (I[a) or its reactive derivative at the amino group or its salt ↓ (I) or a salt thereof (Ia) or a salt thereof (!b) or a salt thereof (Ic) or a salt thereof (Id) or a salt thereof (Is) or a salt thereof or a salt thereof [in the above formula, R1, R2, R3, R4, R5 and R6
are each as defined above, R1 is a protected amino group,
R2 is a protected carboxy group, R4 is a protected carbamoyl (lower) alkyl, and R4 is a carbamoyl (lower) alkyl. ] Starting compound (II[a) can be produced by the following production method.

(IV) (1/) or its salt or its salt 11 (dan (1 (VI) or its salt (I[) or its salt (II[b) or its salt (11[) a) or a salt thereof [in the above formula, R2, R3, R5 and R6 each have the same meaning as above, R7 is an amino or protected amino group,
R7 is a protected amino group.] Hereinafter, the method for producing the object compound of the present invention will be explained in detail.

Compound (I) or a salt thereof is obtained by reacting compound (I) or a reactive derivative thereof at a carboxy group or a salt thereof with a compound <Il[a) or a reactive derivative thereof at an amino group or a salt thereof. It can be manufactured by

Suitable reactive derivatives at the amino group of compound (II[a) include Schiff base-type iminos or their enamine-type derivatives formed by the reaction of compound (n[a) with carbonyl compounds such as aldehydes, ketones, etc. Mutant, compound (I[a) and N,0-bis(trimethylsilyl)acetamide, N-trimethylsilylacetamide,
Silyl derivatives, compounds (I
Examples include derivatives produced by the reaction of I[a) with phosphorus trichloride or phosgene.

Suitable salts of compounds (II) and (I[a) include:
Those exemplified for compound (1) can be mentioned.

Suitable reactive derivatives of the carboxy group of compound (I[) include acid halides, acid anhydrides, activated amides, activated esters, and the like. Suitable examples include acid chlorides, acid azides such as dialkyl phosphates,
Substituted phosphoric acids such as phenyl phosphoric acid, diphenyl phosphoric acid, dibenzyl phosphoric acid, halogenated phosphoric acid, dialkyl phosphorous acids, sulfites, thiosulfuric acids, lower alkanesulfonic acids such as methanesulfonic acid, ethanesulfonic acid, sulfuric acid, Mixed acid anhydrides with acids such as alkyl carbonates, aliphatic carboxylic acids such as pivalic acid, pentanoic acid, impentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, or aromatic carboxylic acids such as benzoic acid, symmetrical Activated amides with acid anhydrides, imidazoles, 4-substituted imidazoles, dimethylpyrazoles, triazoles or tetrazoles, such as cyanomethyl esters, methoxymethyl esters,
dimedeliminomethyl [(C)I3)2N-CH-1 ester, vinyl ester, propargyl ester, p-
Nitrophenyl ester, 2,4-cy=).

Phenyl ester, trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester,
Phenylazophenyl ester, phenylthioester, p-nitrophenylthioester, p-talesylthioester, carboxymethylthioester, pyranyl ester, pyridyl ester, piperidyl ester, 8-
Active esters such as quinolyl thioester, e.g. N, N
-dimethylhydroxylamino, 1-hydroxy-2-
(IH)-pyridone, N-hydroxysuccinimide,
Examples include esters with N-hydroxy compounds such as N-hydroxyphthalimide and 1-hydroxy-6-chloro-IH-benzotriazole. These reactive derivatives can be appropriately selected from among them depending on the type of compound (I[) used.

The reaction is usually carried out in a conventional solvent such as water, acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or any other solvent that does not adversely affect the reaction. carried out in a solvent. These conventional solvents may be used in combination with water.

When compound (II) is used in the reaction in the form of free acid or in the form of a salt, the reaction can proceed with N,N-dicyclohexylcarbodiimide, N-cyclohexyl-N'-morpholinoethylcarbodiimide, N-cyclohexyl-N '-(4-
diethylaminocyclohexyl)carbodiimide, N,
N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide, N-ethyl-N'-(3-
dimethylaminopropyl) carbodiimide, N, N'
-Carbonylbis(2-methylimidazole), pentamethyleneketene-N-cyclohexylimine, diphenylketene-N-cyclohexylimine, ethoxyacetylene, 1-alfoxy-1-chloroethylene, trialkyl phosphite, ethyl polyphosphate, polyphosphoric acid Isopropyl, phosphorus oxychloride, phosphorus trichloride, thionyl chloride, oxalyl chloride, triphenylphosphine, 2-ethyl-
7-hydroxybenzisoxazolium salt, 2-ethyl-5-(m-sulfophenyl)inxazolium hydroxide inner salt, 1-(p-chlorobenzenesulfonyloxy)-6-chloro-IH-benzotriazole, N
, N-dimethylformamide and thionyl chloride, phosgene, phosphorous oxychloride, etc., in the presence of a conventional condensing agent such as the so-called Vilsmeier reagent.

The reaction also includes alkali metal bicarbonates, tri(lower)
May be carried out in the presence of an inorganic or organic base such as alkylamino, pyridine, N-(lower)alkylmorpholine, N,N-di(lower)alkylbenzylamino, etc.
The degree of reaction is not particularly limited, and the reaction is usually carried out under cooling or at room temperature.

The main compound (Ib) or a salt thereof can be produced by subjecting the compound (Ia) or a salt thereof to an amino-protecting group elimination reaction.

Suitable salts of compounds (Ia) and (Ib) include:
Those exemplified for compound (1) can be mentioned.

The elimination reaction can be carried out by hydrolysis, reduction, or a method in which the compound (Ia) in which R1 is acylamino is treated with an iminohalogenating agent or an iminoetherifying agent, and then the obtained substance is hydrolyzed if necessary. It is carried out according to common law. Examples of hydrolysis include methods using acids, bases, hydrazine, and the like.

This method may be selected from among them depending on the type of protecting group to be removed.

Among these methods, hydrolysis using an acid is one of the most common methods. Preferable removal of protective groups such as aralkyl, which may be substituted with alkoxycarbonino, such as aralkyl such as trityl, substituted phenylthio, substituted aralkylidene, substituted alkylidene, substituted cycloalkylidene, etc. It is divorce.

Suitable acids include organic and inorganic acids such as formic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, hydrochloric acid, hydrobromic acid, boron trihalides such as boron trifluoride, boron tribromide, etc. acid or Lewis acid.

The acid can be selected depending on the type of protecting group to be removed. When performing an elimination reaction with an acid, the reaction can be performed in the presence or absence of a solvent. Suitable solvents include water, conventional organic solvents or mixtures thereof.

Elimination reactions using trifluoroacetic acid can be carried out in the presence of anisole. Hydrolysis with hydrazine is commonly used to remove phthaloyl and succinyl type amino protecting groups.

Hydrolysis using a base is preferably used to remove the acyl group; suitable bases include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, etc., such as magnesium hydroxide, calcium hydroxide, etc. alkaline earth metal hydroxides such as alkali metal carbonates such as sodium carbonate and potassium carbonate; alkaline earth metal carbonates such as magnesium carbonate and calcium carbonate; alkali metal carbonates such as sodium bicarbonate and potassium bicarbonate; Hydrogen salts, alkali metal acetates such as sodium acetate and potassium acetate, alkaline earth metal phosphates such as magnesium phosphate and calcium phosphate, alkali metal hydrogen phosphates such as disodium hydrogen phosphate and dipotassium hydrogen phosphate. and trialkylaminos such as trimethylamino, triethylamino, etc.
Pyrin, N-methylpyrrolidone, N-methylmorpholine, 1,5-diazabicyclo[4,3,0]]su-5
-ene 1.4-diazabicyclo[2°2.2]octane, 1.5-diazabicyclo[5°4.0]undecene-
Organic bases such as 5 and the like can be mentioned. Hydrolysis with a base is often carried out in water or a hydrophilic organic solvent or a mixture thereof.

Reductive elimination is generally used to remove protecting groups such as optionally substituted aralkoxycarbonylmethoxycarbonyl, such as trichloroethquinecarbonyl, eg benzyloxycarbonyl.
Suitable reduction methods include reduction with alkali metal borohydrides, such as sodium borohydride, e.g. tin,
Reduction by a combination of metals such as zinc and iron or metal salt compounds such as chromium chloride and chromium acetate with organic or non-corrugated acids such as acetic acid, propionic acid, and hydrochloric acid, and catalytic reduction. I can list the following. Suitable catalysts include, for example, Raney nickel, platinum oxide, elemental palladium II, and the like.

Among protecting groups, acyl groups are generally removed by hydrolysis. In particular, halogen-substituted alkoxycarbonyl and 8
The -quinolyloxycarbonyl group is usually removed by treatment with a heavy metal such as ti4 or zinc.

Among the protecting groups, the acyl group may be treated with an iminohalogenating agent such as phosphorous oxychloride and an iminoetherifying agent such as lower alkanols such as methanol, ethanol, etc., followed by hydrolysis if necessary. It can be removed by

The reaction behavior is not particularly limited and may be appropriately selected depending on the protecting group of the amino group and the type of the elimination method, and it is preferable to carry out the reaction under mild conditions such as cooling or a slight temperature rise.

This invention provides that the protected amino (lower) alkyl and/or the protected carbamoyl (lower) alkyl in R4 is converted into amino ( The scope also includes cases where it can be converted into (lower) alkyl and/or carbamoyl (lower) alkyl.

11.1 Compound (Id) or a salt thereof can be produced by subjecting compound (Ic) or a salt thereof to an elimination reaction of a carboxy protecting group.

Suitable salts of compounds (Ic) and (Id) include those exemplified for compound (I).

This invention is carried out according to conventional methods such as hydrolysis, reduction, etc.

When the protecting group is an ester, it can be removed by hydrolysis. The hydrolysis is preferably carried out in the presence of a base or an acid; suitable bases include, for example, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium and calcium, their hydroxides, carbonates or Bicarbonate, for example trialkylamino such as trimethylamino, triethylamino, pilin, 1,5-diazabicyclo[4,3,0]]non-5-ene 1,4-diazabicyclo[2°2.2]octane, 1 Mention may be made of inorganic bases and organic bases such as .8-diazabicyclo[5°40]undecene-7 and the like. Suitable acids include organic acids, such as formic acid, acetic acid, propionic acid, trifluoroacetic acid, and inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid.

Acidic hydrolysis using trifluoroacetic acid is usually accelerated by the addition of anisole.

The reaction typically involves water, methylene chloride, e.g. methanol,
solvents such as alcohols such as ethanol, mixtures thereof;
or in any other solvent that does not adversely affect the reaction. Liquid bases or acids can also be used as solvents.The reaction temperature is not particularly limited, and the reaction is usually carried out at
It is carried out under cooling or heating.

Reduction includes 4-nitrobenzyl, 2-iodoethyl, 2.
It is preferable to use it for removing a protecting group such as 2.2-trichloroethyl.

Reduction is carried out by conventional methods including chemical reduction and catalytic reduction.

Suitable reducing agents used in the chemical reduction include metals such as tin, zinc, iron or metal compounds such as chromium chloride, chromium acetate and the like and formic acid, acetic acid, propionic acid, trifluoroacetic acid, p - in combination with organic or inorganic acids such as toluenesulfonic acid, hydrochloric acid, hydrobromic acid, etc.

Suitable catalysts used in the catalytic reduction are platinum catalysts such as platinum plates, platinum sponges, platinum black, colloidal platinum, platinum oxide, platinum wire, such as palladium sponges, palladium black, palladium oxide, palladium on carbon, colloidal palladium, palladium. - [Palladium catalyst such as barium acid, palladium-barium carbonate, nickel Mi medium such as reduced nickel, nickel oxide and Raney nickel, reduced cobalt,
Cobalt catalysts such as Raney cobalt, reduced iron, iron catalysts such as Raney iron, copper catalysts such as reduced steel, Raney copper, Ullmann steel, and the like are conventional.

The reduction is usually carried out in water, alcohols such as methanol, ethanol, solvents such as N,N-dimethylformamide, tetrahydrofuran, mixtures thereof, or any other solvent that does not adversely affect the reaction.

Furthermore, when the above-mentioned acids used for chemical reduction are in liquid form, these acids can also be used as solvents.

The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling or heating.

The compound (If> or a salt thereof can be produced by subjecting the compound (Ia) or a salt thereof to an elimination reaction of the carbamoyl protecting group.

Suitable salts of compounds (Is) and (If) include those exemplified for compound (1).

This elimination reaction can be performed in the same manner as Production Method 2.

The method for producing the starting compound will be explained in detail below.

Preparation jI group Δ Compound (V) or a salt thereof can be produced by subjecting compound (IV) or a salt thereof to an oxidation reaction. Suitable salts of compounds (IV) and (Vl) include:
Those exemplified for compound (I) can be mentioned.

Oxidation methods that can be used for this reaction include oxidation using a combination of dimethyl sulfoxide and N,N'-dicyclohexylcarbodiimide and an acid such as orthophosphoric acid or trifluoroacetic acid, oxidation using a chromic anhydride-pyridine complex, etc. can be mentioned.

This reaction is usually carried out in a solvent such as an alcohol such as methanol, ethanol, methylene chloride, benzene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether or any other solvent that does not adversely affect the reaction. It will be done.

The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling or heating.

Compound (III) manufactured by JD Kudan or a salt thereof can be produced by reacting compound (V) or a salt thereof with compound (■) or a salt thereof and subjecting the obtained compound to a reduction reaction.

Suitable salts of compounds (II) and (VW) include those exemplified for compound (I). Reduction methods that can be used in this reaction include, for example, reduction with borane derivatives such as sodium borohydride and sodium cyanoborohydride, metals such as tin, zinc, iron, and the like, as well as, for example, chromous chloride.
Mention may be made of reduction using a combination of a metal salt compound such as monochromic acetate and an organic or inorganic acid such as, for example, acetic acid, propionic acid, hydrochloric acid, and catalytic reduction.

Suitable catalysts include the conventional ones such as Raney nickel, platinum oxide, palladium on carbon and the like.

This reaction is usually carried out in a solvent such as an alcohol such as methanol, ethanol, benzene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether or any other solvent that does not adversely affect the reaction.

The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling or heating.

Production method C Compound (Ha) or a salt thereof can be produced by subjecting compound (II[b) or a salt thereof to an amino-protecting group elimination reaction. Suitable salts of compound (lrb) include those exemplified for compound (1).

This reaction is carried out during the above elimination reaction or during post-treatment of the reaction mixture or reaction product.
The scope also includes the case where the protected carboxyne group represented by is converted to a free carboxy group.

[Production by Fermentation] Among the target compounds of this invention, one specific compound (hereinafter referred to as FR-900490) can be produced by fermentation. The FR-900490 has the following physicochemical properties.

(a) Appearance: White powder (b) Decomposition point: 172-174°C (c) Specific optical rotation: [α]: 3 = +17.5° (C = 1.0. R20)
(d) Molecular weight: 370 [SIMS: m/z 371 (M +
1) ] (e) Elemental analysis (2): C40,55i H6,19: N 20.45 (f)
UV absorption spectrum: ・Terminal absorption (R20
(middle) (g) IR absorption spectrum: KBr: 3400.2920.1680.1620.
1390゜υMax 1100 cm-' (h) IHNMR absorption spectrum: (D20) Sp
pm: 8.01 (LH,s), 7.17 (I
H, s), 4.44 (LH, d, J=5.0Hz>,
4.24 (LH, dd.

J=4.95Hz and 7.91Hz), 3.99(
LH, dd, J = 5.3Hz and 8.2Hz).

3.55 (IH, m), 3.30 (IH, dd
, J=5.3Hz and 15.0Hz), 3.16
(IH, dd.

J:8.2Hz and 15.0Hz), 2°96(I
H, dd, J = 4.95 Hz and ta, 5 Hz).

2.83 (IH, dd, J=7.91Hz and 16
．． 5Hz), 1.16 (3H, d, J=6.6H
z) (i) CNMR absorption spectrum: (D20)1
;ppm: 174.3 (s), 174.3
(s), 174.2 (s).

171.1 (s), 136.4 (d), 1
31.7 (s).

117.5 (d), 60.2 (d), 56
．． 1 (d).

53.6 (d), 51.0 (d), 36.
7 (t).

28.3 (t), 12.5 (q) (j) Solubility: Soluble: Water l! l Soluble: methanol, ethanol insoluble
: Ethyl acetate, chloroform, acetone (k) Color reaction: Positive: ninhydrin reaction, iodine vapor reaction, cerium sulfate reaction, potassium permanganate reaction Negative: ferric chloride reaction, Dragendorff reagent reaction, Molisch reaction, Phosphomolybdic acid reaction (1) Characteristics of the substance: Amphoteric substance (ffi) Thin layer chromatography (silica gel plate): L
U anal plate n-butyl alfur: acetic acid: water 0.27 (2:
1: 2) n-butyl alcohol: ethanol: chloroform: 28X ammonia water 0.22 (2+
2:1:2) 70X isopropyl alfur aqueous solution (contains 7% = 70.28X) 0.25F
R-900490 is Discosia Sp. F-1
1809 (Diseosia sp, F-11809
FR-90049 belonging to the genus Discosia such as ) etc.
0-producing bacteria in a medium, and from the culture, FR-900
It can be manufactured by collecting 490.

Among the FR-900490-producing bacteria belonging to the genus Discosia, Discosia sp. F-11809 is a newly isolated bacteria by the inventors from a soil sample collected at the foot of Mt. Takao, Kyoto Prefecture.

Newly isolated Discosia sp. F-118
The freeze-dried sample of No. 09 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture) on January 25, 1985, with the accession number: Microbiological Research Institute No. 80.
No. 70, and then on February 8, 1986, it was transferred to the same depositary's Budapest Treaty route with a new accession number: FIKEN Article No. 982.

The production of the new compound FR-900490 is not limited to the use of the specific microorganisms described here;
This is for illustration purposes only. This invention also relates to X-rays, UV irradiation, N-methyl-N'-nitro-N
- All FR90 including natural mutants as well as artificial mutants produced from the above-mentioned microorganisms by conventional methods such as treatment with nitrosoquanidine, 2-7 minoprine, etc.
This includes the use of 049G-producing mutant bacteria.

Discosia sp. F-11809 has the following morphological, cultural, and physiological characteristics.

Sexual reproductive forms do not develop on various media, but hyphal conidial forms are abundantly observed on cornmeal/agar media. In addition, leaf pieces inoculated with conidia of strain F-11809 (L
A large number of conidia were formed in the eaf segment). Conidiation mode is holo-budding type (holoplastic)
And the conidia are monogenetic.

The conidial form (conidiophores) is immersed, separated or aggregated in the substrate, forming a flat fawn, papillary, 200-40 mm in diameter.
0P, height 40-80-0 hole opening diameter 20-50Pm
, the conidial wall consists of several layers of dark brown cells.

Conidiophores are not formed. 0 Conidiogenic cells are formed in the lower layer of the inner wall of the conidiophores, and are obverted to piriform or conical in shape, with a length of 7 to 10 P1 and a width of 4 to 5%, tapering to the width of the apex. 2-0 Conidia pale, smooth, cylindrical or S-shaped, 3
It has two partition walls, the base is cut-shaped, and the length is 15 to 22 (~
25)-1 Width 2.5 to 3.5-0 Their apical and proximal cells each have attached threads near the septum. These accessory threads are unbranched, filamentous, and 12-20 (~3
0)-1 width is 0.5 to 1-.

The vegetative hyphae are septated, pale to dark brown, smooth, and branched. The hyphal cells are cylindrical or barrel-shaped, 1.5 to 4 P wide, and do not form chlamydospores, but a dense tissue of seriated hyphae consisting of irregular cells is formed on some media.

Colonies on malt extract agar were widely dispersed and reached a diameter of 8.5 mm after 2 weeks of culture at 25'C. Qcm has been reached. The colony surface is flat and felt-like, with wrinkles in the center, and the oconidia structure, which grows in a circular shape and is light olive gray or olive gray, cannot be formed.The color of the underside is olive black or dark olive. Growth on Hoonmeal agar cultured under the same conditions reached a diameter of 7.5 an. The surface was flat, thin, felt-like, and grayish yellowish brown to brownish gray. The underside was also the same color. Single conidia were located at many locations. dark brown hyphal clusters are formed in or on the surface of the agar medium.

F-11809 strain can grow in a temperature range of 2 to 34°C, and the optimum growth temperature is 24 to 31°C. These temperature data were measured on potato dextrose agar using a temperature gradient incubator (Toyo Kagaku Sangyo Co., Ltd.). This strain can grow at pH 3-9 and exhibits optimum growth at 6-7 pi in YW broth medium (Diff).

Based on the above characteristics, strain F-11809 is a member of the deficient filamentous fungus Discosia Libert.
) seems to belong to the genus. This genus includes Subramanian and Chandra-Reddy [Ka
vaka), 2:57-89.19741, it can be classified into five known species and three comparative species based on conidial characteristics.

According to this criterion, our strain is Discosia strobilina liberto (Discosia 5trobili).
naLibert), but its conidial characteristics (i.e., the position of conidial appendages, etc.) often change depending on culture conditions. Therefore, strain F-11809 was determined to be a strain of the genus Discosia, and Discosia sp.
It was named 11809.

Usually, FR-900490 is produced by culturing FR-900490-producing bacteria in a medium containing an assimilable carbon source and nitrogen source, preferably under aerobic conditions such as shaking culture or submerged culture. be able to.

Preferred carbon sources for the medium are glucose, fructose,
carbohydrates such as glycerin, and starch. Other carbon sources that may be mentioned are lactose, arabinose, xylose, dextrin, molasses and the like.

Preferred nitrogen sources include yeast extract, peptone, gluten flour, cottonseed flour, soy flour, corn steep liquor, dried yeast, etc., as well as ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium phosphate, urea, amino acids, etc. Inorganic and organic nitrogen compounds.

Carbon and nitrogen sources, although advantageously used in combination, do not necessarily need to be used in pure form, as less pure materials containing trace amounts of growth factors and significant amounts of inorganic nutrients are also suitable for use. If desired, inorganic salts such as calcium carbonate, sodium or potassium phosphate, sodium or potassium iodide, magnesium salts, cobalt chloride etc. may be added to the culture medium, if necessary, especially for culture If the medium foams significantly, antifoaming agents such as liquid paraffin, higher alcohols, vegetable oils, mineral oils and silicones may be added.

Deep aerobic culture conditions are the conditions for mass production of FR-
The preferred small volume production of 900490 uses shaking or surface culture carried out in flasks or bottles. Furthermore, when carrying out growth in a large tank, it is preferable to inoculate the bacteria into the production tank using a pre-culture of the microorganism in order to avoid growth delays in the production process of FR-900490. That is, a relatively small amount of culture medium is inoculated with microbial spores or hyphae, the inoculated medium is cultured to first produce a microbial preculture inoculum, and then the cultured preculture inoculum is aseptically transferred to a large tank. The medium producing this preculture inoculum that is desired to be transferred may be substantially the same as the medium used to produce FR-900490, or it may be different.

Agitation and aeration of the culture mixture can be accomplished in a variety of ways. For stirring, use a propeller or similar stirring device, rotate a fermenter, or shake the fermenter.
Aeration may be performed by passing sterile air through the fermentation mixture, which can also be done using various pump devices or by passing sterile air through the medium.

Fermentation is usually carried out at a temperature range of about 20° C. to 40° C., preferably 25° C. to 30° C., for 50 to 100 hours, but the temperature may be changed as appropriate depending on the fermentation conditions and fermentation scale.

Finished production of FR-900490 can be recovered from the culture medium by conventional methods commonly used for recovery of other fermentation products such as antibiotics.

Usually, most of the produced FR-900490 is found in the culture filtrate, and therefore, FR-900490 can be extracted from the filtrate obtained by filtering or centrifuging the culture broth by vacuum concentration, lyophilization, and conventional solvents. Extraction used, pH adjustment,
For example, treatment with conventional resins such as anion or cation exchange resins, nonionic adsorption resins, treatment with adsorbents such as activated carbon, silicic acid, silica gel, cellulose, alumina, etc., crystallization, recrystallization, etc. are used for recovery. be able to.

Based on the above physical and chemical property analysis and other study results for chemical structure confirmation, the chemical structure of FR-900490 was identified and determined as follows.

(2S,3R)-2-[[(2S)-2-amino-3-
carbamoylpropionylcoamino]-3-[[(IS
)-t-carboxy-2-(4-imidazolyl)ethyl]aminocoacetic acid Suitable pharmaceutically acceptable salts of the target compound (I) are:
customary non-toxic salts, such as alkali metal salts such as sodium salts, potassium salts, and alkaline earth metal salts such as calcium salts, magnesium salts;
Ammonium salts, such as trimethylamino salts, triethylamino salts, pyridine salts, pilin salts, dicyclohexylamino salts, organic base salts such as N,N'-dibenzylethylenediamino salts, such as acetate, maleate, tartrate, methane Organic acid salts such as sulfonates, benzenesulfonates, formates, toluenesulfonates, inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, phosphates, or e.g. arginine, aspartic acid. , salts with amino acids such as glutamic acid, and the like.

In the foregoing and following description of this specification, pertinent illustrations of the various definitions encompassed within the scope of this invention are set forth in detail below.

The term "lower" used herein means 1 to 6 carbon atoms, unless otherwise specified.

The appropriate ``protected amino moiety'' of ``r-protected amino (lower) alkyl'' and the appropriate ``protected amino'' herein include an acylamino group or an appropriate substituent such as benzyl or trityl. Examples include amino groups substituted with conventional protecting groups such as al(lower)alkyl, which may have more than one.

Suitable acyl moieties of "acylamino" can include carbamoyl, aliphatic acyl groups and acyl groups having aromatic or heterocycles. Suitable examples of the acyl include lower alkanoyl such as formyl, acetyl, propionyl, butyryl, imbutyryl, valeryl, invaleryl, oxalyl, succinyl, pivaloyl, e.g. methoxycarbonylcarbonyl, propoxycarbonyl, 1-cyclopropylethoxy Lower alkoxycarbonyl such as carbonyl, imbroboxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl, lower alkanesulfonyl such as mesyl, ethanesulfonyl, propanesulfonyl, isopropanesulfonyl, butanesulfonyl, Arenesulfonyl such as benzenesulfonyl, tosyl, aroyl such as benzoyl, toluoyl, xyloyl, naphthoyl, phthaloyl, indancarbonyl, al(
(lower) alkanoyl, such as al(lower) alkoxycarbonyl such as benzyloxycarbonyl and phenethyloxycarbonyl. The above acyl moiety may have at least one or more appropriate substituents such as halogens such as chlorine, bromine, fluorine, and iodine.

Suitable "protected carboxy" may include esterified carboxy and the like.

Suitable examples of the esterified carboxy ester moiety include, for example, acetoxymethylester, propionyloxymethyl ester, butyryloxymethyl ester, valeryloxymethyl ester, pivaloyloxymethyl ester, hexanoyloxymethyl ester, 1(or 2)-acetoxyethyl ester, 1(
or 2 or 3)-acetoxypropyl ester, 1
(or 2 or 3 or 4)-acetoxybutyl ester, 1 (or 2)-propionyloxyether ester, 1 (or 2 or 3)-propionyloxypropyl ester, 1 (or 2)-butyryloxyethyl ester, 1 (or 2)-isobutyryloxyethyl ester, 1 (or 2)-pivaloyloxyethyl ester, 1 (or 2)-hexanoyloxyethyl ester, imbutyryloxymethyl ester, 2-ethylbutyryl Lower alkanoyloxy (lower) alkyl esters such as oxymethyl ester, 3゜3-dimethylbutyryloxymethyl ester, 1 (or 2)-pentanoyloxyethyl ester, lower alkanesulfonyl (lower) such as 2-mesylethyl ester, etc. ) alkyl esters, e.g. 2-iodoethyl ester, 2.2
．． Mono (or di or tri) halo (lower) alkyl esters such as 2-trichloroethyl ester, e.g. methoxycarbonyloxymethyl ester, ethoxycarbonyloxymethyl ester, 2-methoxycarbonyloxyethyl ester, 1-ethoxycarbonyloxyethyl ester , 1-isopropoxycarbonyloxyethyl ester, etc. (7) Lower alkoxycarbonyloxy (lower) alkyl ester, or phthalidylidene (
at least one suitable substituent such as a lower) alkyl ester, etc.
For example, methyl ester, ethyl ester, propyl ester, isopropyl ester, which may have more than one
Lower alkyl esters such as butyl ester, isobutyl ester, t-butyl ester, pentyl ester, hexyl ester, 1-cyclopropylethyl ester;
For example, lower alkenyl esters such as vinyl esters and allyl esters; lower alkynyl esters such as ethynyl esters and propynyl esters; for example, benzyl esters, 4-methoxybenzyl esters, 4-nitrobenzyl esters, phenethyl esters, trityl esters, benzhydryl esters, Examples which may have at least one suitable substituent such as bis(methoxyphenyl)methyl ester, 3,4-dimethoxybenzyl ester, 4-hydroxy-3,5-di-t-butylbenzyl ester, etc. Mono (or di or tri) phenyl (
(lower) alkyl esters such as phenyl esters, 4-chlorophenyl esters, tolyl esters, butyl phenyl esters, xylyl esters, methyl esters, etc. Examples include esterified carboxylic acids such as aryl esters, which may have one or more carboxylic acids.

suitable "lower alkyl" and "amino (lower) alkyl", "protected amino (lower) alkyl 1, 'carbamoyl (lower) alkyl", "protected carbamoyl (lower) alkyl", 1 with suitable substituents; Suitable primary alkyl moieties of "al(lower)alkyl" and "heterocyclic(lower)alkyl" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl. , neopentyl, t-pentyl, hexyl, etc., may be mentioned.

Suitable protecting groups for carbamoyl groups include, for example, 2.4
-Dimethoxybenzyl ester, bis(methoxyphenyl)methyl ester, etc., which may have suitable substituents, e.g. mono(or di- or tri)phenyl (lower)
Al (lower) alkyl esters such as alkyl esters can be mentioned.

Examples of the "appropriate monoaryl moiety of al(lower)alkyl which may have a suitable substituent" as used herein include phenyl, naphther and the like.

Examples of suitable substituents for "al(lower) alkyl which may have one suitable substituent" as used herein include hydroxy and the like.

Appropriate ri+ of "1 heterocyclic (lower) alkyl" here
! The [subcyclic moiety] may have at least one heteroatom selected from nitrogen, sulfur, and oxygen atoms, and may include saturated or unsaturated monocyclic or polycyclic heterocyclic groups. However, preferred heterocyclic groups include, for example, pyrrolyl,
Pyrrolinyl, imidazolyl, pyrazolyl, pyridyl and its N-oxyto, pyrimidinyl, pyrazinyl, pyridazinyl, such as 4H-1,2,4-triazolyl, I
Triazolyl such as H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, unsaturated 3- to 6-membered heterocyclic monomer having 1 to 4 nitrogen atoms such as IH-tetrazolyl, 2H-tetrazolyl, etc. Cyclic groups; For example, saturated 3- to 6-membered heteromonocyclic groups having 1 to 4 nitrogen atoms such as pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl; For example, indolyl, isoindolyl, intridinyl, benzimidazolyl, quinolyl, isoquinolyl, imidazolyl, benzotria Zolyl, e.g. tetracylo[1,5-b
] Unsaturated fused heterocyclic group having 1 to 5 nitrogen atoms such as tetracyclopyridazinyl such as pyridazinyl; for example, oxasilyl, isoxasilyl, for example 1,2,4-oxadiazolyl, 1°3,4-oxadiazolyl , 1, 2.
Unsaturated 3- to 6-membered heteromonocyclic group having 1 or 2 oxygen atoms and 1 to 3 nitrogen atoms such as oxadiazolyl such as 5-oxadiazolyl; for example, 1 or 2 oxygen atoms and 1 to 3 nitrogen atoms such as morpholinyl saturated with 3
~6-membered heteromonocyclic group; for example, an unsaturated fused heterocyclic group having 1 or 2 oxygen atoms and 1 to 3 nitrogen atoms, such as benzoxacylyl, benzoxadiazolyl; for example, thiazolyl, for example 1.2. 4-thiadiazolyl, 1.3
．． An unsaturated 3- to 6-membered heteromonocyclic group having 1 or 2 sulfur atoms and 1 to 3 nitrogen atoms, such as thiadiazolyl such as 4-thiadiazolyl and 1.2.5-thiadiazolyl;
For example, a saturated 3- to 6-membered heteromonocyclic group having 1 or 2 sulfur atoms and 1 to 3 nitrogen atoms, such as thiazolidinyl;
For example, any N-containing heterocyclic group such as an unsaturated fused heterocyclic group having 1 or 2 sulfur atoms and 1 to 3 nitrogen atoms such as benzothiazolyl and benzothiadiazolyl may be used.

Preferred embodiments of the target compound (I) are as follows.

Preferred embodiments of R1 are hydrogen, amino or a protected amino group [more preferably acylamino, most preferably lower alkoxycarbonylamino, ar(lower)alkoxycarbonylamino or lower alkanoylamino], R2 is hydrogen, carboxy or Protected carboxy group [more preferably esterified carboxy group, most preferably lower alkoxycarbonylco, R3 is hydrogen or carboxy, R4 is lower alkyl, amino (lower) alkyl, protected amino (lower)
Alkyl [more preferably acylamino(lower)alkyl, most preferably al(lower)alkoxycarbonylamino(lower)alkylco, carbamoyl(lower)
Alkyl or protected carbamoyl(lower)alkyl, more preferably [bis(4-methoxyphenyl)medelcoyl(lower)alkyl], R is hydrogen or lower alkyl, and R6 is hydrogen, hydroxyphenyl(lower)alkyl or heterocyclic (lower) alkyl [more preferably imidazolyl (lower) alkyl or indolyl (lower) alkyl].

In order to illustrate the usefulness of the target compound (I), some biological properties of representative compounds are explained in detail in the following tests.

Gu Huizhang Huang Cao (2S, 3R)-2-[[(23)-2-amino-3-
carbamoylpropionylcoamino]-3-[[(Is
)-1-carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid [FR-900490] Group Ju Restorative effect on reduced colony-forming ability of bone marrow cells in immunodeficient hosts: Mitomycin treatment of BDF1 mice (female, 8 withdrawals) C(1
mg/kg) was administered intraperitoneally every day for 2 days, and FR-900
490 is administered intraperitoneally daily for 5 days. Mitomycin C
Mice are sacrificed 9 days after the first administration and the colony forming capacity of bone marrow cells (CFU-C) is measured. The above test schedule is shown below.

Administration of mitomycin C-1 ↓ ↓ ↑ ↑ ↑ ↑ ↑ ↑ Penicillin G (100 units/all),
Streptomycin (10oK,/mu) and 5%
Bone marrow cells are collected by inserting α-MEM supplemented with fetal bovine serum (FBS) and draining bone marrow fluid. Bone marrow cells are pelleted by centrifugation (500×g, 10 minutes) and resuspended in α-MEM as described above. Nucleated lJ
B cells were counted with a hemocytometer and the m cells were diluted to 3 lXIO cells/relative. Next, the bone marrow cells (1 relative), FBS (1 can), and 2.2% methyl cellulose in α-MEM (2 mQ
) and L-929 cell culture supernatant (1 hour) are added to a sterile test tube and mixed using an automatic mixer. One volume of Compound 6ii was placed in a 351111 plastic Petri dish and incubated for 7 days in a sufficiently humid atmosphere of 5% CO□ in air.

Prepare three plates for each experiment.

Colonies consisting of 50 or more cells are recorded using an inverted microscope.

The results are shown in Table 1.

Table 1 Rate *p〈0.01 (n=7-9) Rate In control (1) animals, mitomycin C and FR-9
00490 has also not been administered completely.

In the control (2) animals, mitomycin C was also used for FR-
900490 is also not administered and the L-929 cell culture supernatant is not used to culture bone marrow cells.

FR-9QO490 was not completely administered to animals with this rate control <3).

Test 2 Tumor metastasis blocking activity: B-16 melanoma cells were incubated at Roswell Park Memorial Institute (Roswel Park Memo) supplemented with penicillin G (1oo units/mQ), streptomycin (lok/mQ) and 10% FBS.
real institute) (RPMI)-1640
Grow in culture medium and rinse the monolayer with an aqueous trypsin solution.
Hanks (HB) containing the above antibiotics (Ca-Mg free)
S) Add the solution to the monolayer, gently pipette off the monolayer, and wash the cells with the antibiotic-containing HBSrII solution by centrifugation.

Cells are resuspended in the antibiotic-containing HBS solution and stored at low temperature. BDF1 mice (female, 8 weeks old) were injected with B-16 melanoma 2x in the antibiotic-containing HBS solution 0.2-
Inject 105 doses intravenously. FR-900490 is B-16
Administer intraperitoneally once daily on days 1 and 2 before melanoma injection and on days 1 and 4 after injection. Fourteen days after B-16 melanoma injection, mice were sacrificed, lungs were removed, and
Rinse in distilled water containing heparin and determine the number of tumor metastases by counting surface colonies under a dissecting microscope.

The schedule for the above test is shown below.

Administration of B-16 melanoma ↓ ↑　↑　　↑　　　↑　　↑ The results are shown in Table 2.

Blue”.

** p < 0.01 (n:io) Guhui 1 Acute toxicity of FR-900490: A test on the acute toxicity of FR-900490 by intravenous injection was conducted in ddY mice (female, 5 weeks old).
No mortality was observed at mg/kg.

Supervisor K1 Recovery effect on decreased antibody formation ability in immunodeficient hosts: BDF1 mice (female, 8 weeks old) were treated with mitomycin C (1
mg/kg) to 5×to8 human red blood cells (5RBC)
Administer intraperitoneally every day for 3 days starting 4 days before sensitization with 4m.

Test samples are also administered intraperitoneally to mice daily for 5 days starting 4 days before sensitization with sheep red blood cells.

On day 9, mice were killed and the number of direct plaque-forming cells per spleen (PFC-5RBC/spleen) was determined using Cunningham (Cu).
measured using a modified method.

The above test schedule is shown below.

↓ ↓ ↓ ↓ ↓ Maitomai Sheep red blood cells Plaque-forming Syn-C administration
The measurement results of sensitized cells are shown in Table 3.

Table 3 This animal has not received mitomycin C.

The pharmaceutical composition of this invention contains the object compound of this invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, internal or parenteral administration.
For example, they can be used in the form of solid, semi-solid or liquid pharmaceutical preparations. The active ingredient may be mixed with, for example, conventional non-toxic pharmaceutically acceptable carriers for use in tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions and other suitable forms. Carriers that can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, base material, and solid, semisolid or Other suitable carriers for preparing liquid preparations, in addition to which auxiliaries, stabilizers, thickeners, colorants and flavoring agents may be used, the active compound is present in the course of the disease or A sufficient amount is included in the pharmaceutical formulation to produce the desired effect on the condition.

The dose of the target compound depends on the type of disease, patient weight and/or
It may also vary depending on various factors such as age and the route of administration.

The preferred dosage of the target compound is usually 0,1 to 1 per day.
100mg/)tg.

The present invention will be described below with reference to production examples and examples.

Pyridine (
212 mg), trifluoroacetic acid (152 mg), N,
Stir N'-dicyclohexylcarbodiimide overnight.
Add water (101111) and ethyl acetate to the mixture,
Insoluble matter is filtered off.

Separate the organic layer and wash successively with water and aqueous sodium chloride solution. The solution was dried over magnesium sulfate and subjected to column chromatography on silica gel (25 g) using a n-hexane-ethyl acetate mixture (5:1) as eluent.
)-3-year-old xo-methyl 2-tritylaminobutyrate (56
3 mg) as a pale yellow oil.

NMR (CDCl2.8) 7 1.98 (3H.
s), 3.50 (3H.s).

4, 17 (IH.s), 7.1-7.6 (15H
．． Made by m)! JL main (2S)-3-year-old xomethyl 2-tritylaminobutyrate (397 mg) and (23)-2-amino-3-(4
-imidazolyl) methyl propionate dihydrochloride (3
Sodium cyanoborohydride (66,7 m
g) and stir the solution overnight at room temperature. A solution of sodium hydrogen carbonate (400 ml) and water (1011111) was added to the reaction mixture, and the resulting aqueous solution was extracted with chloroform (4 ml). The extract is washed with aqueous sodium chloride solution, dried over magnesium sulphate and evaporated under reduced pressure.

The residue was subjected to column chromatography on silica gel (20 g) using a chloroform-methanol mixture (50:1) as the eluent, and then subjected to C2S. 3B) -3-[[(IS)-t-methoxycarbonyl-2-(4-imidazolyl)ethylcoamino]-2-triple aminobutyrate (141 mg)
) is obtained as a yellow amorphous powder.

Melting point: 55-58℃ IR (X5Fm-4>' 1720.1
195. 1180 am-1HMR (CDC1
3.8 > 80.97 (3H.d.J=6Hz>,
2.6-3.1 (3H, m), 3.13 (3H
,s), 3.41 (11,m), 3.58(
IH. m), 3.68 (3H.s), 5.
50 (2H. Broad).

6, 13 (IH.s), 7.1-7.6 (16
H. m) Mono-p-toluenesulfone of pear 111yu (2S)-3-year-old xomethyl 2-tritylaminobutyrate (2.0Hg) and methyl (28)-2-amino-3-(3-indolyl)propionate methanol (30ff) with acid salt (3.13g)
IQ) to the mixture in sodium cyanoborohydride (
360 mg) and the mixture was stirred at room temperature for 1 day. Dilute the mixture with chloroform and wash successively with water and aqueous sodium chloride solution. The organic layer is separated, dried over magnesium sulfate, and the solvent is evaporated under fJi pressure. The residue was subjected to column chromatography on silica gel (50 g) using a mixture of n-hexane and ethyl acetate (4:1) as the eluent. The desired fractions were collected and evaporated under reduced pressure to obtain (2S,3
R)-3-[[(Is)-1-methoxycarbonyl-2
Methyl -(3-indolyl)ethyl]amino]-2-tritylaminobutyrate (625 mg) is obtained as a pale yellow oil.

NMR (CDCIs, l; )' 0.99 (3
H1d, J'6Hz), 2.93 (IH,t, J-6H
z), 3.06 (3H,s>, 3.20 (2H
,d.

J=6Hz), 3.3-3.6 (IH, m), 3
．． 58 (3H, s).

3.77 (IH, m), 7.1-7.7 (20H
, m), 7.96 (IH, @) 1 Thiobei 1 According to the method of Production Examples 2 and 3, the following compound is obtained.

(1) (2S,3R)-3-[[2-(4-imidazolyl)ethyl]amino]-2-tritylaminobutyric acid methyl NMR<coct3. l;)' 1.11 <2
H, dJ=6Hz), 2.6-3.1 (5H, a+)
, 3.17 (3H,s), 3.48 (LH,m
), 6.74 (IH,s), 7.1-7.6 (1
6H,m)(2)(2S,3R)-3-[[(IS)-
Methyl 1-ethoxycarbonyl-ethyl]aminoco-2-tritylaminobutyrate NMR (CDCIs, δ): 0.97 (3H.
d. J-6Hz), 1.18 (3H.tJ=8Hz)
, 2.6-3.0 <IH, m), 2.93 <2
H.

d. c6Hz), 3.07 (3H.s), 3.2
9 (lH.m).

3, 60 (IH, t.J=6Hz), 4.12 (
2H. q. J=8Hz>.

6, 66 (2H.d.J=8Hz>, 7.06 (
2H. d. J=8Hz).

7, 1-7.6 (15H.m) (3) (2S.3R)-3-methoxycarbonylmethylamino- NMR (CDC1s,8): 1. 07 (3H
．． d. J=6Hz), 3.07 (LH.m), 3.1
4 (3H.s), 3.37 (2H.s), 3.
41 (IH, m>, 3.70 (3H.s),
7.1-7.6 (15H.m>k Week 1 (2S.3R)-3-[[(IS)-1 in ice-salt bath
-Methoxycarbonyl-2-(4-imidazolyl)ethyl]aminoco-2-tritylaminobutyrate (40
0mg) in methylene chloride (101all), boron tribromide (952a+g) in methylene chloride (5m
c) Add the solution dropwise and stir the mixture overnight at room temperature. Filter the pale yellow powder that precipitates during the reaction, wash it with methylene chloride, and dissolve it in water. Dowex 50W solution
X 8 (Product name, Dow Chemical Company) (201111
1) Apply to the column and elute with 2.8% ammonia water. The desired fractions were evaporated under reduced pressure and the residue was crystallized from methanol to give (2S.3R)-2-amino-3-[[
(Is)-1-carboxy-2-(4-imidazolyl)
Ethylcoamino]butyric acid (55 mg) is obtained as a white powder.

Melting point; 226-230°C IR (streak 3-l): 1615, L5
6Q. 1405. 1245 cm-'NMR
(DzO-NaOD,8)' 0.98 (3H
．． d. J=6Hz).

2,57 (IH.m), 2.83 (2H.d.J
=7Hz), 2.97(LH,d.J=8Hz),
3.47 (LH.t.J=7Hz), 6.84 (I
H. s), 7.53 (18, s) 1 (2S.3R) -3-[[(Is)-1-Methoxycarlipo-di-2-(3-indo-di)ethylcoamino-2 A solution of 2-triple aminomethyl acetate (600 mg) in a mixture of methanol (tOml) and 1N hydrochloric acid (2-) is stirred at room temperature for 1 hour and the methanol is evaporated under reduced pressure. Wash the residual aqueous solution with chloroform and add water (15ml
Dilute with 1). Sodium hydroxide (500
mg) and the mixture was stirred at room temperature overnight. Adjust the pH to 3 with IN hydrochloric acid and mix the solution with DOWEX 50WX 8
(H, 20ml) (7) Apply to the column. Wash the column with water and elute with 1.4% aqueous ammonia. The desired fractions were collected and lyophilized to give (2S,3R)-2-amino-a-[[(Is)-1~carboxy-2-(3-indolyl)ethylcoamino]butyric acid (260 mg) as a white powder. get as.

Melting point: 159-163°C NMR (C20.S)' 1. 00 (3H.
d. J=6Hz), 3.20 (IH.m), 3.4-
3.8 (IH.m), 3.8-4.3 (2H,m
>.

7, 2-7.4 (3H, m), 7.57 (IH
．． m), 7.76 (IH.m) Mass (SLMS)' mHz 306
(M”+1>Production Example 7 (2S.3R)-3-[[(Is)-1-ethoxycarbonyl-2-(4-hydroxyphenyl)ethyl]amino]-2-tritylaminomethyl acetate (1, 13g) was treated in the same manner as in Production Example 6 to obtain (2S,3R)-2-amino-3-[[(IS)-1-carboxy-2-(4-hydroxyphenyl)ethylcoamino]butyric acid (375mg).
).

NMR (C20,8)' 0-99 (3H0d, J
=6Hz), 2-61 (IH, m), 2.72 (2
H, d, J=7Hz), 2.93 (LH, d.

J=8Hz), 3.41 (IH, dd,, C6,7
Hz), 6.54 (2H, d, J=8Hz), 7.
00 (2H, d, J=8Hz) 11 encounters 1 According to the method of Production Example 6, the following compound is obtained.

(2S,3R)-2-amino-3-carboxymethylaminobutyric acid NMR (C20,f; )'t,so (3H,d
, J=6Hz), 3.5-4.0 (IH, m), 3
．． 76 (2H, s), 4.16 (IH, a+) Otori JJL and (1) (2S, 3S)-3-methyl-1-tosyl-2-
A solution of D-histidine monohydrochloride (2.45 g) in a mixture of methanol (9,5111 M) and IN aqueous sodium hydroxide solution (9,1 ffl11) was stirred at room temperature for 30 minutes. 5.23g
) and sodium hydroxide (2.55 g). The solution is refluxed for 5 hours and the solvent is evaporated under reduced pressure. The residue is dissolved in water and adjusted to ptts with IN hydrochloric acid. This solution is applied to a column of "Diaion HP-20" (trade name, manufactured by Mitsubishi Chemical Industries, Ltd.) (xooma), and the column is washed with water. Elution with methanol-water (,1:1) yielded (25
,3R)-3-[[(IR)-1-carboxy-2-(
Mixed production of 4-imidazolyl)ethyl]amino]-2-tosylaminobutyric acid and (2S,3R)-2-[[(IR)-1-carboxy-2-(4-imidazolyl)ethyl]aminoco-3-tosylaminoacetic acid (2.47 g) was obtained. (2) Mixed product obtained in Production Example 9 (1) (2.47 g)
and liquid ammonia (30ml), -78
Sodium (1.25 g) is added at °C and the mixture is stirred at the same temperature for 5 hours. Ammonium chloride is added to this mixture until the blue color disappears, and then the ammonia is evaporated at room temperature. The residue is diluted with water and adjusted to pH 4 with IN hydrochloric acid. Add the solution to DOWEX 50W x 8 (30m11
) and wash the column with water. Next, 2.8
% aqueous ammonia and evaporate the eluate under reduced pressure. The residue was dissolved in pyridine-acetate buffer (0.01M, pH
4,5) and Whatman CM52 (300+nQ
') column. Wash the column with the same buffer and
Pyridine-acetate buffer (0,01-0,3M, p) 1
Elute in step 4.5). Both fractions containing the target compound were collected and evaporated under reduced pressure, and the residue was solidified with methanol to give (2S
, 3R)-2-Amino-3-[[(IR)-1-carboxy-2-(4-imidazolyl)ethyl]aminocobutyric acid (584 mg) is obtained as a tan powder.

Melting point: 168-171°C (decomposition) NMR (C20, δ): 1.25 (3H, d, J
=7Hz), 2.9-3.6 (3H, m), 3.
03 (2H, d, J: 6Hz), 3.68
(IH, d.

J=3)1z), 7.26 (IH,s), 8.4
6 (IH,s)Mass (SIMS) ' m/z
257 (M +1>Tu m (2S, 3R)-2-amino-3-[[(IS)-1-
Carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid (51 mg) and triethylamino (0,14
1d) in water (1)-dioxane (1 mQ) was added t-butoxycarbonyl-L-aspartic acid p.
-Nitrophenyl (200 liters) is added and the mixture is stirred at room temperature. After 15 hours, a further 200 mg of p-nitrophenyl t-butoxycarbonyl-acid and triethylamino (0.14111Q) are added and the mixture is stirred at room temperature for 4 hours. Furthermore, 200m p-nitrophenyl t-butoxycarbonyl-aspartate
g and triethylamino (0.141119) are added and the mixture is stirred at room temperature for 4 hours.

The reaction mixture is diluted with water (IV), neutralized with IN-hydrochloric acid, washed with ethyl acetate (10 mQ) and the aqueous layer is evaporated under reduced pressure.

The residue was dissolved in 75% trifluoroacetic acid aqueous solution (5 ml
) and stir the solution at room temperature for 3 hours. After evaporating the solvent under reduced pressure, the residue is dissolved in water (5%) and adjusted to pH 7 with IN aqueous sodium hydroxide solution. The solvent was evaporated under reduced pressure and the residue was dissolved in pyridine-acetate buffer (0,01
M, pus, o). Whatman CM solvent
52 (trade name, Whatman) (50ml!) and wash the column with the same buffer (70ml).
Pyridine-acetate buffer (0.01-0.1M, pH 5
,0). Both fractions containing the target compound are collected and the solvent is evaporated under reduced pressure. Residue Amberlite IRA4
00 (Product name, Rohm and Haas) (OHOH-
1LO), and the column was filled with water (50+1111
) and elute with 4% acetic acid aqueous solution (20 Makoto). The solvent was evaporated under reduced pressure, and the residue was subjected to column chromatography on silica gel (3 g) using chloroform-methanol-concentrated aqueous ammonia solution (5:3:1) as eluent.
2S,3R)-2-[[(2S)-2-amino-3-carbamoylpropionyl]aminoco-3-[[(IS)
-1-carboxy-2-(4-imidazolyl)ethyl]
Amino]butyric acid (19.8 mg) is obtained.

NMR (D20-NaOD, δ): L, 14
(38, d, J=7Hz), 2.78(1)1. d,
J=7Hz), 2.82 (IH, d, J-6Hz
), 3.19 (IH, d, J=8Hz), 3.
24 (IH, d, J=6Hz>, 3.54 (18
, m>, 3.97 (IH, dd, J=6.8H
z>, 4.08 (LH, dd, J=6.7Hz),
4.43 (LH, d, J: 5Hz>.

7.15 (LH,s), 7.93 (IH,s)
This compound is Discosia sp. F-1180
It corresponds to FR-900490, which was produced by culturing 9.

Togotayu (2S, 3R)-2-amino-3-[[(IS)-1-
Carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid (700 mg) and triethylamino (1,29
N-hydroxysuccinimide ester of t-butoxycarbonyl-di-alanine is added to a mixture of N-hydroxysuccinimide ester of tert-butoxycarbonyl-di-alanine and the mixture is stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure and the residue
% trifluoroacetic acid aqueous solution (20111Q). The solution is kept at room temperature overnight and the solvent is evaporated under reduced pressure. DOWEX 50W x 8 (30IQ) residue
) and wash the column with water. Elute with 2.8% aqueous ammonia. The desired fractions are collected and evaporated under reduced pressure. The residue was poured onto a column of Whatman CM52 (150 ml!) and pyridine-acetate buffer (0, OIM-0
, 14M, pH 4,9). The desired fractions are collected and evaporated under reduced pressure. The residue was dissolved in water and lyophilized to give (2S,3R)-2-[[(2S)-2-aminopropyl dirucoaminocor a-[[(ts)-1-carboxy-2-(4-imidazolyl)]. ) Ethyl]amino]butyric acid (580 mg) is obtained.

Melting point: 155-157°C (decomposition) NMR (D20.δ): 1.14 (3H, d, J
=7Hz), 1.23(3H,d,J=6Hz>,
3.27 (2H, m), 3.60 (IH, m).

3.9-4.3 (2H, m), 4.50 (IH,
d, J=5Hz) Mass (SIMS): m/z
328 (M”+1)
Carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid (400 mg) and triethylamino (0.70 mg)
water (tomQ)-dioxane (tOII) with rnQ)
To the middle compound was added (2S)-6-benzyloxycarbonylamino-2-t-butoxycarbonylaminohexanoic acid (894 mg) at 5°C, and the mixture was stirred in a water bath for 3 hours. The volatile solvents are evaporated under reduced pressure and the residue is adjusted to pH 4 with IN hydrochloric acid. The mixture is applied to a Diaion HP-20J (8QmQ) column. After washing the column with water, it is eluted with methanol-water (3N-8:2), and the desired fractions are collected and evaporated under reduced pressure.Residue was solidified with a methanol-ether mixture to form (2S,3R)-
2-[[(23)-6-Benzyloxycarbonylamino-2-t-butoxycarbonylaminohexanoylcoamino]-3-[[1s)- to -carboxy-2-(4
-imidazolyl)ethyl]amino]butyric acid (786mg)
is obtained as a white powder.

NMR (D20.S)' 1.26 (3H1d9
J=7Hz>, 1.3-1.8 (6H, m>, 1.4
1 (9H, s), 3.0-3.3 (2H, m>.

3.45 (2H, d, J=7Hz), 3.70 (
IH, m>, 3.9-4.3 (2H, m), 4.5
0 (LH, d, J=5Hz), 5.09 (28,
s).

7.38 (5H,s), 7.41 (LH,s
), 8.62 (IH,s)Tu 1di± (2S,3R)-2-amino-3-[[(IS)-1-
Carboxy-2-(3-indolyl)edelcoaminocobutyric acid (207 mg) and triethylamino (0.33 mQ
) and water (2ml)-dioxane (5-), (2S)-2-benzyloxycarbonylamino-
N-hydroxysuccinimide ester of 3-[N-[bis(4-methoxyphenyl)methyl]carbamoyl]propionic acid (560 mg) is added at 5°C and the mixture is stirred in a water bath for 2 hours. The volatile solvents are evaporated under reduced pressure and the residual solution is adjusted to pH 4 with IN hydrochloric acid. The precipitate was collected by filtration, and (2S,3R)-2-[[(2S)-2-benzyloxycarbonylamino-3-[N-[bis(4-methoxyphenyl)methyl]-carbamoyl]propionyl]amino]- 3-[C(tS)-1-carboxy-2-
(3-indolyl)ethyl]amino]butyric acid (726 mg
) is obtained as a white powder.

] d1 mutual (2R,3S)-2-amino-3-[[(IS)-1-
Ruboxy-2-(4-imidazolyl)ethyl]aminocobutyric acid (200 mg) and triethylamino (0,
3811111) of water (1,5ff1m)-dioxane (51111), (28)-2-benzyloxycarbonylamino-3-[N-[bis(4-methoxyphenyl)methyl]carbamoyl]propionic acid N
-Hydroxysuccinimide ester (5J16III
g) is added and the solution is stirred at room temperature overnight. The volatile solvent is removed under reduced pressure and the remaining aqueous solution is adjusted to pH 4 with IN hydrochloric acid. The precipitate was collected by filtration, vacuum dried, and (2R,3S
)-2-[[(2S)-2-benzyloxycarbonylamino-3-[N-[bis(4-methoxyphenyl)methyl]carbamoyl]propionyl]amino]-s-[
[:1s)-x-carboxy-2-(4-imidazolyl)ethyl]aminocobutyric acid (685 mg) is obtained as a white powder.

Fire Jl (2S)-2-amino-3-[[(Is)-1-carboxy-2-(4-imidazolyl)ethylcoamino]propion H (265 mg) and triethylamino (0
N-hydroxysuccinimide ester of (2S)-2-benzyloxycarbonylamino-methoxyphenyl)methylcocarbamoylcobropionic acid (771
mg) in an ice water bath. The mixture is stirred at room temperature for 3.5 hours and evaporated under reduced pressure to give a white residue, to which water (4011!) is added. The suspension was placed in an ice water bath.
Adjust to pH 3-4 with 6N hydrochloric acid.

The precipitate was collected by filtration, washed with water (6011111), and dried under reduced pressure to obtain (2S)-2-[[(2S)-2-benzyloxycarbonylamino-3-[N-[bis(4-methoxyphenyl)]. methyl]carbamoyl]propionyl]
amino]-3-[[(15)-1-carboxy-2-(
A white powder (772 mg) of 4-imidazolyl)ethyl]aminocopropionic acid is obtained.

mヱ According to the method of Example 4-6, the following compound is obtained.

(1)(2S.3R)-2-[:[(2S)-2-benzyloxycarbonylamino-3-[N-[His(4-
methoxyphenyl)methyl]carbamoyl]propionyl]amino]-3-[[(IR)-1-carboxy-2
-(4-Imidazolyl)ethyl]amino]butyric acid (2>(2S,3R)-2-[[(2S)-2-benzyloxycarbonylamino-3-[N-[bis(4-methoxyphenyl)] Methyl]carbamoyl]propionyl]amino]-3-[C2-(4-imidazolyl)ethylcoaminocobutyrate methyl(3)(2S.3R)-2-[[(2R)-2-benzyloxycarbonylamino-3-[N -[bis(4-methoxyphenyl)methyl]carbamoyl]propionyl]amino]-3-[: )-2-[[:(2S)-2-benzyloxycarbonylamino-3-[N-[bis(4-
methoxyphenyl)methylcocarbamoylcopropionyl]amino]-3-[[(IS)-1-carboxy-2
-(4-hydroxyphenyl)ethyl]amino]butyric acid (5)(2S,3R)-2-[[(2S)-2-benzyloxycarbonylamino-3-[N-[his(4-methoxyphenyl)methyl ]Carbamoyl]propionyl]amino]-3-carboxymethylaminobutyric acid ) methyl]carbamoyl]propionylcoamino]-3-[[(IS)-1-carboxy-2-(3-
A solution of indolyl)ethyl]amino]butyric acid (800 mg) in acetic acid (20 fflQ ) was prepared under moderate pressure (3 atm).
The mixture was hydrogenated for 8 hours at room temperature in the presence of 10% palladium on carbon (120 mg). After removing the catalyst, the solution was evaporated under reduced pressure and the residue was solidified with water to give (2S,3
R)-2-[[(2S)-2-amino-3-[N-[bis(4-methoxyphenyl)methyl]carbamoyl]propionyl]amino]-3-[[(ls)-1-carboxy-2 -(3-indolyl)ethyl]amino]butyric acid (
460 mg) as a tan powder.

K-Seihan (2S, 3R)-2-[[(2S)-6-benzyloxycarbonylamino-2-t-butoxycarbonylaminohexanoyl[[(IS)-1-carboxy-2-(4- imidazolyl)ethyl]amino]butyric acid (720 mg) and 3
Stir a 0% hydrogen bromide solution in acetic acid (201+111) at room temperature for 3 hours, then pour into ice water (40 mm). Wash the aqueous solution with ether and apply DOWEX 50WX 8 (H”
, 15 relatives) column. Wash the column with water, 2.
Elute with 8% ammonia water. Collect the desired fractions and evaporate under reduced pressure. The residue was dissolved in water and lyophilized to give (2
S. 3R)-2-[[(28)-2.6-diamithexanoyl]amino]-3-[[(1s)-1-carboxy-2-(4-imidazolyl)ethyl]ami/]butyric acid (
399 mg) as a pale yellow powder.

Melting point i 175-179℃ Mass (SXMS): m/z 385
(M”+1)Nt'lR (D20, δ): 1
．． 10 (3H.d.J=7Hz), 1.3-2. O
(6H.m), 2.8-3.4 (51,m>, 3
．． 6-3.8 (2H.m).

4, 55 (2H.d.J=7Hz), 7.06 (
LH. s), 7.83 (LH.s) Yi J Jrecocarbamoylcoprobionyl]aminoco-3-[[(1s)-1-carboxy-2
-(4-imidazolyl)ethyl]amino]butyric H (6
35 mg ) and 30% hydrogen bromide vinegar i11i! (5
The solution (11LQ) was stirred at room temperature for 2 hours, then diluted with ice water (20mL
11) and ethyl acetate (20ml). Separate the aqueous layer and add DOWEX 50WX8 (H”
, 20111Q') column. Wash the column with water and elute with 2.8% aqueous ammonia. Collect the desired fractions and evaporate under reduced pressure. The residue was dissolved in water (50 mm) and lyophilized to give (2R.3S)-2-[[(25)
-2-amino-3-carbamoylpropionyl]amino]-3-[[(IS)-1-carboxy-2-( 4-
imidazolyl) ether] aminocobutyric acid (190ng
) is obtained as a white powder.

Melting point: 120-123℃ (decomposition) Mass (SIMS)' m/z 371 (M
+1) NMR (D20.8): 1.63 (3H
．． d. J=7Hz), 2.87(2H.d.J=71(
z), 3.0-3.3 (21, m), 3.43 (
IH. m), 3.9-4.4 (2H.m), 4.
45 (IH.d.

J=4Hz), 7.21 (IH.s), 8.07
(IH.s) or Jl[[11 A 30% solution of hydrogen bromide in acetic acid (4.0 mQ) was added to (2S)-2-[[(2S)-2-benzyloxycarbonylamino-3] in an ice-water bath. -[N-[bis(4-methoxyphenyl)methylcocarbamoyl]propionyl]amino]-a-[[(IS)-1-carboxy-2-(4-
imidazolyl)ethyl]amino]propionic acid (772
sag). The mixture was stirred for 15 minutes, and to this was added roughly a 4.0 μl solution of hydrogen bromide in acetic acid (25111) at room temperature.
and ethyl acetate (251111), separate the aqueous layer and extract the remaining organic layer with water (30111). Combine the water layers, DOWEX 50WX8 (H, 201
11), water (200 ml) and 1
．． Elute with 4% aqueous ammonia (250111Q).

Fractions containing the target compound are combined and evaporated under reduced pressure.

The residue is dissolved in water (3G119) and filtered. The filtrate was lyophilized to yield (2S)-2-[[(2S)-2-amino-3-carbamoylpropionylcoamino]-3-[
[(1S)-1-carboxy-2-(4-imidazolyl)ethylcoaminocobropionic acid white powder (315m
g) is obtained.

NMR (D20.ε): 8.05 (LH,s),
7.19 (IH, s).

3.7-4.6 (3H, m), 2.7-3.4 (
6H, m) Mass (SIMS): m/z 35
7 (M"+1) Dog shoes and ■ According to the method of Examples 10 and 11, the following compound is obtained.

(1) (2S,3R)-2-[[(2S)-2-amino-3-carbamoylpropionylcoamino]-3-[[
(IR)-1-carboxy-2-(4-imidazolyl)
Ethyl]amino]butyric acid Melting point: 171-173°C NMR (D20.S)' 1.26 (3H, d,
J=7Hz), 2-8-3.0 (2H, a+), 3
．． 0-3.3 (2H, m), 3.47 (LH, m
).

3.9-4.3 (2H, m), 4.38 (IH,
d, J=4Hz).

7.20 (IH,s), 8.04 (IH,s)M
ass (SIMS): m/z 371
(M”+1)(,ri(2S,3R)-2-[[(2S
)-2-Amino-3-carbamoylpropionyl]amino]-3-4[2-(4-imidazolyl)ethyl]amino]butyric acid Melting point: 110-114°C (decomposition) NMR (D20.S >' 1.26 ( 3H,d,
J=7Hz), 2.70 (2H, m), 2.8-3
．． 3 (3H, m), 3.4-3.7 (2H, m)
.

3.92 (LH, m), 4.51 (IH, d, J
=4Hz), 7.11(LH,s), 7.80(
IH,s)Mass (SIMS): m/z 32
7 (W +1) (3) (2S, 3R)-2-[[(2
R)-2-amino-3-carbamoylpropionylcoamino]-3-[:[(IS)-1-carboxy-2-(
4-Imidazolyl)ethyl]amino]butyric acid Melting point: 18
1-185℃ NMR (D20.ε): 1.15 (3H, d, J
=7Hz), 2.90 (2H, d, J=6Hz),
3.24 (2H, m), 3.50 (IH, dd.

J=6.5Hz), 4.01 (IH, dd, J=6
．． 7Hz), 4.28 (LH, t, J=7H2),
4.52 (IH, d, J=5H2), 7.19 (I
H,s), 8.04 (IH,s)Mass (SI
MS): m/z 371 (M”+1>(4)(2
S, 3R)-2-[[(2S)-2-amino-3-carbamoylpropionylcoamino]-s-[[(1s)-
-Carboxy-2-(4-hydroxyphenyl)ethyl]amino]butyric acid Melting point: 170-173°C (decomposed) NMR (D20,8)' 1.11 (3H1d,
J=7Hz), 2-8-3.0 (38, m), 3.
20 (IH, m), 3.53 (LH, m),
4.00 (IH, m), 4.24 (IH, d
d, J=6. 8Hz), 4.47<IH, d, J
=5Hz), 6.93 (2H, d, J=8Hz)
, 7.26 (IH, d, J=8Hz) Mass (5HMS): m/z 397 (M
+1>(5)(2S,3R)-2-[[(2S)-2
-amino-3-carbamoylpropionyl]amino]-
3-Carboxymethylaminobutyric melting point: 146-15
0°C NMR (D20.8) = 1.33 (3H, d, J
=7Hz>, 2-7-3-0 (2H, m), 3.4
0 (2H, s), 4.03 (IH, dd, J=5
゜7Hz>, 4.28 (LH, m), 4.55
(IH, d, J=5Hz)Mass (SXMS)
: m/z 291 (M”+1) or 11 (2S, 3R)-2-[[(2S)-2-benzyloxycarbonylamino-3-[N-[: bis(4-methoxyphenyl)medel ]carbamoyl]propionyl]amino]-3-[[2-(4-imidazolyl)ethyl]amino]methyl butyrate (1,40 g) in methanol (501
11)-1N aqueous sodium hydroxide solution (25+1111
) The solution was stirred at room temperature for 30 minutes and adjusted to pH 7 with IN hydrochloric acid.
Adjust to. The precipitate was collected by filtration, and (2S,3R)-2-[
(2S)-2-benzyloxycarbonylamino-3-
[N-[bis(4-methoxyphenyl)methyl]carbamoyl]propionyl]amino]-3-[[2-(4-
Imidazolyl)ethylcoaminocobutyric acid (1.16 g) is obtained as a white powder.

Trap Gogo B ls, +R)-2-[[1s)-2-amino-3-[N
-[bis(4-methoxyphenyl)methyl]carbamoyl]propionyl]amino]-3-[[(IS)-1-
carboxy-2-(3-indolyl)ethyl]amino]
Butyric acid (427mg) trifluoroacetic acid (311)-
Anisole (0,5 ml!) - ethanedithiol (0
, 1ffLI+) mixture was stirred overnight at room temperature, poured into a mixture of water (1oma) and ethyl acetate (10mQ), the aqueous layer was separated, and DOWEX 50WX 8 (H
20ml) Fill a column, wash the column with water, and elute with 2.8% aqueous ammonia.

The desired fractions are collected and evaporated under reduced pressure. Water was added to the residue and the mixture was lyophilized to give (2S,3R)-2-[[(
28)-2-amino-3-carbamoylpropionyl]
amino]-3-[[(13)-1-carboxy-2-(
3-Indolyl)ethyl]amino]butyric acid (85 mg) is obtained as a light brown powder.

Melting point: 65-70℃ Mass (SIMS): m/z 420 (11
+1) NMR (DzO, ε): 0.90 (3H,
d, J=6Hz), 2.6-2.9(3H,m),
3.50 (IH, m), 3.9-4.3 (3H,
m).

4.35 (IH, d, J=4Hz>, 7.2-7.
A methanol solution of methyl 14(1)(25)-1-tosyl-2-aziridinecarboxylate (1,04 g) manufactured by 9 (5H, m) was added to an IN aqueous solution of sodium hydroxide (4, 1mt) in addition to
The mixture is stirred at the same temperature for 50 minutes.

L-histidine (7.11 g) and sodium hydroxide (730 mg) were added to the resulting solution, and the mixture was diluted with 1.
Reflux for 5 hours. After evaporating the solvent under reduced pressure, the residue is dissolved in water and adjusted to pH 4 with 2N sulfuric acid. This solution is applied to a column of 1 Diaion H1"20 J (150 liters) and the column is washed with water. Water-methanol mixture (1:
Elute at 1.4QQml). The eluate was evaporated under reduced pressure, and the residue was chromatographed on a silica gel (long) column using chloroform-methanol 28% aqueous ammonia (5:3-1).
)-a-[[(IS)-1-carboxy-2-(4-imidazolyl)ethyl]amino]-2-tosylaminopropionic acid (485 mg) is obtained.

NMR (DzO-NaOD, ε): 2.21 (3
H,s), 1.7-2.1(4H,m), 3.4
4 (IH, t, J=6Hz), 3.74 (IH
, dd.

J=4. 9Hz), 6.86 (11,s),
7.41 (2H, d.

J=9Hz), 7.74 (2H,d, J=9Hz
>, 7.84 (IH, 5) (2) (23) -3-
[[(Is)-1-carboxy-2-(4-imidazolyl)ethyl]amino]-2-tosylaminopropionic acid (450 mg) was treated according to the method of Production Example 9 (2) to obtain (2S). -2-Amino-3-[(Is)-1-carboxy-2-(4-imidazolyl)ethyl]aminocopropionic acid (265 mg) is obtained.

Melting point: 215-220°C (decomposition) NMR (DzO,S)' 3.0-3.2 (4H
9m), 3.52 (IH9t.

J=6.5)1z), 3.82 (IH, dd, J=
5.5.6.5Hz).

7.26 (l)1. s), 8.47 (LH, s)
Tue】mouth 1 German (2S, 3R)-2-[[(2S)-2-amino-3-
carbamoylpropionylcoamino]-3-[[(13
)-1-carboxy-2-(4-imidazolyl)ethyl]aminocobutyric acid (500 mg), triethylamino (0
, 38111''), methanol (3oma) and water (6ffl11) in a mixture of acetic anhydride (0.13mm
) and stirred at room temperature for 20 hours. The solvent is removed under reduced pressure and the residue is dissolved in water and adjusted to pH 3 with IN hydrochloric acid. Add this solution to DOWEX 50WX8 (H”, 10mm
) and wash with water.

Elute with 2% aqueous ammonia, collect the desired fractions, and evaporate under reduced pressure. The residue is dissolved in a mixture of acetic acid (28,61111") and water (500 ml) and lyophilized.

The residue was solidified with methanol to give (2!S,3R)-2-
[[(28)-2-acetylamino-3-carbamoylpropionylkoamino]-3-[[l5)-1-carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid (500 mg) is obtained.

Melting point: 205-208°C (decomposition) NMR (DzOlS)' 1.24 (3H, d,
J=6Hz>, 2.05 (3H, s), 2.7-
2.9 (2H, m), 3.4-3.5 (3H,
m).

3.68 (IH, m), 4.12 (IH, m)
, 4.50 (IH, d.

J=4Hz), 7.38 (LH,s), 8.
55 (IH,s)Mass (SIMS): m/
z 413 (M”+1) shake 1st 1u Pyridine (11゜2otc) methylene chloride (300f
fl+1) solution at 10-15°C.
, 32g). After stirring at room temperature for 1 hour, the solution was cooled to 8°C and added with N-(t-butoxycarbonyl)ethanolamino (2,0 g) in methylene chloride (20
+11Q) #Drop the liquid. The mixture is stirred at the same temperature for 3 hours and filtered through Celite. The filtrate is evaporated under reduced pressure to give a brown oil. This oily methanol (
46111) solution with sodium hydrogen carbonate (2.11 g)
, (29)-2-amino-3-(4-imidazolyl)propionate dihydrochloride (4.54 g) and sodium cyanoborohydride (833 mg) are added and the mixture is stirred at room temperature overnight. A solution of sodium bicarbonate (3.15 g) in water (tsomu) is added to the mixture, and the solution is extracted with chloroform (100 mQ). The organic layer is washed with aqueous sodium chloride solution and dried over magnesium sulfate. The solvent was evaporated under reduced pressure and the residue was chromatographed on silica gel (50 g) using a chloroform-methanol mixture (roll) as eluent to give N-(t-
Butoxycarbonyl methoxycarbonyl)ethyl]amino]ethylamino (757mg)
Obtained as a pale yellow oil.

NMR (CDCIs.l; )' 2-62 (
2H,m), 2-86 (2H.m).

3, 20 (21 (, m), 3.53 (IH.m)
, 3.72 (3H.s).

6, 83 (IH.s), 7.57 <IH. s)1
SangoB N-(t-butoxycarbonyl)-2-[[(IS)-
t-Methoxycarbonyl-2-(4-imidazolyl)ethyl]amino]ethylamino (750 mg) was added to methanol (10 ml), water (5 mQ), trifluoroacetic acid (
5I111) and stir the solution at room temperature for 2 hours. After removing the solvent, the residue was dissolved in water (511F11)
and adjust the solution to pH 7 with IN sodium hydroxide. Add IN sodium hydroxide (81111) to this solution.
) is added and the mixture is stirred at room temperature for 24 hours. The resulting solution was adjusted to pH 3 with IN hydrochloric acid and diluted with DOWEX 50.
Add carbon to a WX8 (20mm) column. Wash the column with water and elute with 2.8% ammonia water. The desired fractions were collected and evaporated under reduced pressure to give 2-[[(Is)-
1-Carboxy-2-(4-imidazolyl)ethyl]amino]ethylamino (448 mg) is obtained.

NMR (D20.δ): 2.75 (LH.,
J=6.5Hz), 2.8-3.0<28. m),
3.28 (IH.t.J=5Hz), 3.37<
IH. t.

J=6. 5Hz) straight day According to the method of Example 4-6, the following compound is obtained.

(1) N-[(23)-2-benzyloxycarbonylamino-3-[N-[bis(4-methoxy7 enyl)
methyl]carbamoyl]propionyl]-2-[[(I
s)-1-carboxy-2-(4-imidazolyl)ethyl]amino]ethylamino(2)(2S,3R)-3-[[(IS)-1-carboxy-2-(4-imidazolyl)ethyl] Amino]-2
-[[3-[-[bis(4-methoxyphenyl)methyl]carbamoylcopropionyl]amino]butyric acid Higashigogo] According to the method of Examples 10 and 11, the following compound is obtained.

(1) N-[(2S)-2-7mi/-3-force Lbamoylpu-pi-sai-niru]-z-[[(1s)-t-carboxy-
2-(4-imidazolyl)ethyl]amino]ethylamino Melting point + 105-111°C (decomposition) NMR (D20.8): 2-66 (2H, t, J
=5Hz), 2.90(LH, dd, J=4.7.5
Hz>, 3.05-3.20 (2H, a+).

3.53 (2H, t, J=5Hz), 3.82 (
2H, m), 4.03 (IH, dd, J=4.7.5
Hz), 7°04 (IH, s), 7.85 (IH
,5) (2)(2S,3R)-2-[[3-carbamoylpropionylkoamino]-3-[[(IS)-1-carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid Melting point: 130-137℃ (decomposition) NMR (DzO9E)' 1-11 (LH, d,
J=7Hz>, 2.5-2.8 (4H, m), 3
．． 1 to 3.4 (2H, m), 4.07 (L
H,t.

J=6Hz), 4.50 (111,d, J=5Hz
), 7.30 (IH,s).

8.24 (LH,s) Trap 5'' Soluble starch (1%), cornstarch (1%), glucose (1%), cottonseed flour (0.5%), dried yeast (0,
Seed medium (8G1d) containing (5%), Cornstabri's force (0,5%), calcium carbonate (0,2%) (
vi) into each of 30 Erlenmeyer flasks of 250111 volume and sterilized at 120° C. for 30 minutes. Slant culture of Discosia sp. F-1L809! A loopful of platinum was inoculated into each medium and heated at 25°C using a rotary shaker (200 rpm) with a 3 inch slow speed.
Culture for 72 hours.

The resulting 2400m seed culture was heated to 120°C in advance.
A production medium with the same composition as the seed medium in a 2001-volume jar fermenter (inoculated into 160 jars, aeration volume 16011 minutes, stirring speed 300 rpm, 3
Incubate at 0°C for 96 hours.

The culture broth obtained in this way was mixed with diatom ± (4 kg).
). The filtrate is applied to an activated carbon column (301), the column is washed with deionized water (904) and eluted with 60% aqueous acetone (90j2).

The eluate was concentrated under reduced pressure to 60e, and then transferred to DOWEX 50W-
Wash the column with deionized water and elute with 1.5% aqueous ammonia (11). Concentrate the eluate to dryness under reduced pressure. Then, the mixture was subjected to column chromatography using silica gel (trade name: Kiesselgel 60, manufactured by Merck & Co., Ltd., 70-230 mesh, 1.8J).

The column was heated with 80% aqueous isopropyl alcohol (1°8J2) containing 0.28% aqueous ammonia and 0.28%
Wash with 75% aqueous isopropyl alcohol containing aqueous ammonia and elute with 70% aqueous isopropyl alcohol containing 0.28% aqueous ammonia. Active fraction (2,51)
was concentrated under reduced pressure to 1001111, and silica gel (Kieselgel 60.70-230 mesh, 900I1111
) Subject to column chromatography.

The column was washed with a mixture (1,81) of n-butyl alcohol, acetic acid and deionized water (3:1:2), and washed with a mixture (1,81) of n-butyl alcohol, acetic acid and deionized water (C2:1:2).
, 8j. The active fraction is neutralized with 6N aqueous sodium hydroxide solution and concentrated under reduced pressure to a concentration of 10011111. This solution was subjected to silica gel (60011Q) column chromatography using a mixture of n-butyl alcohol-ethyl alcohol-chloroform-28% ammonia water (2:
2:1:2). The active fraction (700 mQ) was concentrated under reduced pressure to 400 mQ and applied to an activated carbon column. The column was washed with deionized water (400 mQ) and eluted with 60% acetone aqueous solution (40011111). 3Qml of eluate
Concentrate and lyophilize to obtain 1.5 g of white powder of FR-900490.

Claims (1)

[Claims] 1. Formula: ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R^1 is hydrogen, amino or a protected amino group, R^2 is hydrogen, carboxy or protected carboxy group, R^3 is hydrogen, carboxy or protected carboxy group, R^4 is lower alkyl, amino (lower) alkyl, protected amino (lower) alkyl, carbamoyl (lower) alkyl or protected carbamoyl (
lower) alkyl, R^5 is hydrogen or lower alkyl, R
^6 is hydrogen, alkyl (lower) alkyl or heterocyclic (lower) alkyl which may have an appropriate substituent, and salts thereof. 2, R^1 is hydrogen, amino or acylamino, R^2
is hydrogen, carboxy or esterified carboxy group, R^3 is hydrogen, carboxy or esterified carboxy group, R^4 is lower alkyl, amino (lower)
Alkyl, acylamino (lower) alkyl, carbamoyl (lower) alkyl or [bis(4-methoxyphenyl)methyl]carbamoyl (lower) alkyl, R^5 is hydrogen or lower alkyl, R^6 is hydrogen, hydroxyphenyl (lower) A compound according to claim 1 which is alkyl, imidazolyl(lower)alkyl or indolyl(lower)alkyl. 3, R^1 is hydrogen, amino, lower alkoxycarbonylamino, al(lower) alkoxycarbonylamino or lower alkanoylamino, R^2 is hydrogen, carboxy or lower alkoxycarbonyl, R^3 is hydrogen or carboxy, R^4 is lower alkyl, amino (lower) alkyl, al (lower) alkoxycarbonylamino (lower) alkyl, carbamoyl (lower) alkyl or [
Bis(4-methoxyphenyl)methyl]carbamoyl (
The compound according to claim 2, which is (lower) alkyl. 4. The compound according to claim 1, wherein R^6 is imidazolyl (lower) alkyl. 5, R^1 is hydrogen, amino or acylamino, R^2
is hydrogen, carboxy or esterified carboxy group, R^3 is hydrogen, carboxy or esterified carboxy group, R^4 is lower alkyl, amino (lower)
The compound according to claim 4, wherein alkyl, acylamino(lower)alkyl, carbamoyl(lower)alkyl or [bis(4-methoxyphenyl)methyl]carbamoyl(lower)alkyl, R^5 is hydrogen or lower alkyl. . 6, R^1 is hydrogen, amino, lower alkoxycarbonylamino, al(lower) alkoxycarbonylamino or lower alkanoylamino, R^2 is hydrogen, carboxy or lower alkoxycarbonyl, R^3 is hydrogen or carboxy, R^4 is lower alkyl, amino (lower) alkyl, al (lower) alkoxycarbonylamino (lower) alkyl, carbamoyl (lower) alkyl or [
Bis(4-methoxyphenyl)methyl]carbamoyl (
6. The compound according to claim 5, which is (lower) alkyl. 7, R^1 is amino, R^2 is carboxy, R^3 is carboxy, R^4 is carbamoyl (lower) alkyl, R
7. The compound according to claim 6, wherein ^5 is lower alkyl. 8, (2S,3R)-2-[[(2S)-2-amino-
3-carbamoylpropionyl]amino]-3-[[(
The compound according to claim 7, which is 1S)-1-carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid. 9. Formulas: ▲Mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1 is hydrogen, amino or a protected amino group, R^2 is hydrogen, carboxy or a protected carboxy group, R^3 is hydrogen, carboxy or protected carboxy group, R^4 is lower alkyl, amino (lower) alkyl, protected amino (lower) alkyl, carbamoyl (lower) alkyl or protected carbamoyl (
lower) alkyl, R^5 is hydrogen or lower alkyl, R
(1)
FR-900490-producing bacteria belonging to the genus Discosia,
FR-900490 can be obtained by culturing it in a medium and collecting FR900490 from the resulting culture, or formula (2): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1 and R^4 each has the same meaning as above]
A compound represented by the formula or its reactive derivative at the carboxy group or a salt thereof is represented by the formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^2, R^3, R^5 and R^6 are each Synonymous with the above] or a reactive derivative thereof in an amino group or a salt thereof to form a compound having the formula: ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R^1, R^2, R^3, R^4, R^5 and R^6 each have the same meaning as above], or obtain a compound or its salt represented by formula (3): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [ In the formula, R^2, R^3, R^4, R^5 and R^6
each has the same meaning as above, and R^1_a is a protected amino group] or a salt thereof is subjected to an amino-protecting group elimination reaction to form the formula: ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R^2, R^3, R^4, R^5 and R^6
have the same meanings as above] or obtain a compound or a salt thereof, or (4) Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1, R^3, R^4, R^5 and R^6
each has the same meaning as above, and R^2_a is a protected carboxy group] or a salt thereof is subjected to an elimination reaction of the carboxy protecting group to form the formula: ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R^1, R^3, R^4, R^5 and R^6
have the same meanings as above], or obtain the compound or its salt represented by formula (5): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1, R^2, R^3 , R^5 and R^6
each has the same meaning as above, and R^4_a is protected carbamoyl (lower) alkyl] or a salt thereof is subjected to an elimination reaction of the carbamoyl protecting group to form the formula: ▲ Numerical formula, chemical formula , tables, etc. ▼ [In the formula, R^1, R^2, R^3, R^5 and R^6
each has the same meaning as above, and R^4_b is carbamoyl (
(lower) alkyl] or a salt thereof, the formula is: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1, R^2, R^3, R ^4, R^5 and R^6 each have the same meaning as above] or a method for producing a salt thereof. 10. Formulas: ▲ Numerical formulas, chemical formulas, tables, etc.▼ [In the formula, R^2 is hydrogen, carboxy or a protected carboxy group, R^3 is carboxy or a protected carboxy group, R^5 is hydrogen or lower alkyl, R^6 is hydrogen, hydroxyphenyl (lower) alkyl, imidazolyl (lower) alkyl or indolyl (lower) alkyl] and salts thereof, provided that the compound represented by the formula (2S, 3R)-2-amino-3-[[(1S)-1-carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid,
(2R,3S)-2-amino-3-[[(1S)-1-
Carboxy-2-(4-imidazolyl)ethyl]amino]butyric acid or (2S)-2-amino-3-[[(1S)
-1-carboxy-2-(4-imidazolyl)ethyl]
shall not be amino]propionic acid. 11. Formulas: ▲Mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1 is hydrogen, amino or a protected amino group, R^2 is hydrogen, carboxy or a protected carboxy group, R^3 is hydrogen, carboxy or protected carboxy group, R^4 is lower alkyl, amino (lower) alkyl, protected amino (lower) alkyl, carbamoyl (lower) alkyl or protected carbamoyl (
lower) alkyl, R^5 is hydrogen or lower alkyl, R
^6 is hydrogen, alkyl (lower) alkyl or heterocyclic (lower) alkyl which may have a suitable substituent] or a pharmaceutically acceptable salt thereof. 12. Microorganism Discosia sp. F-11809
(Discosia sp.F-11809).