JPH10310599A - Phebestin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound comprising a specific phebestin derivative, having inhibiting activity on aminopeptidase N and expected in applications as an anticancer agent or an immunopotentiator and further as an analgesic-retaining and an enhancing agent, etc. SOLUTION: This new phebestin derivative (salt) is represented by formula I [R is a (substituted)lower hydrocarbon; X and Y are each an amino acid residue, except when R is phenyl group and X is valine residue and Y is phenylalanine residue] and has inhibiting activity on aminopeptidase N which participates in tumor metastasis or immune system. Thereby, the compound is expected in use as an anticancer or an immunopotentiator and further use as an analgesic-retaining and enhancing agent, etc. The compound is obtained by reacting a protected dipeptide compound represented by formula H (P1 is a protecting group of carboxyl group; Ps is a protecting group of side chain functional group) with a carboxylic acid compound represented by formula III (P3 is a protecting group of amino group) and deprotecting the reaction product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel synthetic peptide having an inhibitory activity on aminopeptidase N. Since aminopeptidase N is involved in cancer metastasis or the immune system, the peptide of the present invention having aminopeptidase N inhibitory activity is expected to be used as an anticancer agent or an immunostimulant. Furthermore, use as an analgesic sustaining and potentiating agent is expected.

[0002]

2. Description of the Related Art Provestin, bestatin, leucistin and the like are known as low molecular weight peptides derived from natural products which inhibit aminopeptidase N. These are monoamino acids, dipeptides and tetrapeptides having 3-amino-2-hydroxy fatty acids at the N-terminus.
In addition, febestin, ie, (2S, 3R) -3-amino-2-hydroxy-4-phenylbutanoic acid-Val-Phe, was isolated from a strain belonging to the genus Streptomyces, and febestatin had an inhibitory activity of aminopeptidase N. (Japanese Patent Application Laid-Open No. 9-20794).

[0003]

Inhibitors of aminopeptidase N, such as probetin, bestatin and leuhistin, have low specificity for aminopeptidase N and insufficient inhibitory activity against aminopeptidase N. Had problems.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a febestatin derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof has an excellent inhibitory activity on aminopeptidase N. Found to have. That is, the present invention provides: Equation (1)

[0005]

Embedded image [Wherein, R represents a saturated or unsaturated lower hydrocarbon group which may have a substituent, and X and Y represent amino acid residues.
However, the case where R is a phenyl group, X is a valine residue, and Y is a phenylalanine residue is excluded. A novel vestestin derivative represented by the formula: or a pharmacologically acceptable salt thereof; R is a phenyl group which may have a substituent, a cycloalkyl group having 3 to 9 carbon atoms which may have a substituent, an alkyl group having 1 to 8 carbon atoms which may have a substituent, or a substituent. 2. The compound according to the above 1, which is an alkenyl group having 2 to 8 carbon atoms, or a pharmaceutically acceptable salt thereof; 3. The compound according to the above 1, wherein R is a phenyl group, a cyclohexyl group, or a lower alkyl group having 1 to 4 carbon atoms, or a pharmaceutically acceptable salt thereof; X is an amino acid residue having a basic side chain;
Or the compound according to 3 or a pharmacologically acceptable salt thereof; R is a cyclohexyl group, X is valine, histidine,
5. the compound of the above 1, wherein Y is a residue of phenylalanine, histidine or proline, or a pharmaceutically acceptable salt thereof; 6. A medicament comprising the compound according to any one of the above 1 to 5 or a pharmacologically acceptable salt thereof as an active ingredient; An aminopeptidase N inhibitor comprising the compound according to any one of the above 1 to 5 or a pharmacologically acceptable salt thereof as an active ingredient. Furthermore, since the compound of the present invention has an inhibitory activity on aminopeptidase N, it is expected to be used as an anticancer agent, an immunostimulant, or an analgesic sustaining and enhancing agent.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a saturated or unsaturated lower hydrocarbon group which may have a substituent is preferably phenyl which may have a substituent or carbon number which may have a substituent. A cycloalkyl group having 3 to 9 carbon atoms, an alkyl group having 1 to 8 carbon atoms which may have a substituent, or 2 to 8 carbon atoms;
An alkenyl group of Examples of these substituents include an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, a phenyl group, a halogen (fluorine, chlorine, bromine, and iodine), an amino group, a nitro, a hydroxyl group, a mercapto group, and the like. Is mentioned. Examples of the saturated or unsaturated lower hydrocarbon group which may have a specific substituent in the present invention include:
For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, 3-methylbutyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methyl Heptyl, 3-methylheptyl, 3-ethylheptyl, chloromethyl, dichloromethyl, 1-bromoethyl, 2-bromoethyl, 2-aminoethyl, 3-hydroxypropyl, 2-hydroxypropyl, 1-
C1-C8 alkyl groups which may have a substituent such as hydroxypropyl, 2-nitrohexyl, 2-mercaptoethyl, benzyl, 2-phenylethyl, 1-phenylethyl; cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl , Cycloheptyl, 4-methylcyclohexyl, 3-chlorocyclohexyl, cyclohexylmethyl, etc., which may have a substituent; and a cycloalkyl group having 3 to 9 carbon atoms; phenyl, 4-isopropylphenyl, 2,6-
Dimethylphenyl, 3,5-dimethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,
2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromo-2-chlorophenyl,
A phenyl group which may have a substituent such as 4-bromophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 4-iodophenyl A alkenyl group having 2 to 8 carbon atoms which may have a substituent such as vinyl, 1-butenyl, 1-pentenyl, 2-hexenyl and the like. In the present invention, an unsubstituted phenyl group, a cyclohexyl group, and a lower alkyl group having 1 to 4 carbon atoms are particularly preferable. The configuration of the 2-position carbon atom substituted by RCH ₂ — and the 3-position carbon atom substituted by —OH of the 3-amino-2-hydroxy fatty acid moiety of the formula (1) may be S, R or SR. Is (2S, 3R)
An arrangement is preferred.

The amino acid residue in X or Y in the formula (1) generally includes α-amino acid residues, but need not necessarily be α-amino acids. β-
Β-amino acids such as alanine may be used. Specific examples of these amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), and threonine (Th).
r), cysteine (Cys), methionine (Met), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), phenylalanine (P
he), tyrosine (Tyr), histidine (His), tryptophan (Try), homophenylalanine (Hph), proline (Pro), oxyproline (Hypro), cyclohexylalanine (Ch) and the like. The configuration of these amino acids may be D, L or DL, but the L configuration is preferred.

The amino acid residue in X is preferably an amino acid residue having a basic side chain such as arginine, lysine, hydroxylysine or histidine, and valine,
The residue of phenylalanine is preferred. Particularly, in the present invention, valine (Val), phenylalanine (Phe), histidine (His) or arginine (Arg) are preferable as the amino acid residue in X, and histidine (His) or arginine (Arg) is more preferable.
The residue of Preferred amino acid residues in Y include phenylalanine (Phe), histidine (His) and proline (Pro) residues, more preferably phenylalanine (Phe) residues. The festestine derivative of the present invention can also be used as a pharmacologically acceptable salt with an acid. These salts include inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid; and organic acids such as p-toluenesulfonic acid. And the like.

[0009] In addition to the above amino acids, the abbreviations used in the present specification for amino acid residues, protecting groups and others are as follows. AP-N: aminopeptidase N Leu-AP: leucine aminopeptidase AP-A: aminopeptidase A AP-B: aminopeptidase B AHPA: (2S, 3R) -3-amino-2-hydroxy-4-phenylbutanoic acid AHCA : (2S, 3R) -3-amino-4-cyclohexyl-2-hydroxybutanoic acid PenAhp: (2S, 3R) -3-amino-2-hydroxyoctanoic acid EtAhp: (2S, 3R) -3-amino-2 -Hydroxypentanoic acid Boc: t-butoxycarbonyl Bzl: benzyl Me: methyl Z: benzyloxycarbonyl Ph: phenyl cHx: cyclohexyl DMF: N, N-dimethylformamide

Equation (1)

Embedded image Specific examples of typical compounds among the compounds represented by are shown in the following table.

[0011]

[Table 1]

[0012]

[Table 2]

[0013]

[Table 3] Note: The configuration of each amino acid may be D, L or DL.

The compound of the present invention represented by the formula (1) is produced by a method generally used in the field of peptide chemistry. For example, it can be produced by the method of the following reaction formula (A) and reaction formula (B).

Reaction formula (A)

Embedded image

In the above reaction formula, R, X and Y have the same meaning as described above. P3 represents an amino-protecting group. The step represented by the reaction formula (A) will be described in more detail. The compound represented by the formula (A1), which is a starting compound, can be produced by a known method. For example, AHPA (R =
Phenyl group) can be obtained from Z-phenylalanine by a method described in the literature [J. Med. Chem., 20, 513 (19
77)). AHCA (R =
The cyclohexyl group), PenAhp (R = n-butyl group) or EtAhp can be obtained from aspartic acid by the method described in the literature [Tetrahedron Lett., 34, 7].
557 (1993)]. The above-mentioned PenAhp and EtAhp can be obtained by the method described in another literature [J. Antibiotics, 49, 281 (199
6) or Peptide Chemistry 1995, 145
(1996)]. Other compounds represented by the formula (A1) can be produced in the same manner.

The compound represented by the formula (A2) has the formula (A)
The amino group of the compound represented by 1) may be protected with a protecting group P3 according to a conventional method. For example, when P3 is a Boc group, it may be reacted with di-t-butyl dicarbonate in the presence of triethylamine. When P3 is a Z group, S-benzyloxycarbonyl-
It may be reacted with 4,6-dimethyl-2-thiopyrimidine. The compound of the formula (A2) having another protecting group can also be produced by a conventional method.

Reaction formula (B)

Embedded image

In the above reaction formula, R, X and Y have the same meaning as described above. P1 represents a carboxyl protecting group,
P2 and P3 represent a protecting group for an amino group. X (Ps)
And Y (Ps) indicate that the functional groups on the side chains of amino acid residues X and Y are protected by a protecting group (Ps) as necessary. The step represented by the reaction formula (B) will be described in more detail. In order to produce the compound represented by the formula (B2) from the compound represented by the formula (B1) which is the other starting compound, the compound represented by the formula (B1) and the protected amino acid (P2
-X (Ps) -OH) may be condensed by a peptide bond forming reaction. The peptide bond formation reaction includes an acid halide method, hydroxysuccinimide ester, a substituted and unsubstituted phenyl ester, an active ester method using a thiophenyl ester, etc., dicyclohexylcarbodiimide,
1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or a method using them and a condensation auxiliary such as 1-hydroxybenzotriazole, N-hydroxysuccinimide, diphenylphosphoric azide, Bop reagent, PyBop reagent, Amides used in peptide chemistry such as a method using a condensing agent such as -ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline, a mixed acid anhydride method using ethyl chloroformate, isobutyl chloroformate or the like, and azide method What is necessary is just to select suitably from among formation reactions.

Further, as the solvent used in the reaction, a solvent used in ordinary peptide chemistry can be used. For example, tetrahydrofuran, ethers such as dioxane, halogenated hydrocarbons such as dichloromethane and chloroform, dimethylformamide, amides such as dimethylacetamide, nitriles such as acetonitrile,
It may be used alone or as a mixed solvent among esters such as ethyl acetate and ketones such as acetone and methyl ethyl ketone. Next, the protecting group P2 of the amino group of the compound represented by the formula (B2) is removed to produce a compound represented by the formula (B3). The removal of the protecting group P2 may be performed by a conventional method. For example, when the protecting group P2 is a Boc group, the protecting group P2 may be removed using a deprotection reagent such as a hydrochloric acid-dioxane solution or trifluoroacetic acid. In the case of Z group,
It may be removed by catalytic hydrogenation in the presence of a Pd catalyst. Other protecting groups can be removed by a conventional method.

Next, the compound represented by the formula (A2) produced by the reaction formula (A) and the compound represented by the formula (B3) are prepared using the same method for forming a peptide bond as when the compound represented by the formula (B2) is obtained. To give the compound of formula (B4). The compound represented by the formula (1) can be produced by removing the protecting groups P1, P3 and Ps of the compound represented by the formula (B4) according to a conventional method. For example, when the protecting groups P3 and Ps are Z groups and the protecting group P1 is a benzyl ester, all of the protecting groups may be removed at once by catalytic hydrogenation in the presence of a Pd catalyst.
oc group, X (Ps) = X and Y (Ps) = Y (ie,
When it is not necessary to protect the functional groups on the side chains of the amino acid residues X and Y), when the protecting group P1 is a benzyl ester,
The protecting group P1 may be removed by catalytic hydrogenation in the presence of a Pd catalyst, and the Boc group of the protecting group P3 may be removed using a deprotecting reagent such as a hydrochloric acid-dioxane solution or trifluoroacetic acid. When P1 is a lower alkyl ester type protecting group, it may be removed by alkali saponification using about 1M sodium hydroxide solution. Other protecting groups can be removed by a conventional method. The compound of the formula (1) thus obtained may be converted, if necessary, to a pharmacologically acceptable salt. The conversion into a salt is carried out by converting the compound of the formula (1) into an inorganic acid such as hydrochloric acid or nitric acid, or p-
It can be carried out by treating with an organic acid such as toluenesulfonic acid by a conventional method.

The febestin derivative of the formula (1) or a pharmacologically acceptable salt thereof of the present invention exhibits an enzyme inhibitory activity, particularly an excellent inhibitory activity against aminopeptidase N, and is useful as an anticancer agent or an immunostimulant. Applications are expected. Accordingly, a further aspect of the present invention is a drug, particularly an aminopeptidase N inhibitor, comprising a phevestin derivative or a pharmaceutically acceptable salt thereof and a pharmaceutical additive. The pharmaceutical additives are not particularly limited, and those generally used can be used. Although the ratio of the active ingredient (febestin derivative or its salt) in the above-mentioned medicines, particularly the inhibitor composition, varies depending on the dosage form and the like, it cannot be said unconditionally.
It can be used in a wide range from about 05 to 99% by weight, usually about 0.1 to 50% by weight for injections, and about 1 to 60% by weight for other preparations. The balance is a pharmaceutical additive.

Administering an effective amount of a febestin derivative or a pharmacologically acceptable salt thereof to a warm-blooded animal, including humans, usually orally or parenterally such as intravenously, intradermally, or intramuscularly. Can inhibit aminopeptidase N in the living body. The dosage depends on the subject to be administered,
It varies depending on the administration route and the like.
It is 50 mg / Kg / day, preferably 0.5-100 mg / Kg / day, more preferably 1-50 mg / Kg / day.

The preparation for administration may be a conventionally used dosage form. In the case of oral administration, tablets, granules, capsules and the like molded together with a pharmaceutical excipient such as a common excipient such as starch are used. In the case of parenteral administration, ordinary injections formulated with pharmaceutical additives such as physiological saline, dissolving agents and the like are used.

[0025]

Industrial Applicability According to the present invention, it is represented by the formula (1), which is expected to be used as an active ingredient of an anticancer agent or an immunostimulant, having an enzyme inhibitor having an enzyme specificity, particularly an aminopeptidase N inhibitory action. Febestin and its derivatives are obtained.

[0026]

EXAMPLES The present invention will be described below in more detail with reference to examples and test examples, but the present invention is not limited thereto. Reference Example 1 Boc-L-valyl-L-phenylalanine benzyl ester
Ester synthesis Boc-L-valine 358.4mg (1.65mmol), phenylalanine benzyl ester · p-toluenesulfonate 642.
2 mg (1.5 mmol) and 1-hydroxybenzotriazole
Dissolve 252.5 mg (1.65 mmol) of monohydrate in 6.5 ml of DMF,
Under ice cooling, 0.235 ml (1.65 mmol) of triethylamine and 317.8 mg (1.65 mmol) of water-soluble carbodiimide monohydrochloride were added, and the mixture was stirred for 2 hours and further stirred at room temperature for 20 hours. After completion of the reaction, 50 ml of a 10% aqueous citric acid solution was added, and the mixture was extracted twice with 50 ml of ethyl acetate. Wash the organic layer with 50 ml of 10% aqueous citric acid solution.
The mixture was washed twice with 50 ml of a 4% aqueous solution of sodium hydrogencarbonate and once with 50 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain crude Boc-L-valyl-L-.
684.7 mg of phenylalanine benzyl ester were obtained. Chloroform was added to the crude product to dissolve it, and hexane was added to the crude product to crystallize at a low temperature. The crystals were collected by filtration, washed with hexane, and dried to obtain 644.0 mg of Boc-L-valyl-L-phenylalanine benzyl ester as white crystals.
I got 94.7% yield. FAB-MS m / Z 455 (M + H) +.

Reference Example 2 Boc-AHPA-L-valyl-L-phenylalanine
Synthesis of benzyl ester Boc-L-valyl-L-phenylalanine benzyl ester (530.0 mg, 1.17 mmol) was added to 4N hydrochloric acid-dioxane (5 ml).
, And stirred at room temperature for 1 hour.
A white powder of L-valyl-L-phenylalanine benzyl ester hydrochloride was obtained. 250 mg (0.64 mmol) of this
In addition, Boc-AHPA 207.7 mg (0.70 mmol), 1-hydroxybenzotriazole monohydrate 107.8 mg (0.70 mmol
l) and 2.5 ml of DMF were added and dissolved, and 0.0985 ml (0.70 mmol) of triethylamine and 147.6 mg (0.77 mmol) of water-soluble carbodiimide monohydrochloride were added under ice-cooling, followed by stirring for 2 hours.
Stirred at room temperature for another 16 hours. After completion of the reaction, 50 ml of a 10% aqueous citric acid solution was added, and the mixture was extracted twice with 50 ml of ethyl acetate.
The organic layer was washed once with 50 ml of a 10% aqueous citric acid solution, twice with 50 ml of a 4% aqueous sodium hydrogen carbonate solution and once with 50 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and evaporated to remove crude Boc. Thus, 414.6 mg of -AHPA-L-valyl-L-phenylalanine benzyl ester was obtained. Chloroform was added to the crude product to dissolve it, and hexane was added thereto.
Crystallized at low temperature. The crystals were collected by filtration, washed with hexane, and dried to obtain white crystals of Boc-AHPA-L.
-Valyl-L-phenylalanine benzyl ester 380.
8 mg were obtained. 94.3% yield. FAB-MS m / Z 632 (M + H) +.

Reference Example 3 AHPA-L-valyl-L-phenylalanine (Fevee
Palladium black 50m in the synthesis Boc-AHPA-L- valyl -L- phenylalanine benzyl ester 337.1mg (0.53mmol) of cystine)
g, 3 ml of acetic acid and 3 ml of methanol were added, and the system was replaced with hydrogen and then catalytically reduced at room temperature for 1 hour. After the catalyst was filtered off, the filtrate was evaporated to remove white powder of Boc-AHPA-.
270.8 mg of L-valyl-L-phenylalanine were obtained. To this was added 2.5 ml of trifluoroacetic acid, and after stirring at room temperature for 1 hour, the solvent was distilled off to obtain 204.9 mg of AHPA-L-valyl-L-phenylalanine. This was purified by high performance liquid chromatography (HPLC) (column: CAPCELL
PAK C18, SHISEIDO, 20 × 250 mm, eluent: acetonitrile: 0.1% trifluoroacetic acid = 6: 19, flow rate: 8 ml / min, detection wavelength: 210 nm), and AHPA-L-
The fraction containing valyl-L-phenylalanine was concentrated to dryness. This was dissolved in 3 ml of methanol, applied to a column of Sephadex LH-20 (550 ml) swollen with methanol, and developed with methanol. The fraction containing AHPA-L-valyl-L-phenylalanine was concentrated under reduced pressure to obtain 190.1 mg of a white powder. 88.5% yield. FAB-MS m / Z 442 (M + H) +.

Example 1 Boc-L-valyl-L-proline benzyl ester
478.0 mg (2.2 mmol) of synthetic Boc-L-valine, 439.4 mg (1.82 mmol) of L-proline benzyl ester monohydrochloride and 1-
337.6 mg of hydroxybenzotriazole monohydrate (2.2 m
mol) and dissolved in ice-cooling with 0.308 ml (2.2 mmol) of triethylamine and water-soluble carbodiimide.
After 421.7 mg (2.2 mmol) of hydrochloride was added and stirred for 2 hours, the mixture was further stirred at room temperature for 20 hours. After completion of the reaction, 10% aqueous citric acid solution
50 ml was added, and the mixture was extracted twice with 50 ml of ethyl acetate. Organic layer
Washed once with 50 ml of 10% aqueous citric acid solution, twice with 50 ml of 4% aqueous sodium hydrogen carbonate solution and once with 50 ml of saturated saline,
After drying over anhydrous sodium sulfate, the solvent was distilled off to obtain 788.0 mg of crude Boc-L-valyl-L-proline benzyl ester as a candy. The crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 1)) to obtain 710.9 mg of candy L-proline benzyl ester. 96.7% yield. FAB-MS m / Z 405 (M + H) +.

Example 2 Boc-AHPA-L-valyl-L-proline benzyl
Synthesis of esters Boc-L-valyl-L-proline benzyl ester 34
To 3.8 mg (0.85 mmol) of 3.5 N hydrochloric acid-dioxane (3.5 ml) was added, and the mixture was stirred at room temperature for 1 hour, and then the solvent was distilled off to obtain L-valyl-L-proline benzyl ester hydrochloride as a white powder. Boc-AHPA 1 was added to 140.4 mg (0.41 mmol) of this L-valyl-L-proline benzyl ester hydrochloride.
To 33.4 mg (0.45 mmol) and 68.9 mg (0.45 mmol) of 1-hydroxybenzotriazole monohydrate were added and dissolved 1.5 ml of DMF, and the mixture was dissolved in ice-cooled water-soluble carbodiimide monohydrochloride 86.3.
mg (0.45 mmol) was added, and after stirring for 15 minutes, triethylamine was added.
063 ml (0.45 mmol) was added and the mixture was stirred for 2 hours. Further, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, 10% aqueous citric acid solution
50 ml was added, and the mixture was extracted twice with 50 ml of ethyl acetate. Organic layer
Washed once with 50 ml of 10% aqueous citric acid solution, twice with 50 ml of 4% aqueous sodium hydrogen carbonate solution and once with 50 ml of saturated saline,
After drying over anhydrous sodium sulfate, the solvent was distilled off to obtain 231.3 mg of crude Boc-AHPA-L-valyl-L-proline benzyl ester. The crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 1)) to obtain Boc-AHP as a white powder.
AL-Valyl-L-proline benzyl ester 203.3m
g. 85.1% yield. FAB-MS m / Z 582 (M + H) +.

Example 3 Synthesis of AHPA-L-valyl-L-proline To 150.2 mg of Boc-AHPA-L-valyl-L-proline benzyl ester, 25 mg of palladium black, 1.5 mg of acetic acid and 1.5 ml of methanol were added. After hydrogen replacement, at room temperature
Catalytic reduction was carried out for hours. After the catalyst was separated by filtration, the filtrate was evaporated to remove the solvent, thereby obtaining white powder Boc-AHPA-L-valyl-L-.
134.4 mg of proline were obtained. 1 ml of trifluoroacetic acid
After stirring at room temperature for 1 hour, the solvent was distilled off to obtain 109.1 mg of crude AHPA-L-valyl-L-proline.
This crude product was purified by HPLC (column: CAPCELL P
AK C18, SHISEIDO, 20 × 250 mm, eluent: acetonitrile: 0.1% trifluoroacetic acid = 4: 21, flow rate: 8 ml / min., Detection wavelength: 210 nm), and AHPA-L-
The fraction containing valyl-L-proline was concentrated to dryness. This was dissolved in 2 ml of methanol, applied to a column of Sephadex LH-20 (550 ml) swollen with methanol, and developed with methanol. AHPA-L-Valyl-L
-The fraction containing proline was concentrated under reduced pressure to obtain 90.4 mg of a white powder. Yield 89.1%. FAB-MS m / Z 392 (M + H) +.

Example 4 of Boc-L-valyl-L-histidine methyl ester
521.5 mg (2.4 mmol) of synthetic Boc-L-valine, 518.4 mg (2.0 mmol) of L-histidine methyl ester dihydrochloride, 367.8 mg (2.4 mmol) of 1-hydroxybenzotriazole monohydrate
And benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide (Bop
(Reagent) 1063.2 mg (2.4 mmol) was dissolved in DMF (7 ml), triethylamine (0.98 ml, 7.0 mmol) was added under ice cooling, and the mixture was stirred for 1 hour and further stirred at room temperature for 16 hours. After the reaction, 4
50 ml of sodium hydrogencarbonate and 50 ml of ethyl acetate.
Extracted times. The organic layer is treated with a 4% aqueous sodium hydrogen carbonate solution 50
After washing twice with 50 ml of water, once with 50 ml of water and once with 50 ml of saturated saline, drying over anhydrous sodium sulfate and distilling off the solvent, 774.3 mg of crude Boc-L-valyl-L-histidine methyl ester was obtained. Obtained. This crude product was purified by column chromatography (silica gel, chloroform: methanol (10
0: 3)) to obtain 553.0 mg of Boc-L-valyl-L-histidine methyl ester as white powder. yield
75.0%. FAB-MS m / Z 369 (M + H) +.

Example 5 Boc-AHPA-L-valyl-L-histidine methyl
Synthesis of esters Boc-L-valyl-L-histidine methyl ester 51
To 3.6 mg (1.39 mmol) was added 7 ml of 4N hydrochloric acid-dioxane,
After stirring at room temperature for 1 hour, the solvent was distilled off to obtain L-valyl-L-histidine methyl ester dihydrochloride as a white powder. Boc-AH was added to 178.9 mg (0.52 mmol) of these.
PA 177.5 mg (0.60 mmol), 1-hydroxybenzotriazole monohydrate 92.1 mg (0.6 mmol), benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide salt 266.8 mg (0.6 mmol) and DM
F 2 ml was added and dissolved, and triethylamine 0.24 was added under ice cooling.
After adding 5 ml (1.75 mmol) and stirring for 1 hour, the mixture was stirred
Stirred for hours. After completion of the reaction, 4% sodium bicarbonate 50
Then, the mixture was extracted twice with 50 ml of ethyl acetate. 4 organic layers
The mixture was washed once with 50 ml of a 50% aqueous sodium hydrogen carbonate solution and once with 50 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain crude Boc-AHPA-L-valyl-.
299.2 mg of L-histidine methyl ester were obtained. This crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 6)) to obtain 230.6 mg of Boc-AHPA-L-valyl-L-histidine methyl ester as a white powder. Yield 81.1%. FAB-MS m / Z 546 (M + H) +.

Example 6 Synthesis of AHPA-L-valyl-L-histidine To 216.4 mg (0.40 mmol) of Boc-AHPA-L-valyl-L-histidine methyl ester was dissolved by adding 3.96 ml of methanol, and 1M hydroxylation was performed. 0.87 ml of sodium aqueous solution (0.79 m
mol) and stirred at room temperature for 3.5 hours.
083 ml, and the solvent was distilled off to obtain crude Boc-AHP.
350.2 mg of AL-valyl-L-histidine were obtained. 2.5 ml of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 1 hour, and then the solvent was distilled off to obtain crude AHPA-L-valyl-L-histidine / 1 trifluoroacetate. This crude body is HPL
C purification (column: CAPCELL PAK C18, SH
ISEIDO, 20 × 250mm, eluent: acetonitrile: 0.1
% Trifluoroacetic acid = 9: 91, flow rate: 8 ml / min., Detection wavelength: 210 nm), and the fraction containing AHPA-L-valyl-L-histidine was concentrated to dryness. This was dissolved in 2 ml of methanol, applied to a column of Sephadex LH-20 (550 ml) swollen with methanol, and developed with methanol. AHPA-L-Valyl-L-histidine-1
The fraction containing trifluoroacetate is concentrated under reduced pressure,
166.8 mg of a white powder were obtained. Yield 78.3%. FAB-MS m / Z 432 (M + H) +.

Example 7 Boc-L-phenylalanyl-L-histidinemethyl
Synthesis of ester Boc-L-phenylalanine 636.5 mg (2.4 mmol), L-
Histidine methyl ester dihydrochloride 518.3mg (2.0mmo
l), 1-hydroxybenzotriazole monohydrate 367.
6 mg (2.4 mmol) and 1063.2 mg (2.4 mmol) of benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide were dissolved in 7 ml of DMF, and 0.98 ml (7.0 mmol) of triethylamine was added under ice-cooling for 1 hour. After stirring, the mixture was further stirred at room temperature for 16 hours. After the reaction, 4
50 ml of sodium hydrogencarbonate and 50 ml of ethyl acetate.
Extracted times. The organic layer is treated with a 4% aqueous sodium hydrogen carbonate solution 50
The mixture was washed twice with 50 ml of water, once with 50 ml of water and once with 50 ml of saturated saline, dried over anhydrous sodium sulfate, and evaporated to remove crude Boc-L-phenylalanyl-L-histidine methyl ester. mg was obtained. The crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 3)) to give Boc-
L-phenylalanyl-L-histidine methyl ester
779.3 mg were obtained. 93.6% yield. FAB-MS m / Z 417 (M + H) +.

Example 8 Boc-AHPA-L-phenylalanyl-L-hist
Synthesis of gin methyl ester Boc-L-phenylalanyl-L-histidine methyl ester 735.8 mg (1.77 mmol) was added to 4N hydrochloric acid-dioxane 10
Then, the mixture was stirred at room temperature for 1 hour, and the solvent was distilled off to obtain a white powder of L-phenylalanyl-L-histidine methyl ester dihydrochloride. 194.6mg of this
(0.5 mmol) to 177.4 mg (0.6 mmol) of Boc-AHPA, 1
92.3 mg of hydroxybenzotriazole monohydrate (0.6
mmol), benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide salt 26
5.9 mg (0.6 mmol) and 2.5 ml of DMF were added to dissolve, 0.245 ml (1.75 mmol) of triethylamine was added under ice cooling, and the mixture was stirred for 1 hour and further stirred at room temperature for 16 hours. After completion of the reaction, 50 ml of 4% sodium hydrogen carbonate was added, and ethyl acetate 50 ml was added.
Extracted twice with ml. The organic layer was washed once with 50 ml of a 4% aqueous sodium hydrogen carbonate solution and once with 50 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and evaporated to remove crude B.
321.4 mg of oc-AHPA-L-phenylalanine-L-histidine methyl ester was obtained. The crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 5)) to obtain a white powder Bo.
262.5 mg of c-AHPA-L-phenylalanyl-L-histidine methyl ester were obtained. 88.3% yield. FAB-MS m / Z 594 (M + H) +.

Example 9 Synthesis of AHPA-L-phenylalanyl-L-histidine
Methanol 3 for forming Boc-AHPA-L- phenylalanyl -L- histidine methyl ester 225.2mg (0.38mmol).
8 ml was added and dissolved, and a 1 M aqueous solution of sodium hydroxide was added.
After adding 834 ml (0.83 mmol) and stirring at room temperature for 3.5 hours, 0.078 ml of trifluoroacetic acid was added and the solvent was distilled off to obtain crude B
351.3 mg of oc-AHPA-L-phenylalanyl-L-histidine were obtained. To this was added 2.5 ml of trifluoroacetic acid, and after stirring at room temperature for 1 hour, the solvent was distilled off to obtain crude AHPA-L.
-Phenylalanyl-L-histidine / 1 trifluoroacetate was obtained. This crude product was purified by HPLC (column: CA
PCELL PAK C18, SHISEIDO, 20 × 250mm
Eluent: acetonitrile: 0.1% trifluoroacetic acid = 3:22, flow rate: 8 ml / min., Detection wavelength: 210 nm)
The fraction containing AHPA-L-phenylalanyl-L-histidine was concentrated to dryness. 2 ml of this
Sephadex LH dissolved in methanol and swollen with methanol
It was applied to a -20 (550 ml) column and developed with methanol. The fraction containing AHPA-L-phenylalanyl-L-histidine / 1-trifluoroacetate was concentrated under reduced pressure to obtain 165.2 mg of a white powder. Yield 73.4%. FAB-MS m / Z 480 (M + H) +.

Example 10 Boc-L-histidyl (Nπ-benzyloxymethy
B) Synthesis of L-phenylalanine benzyl ester Boc-L-histidyl (Nπ-benzyloxymethyl) monohydrate 592.1 mg (1.5 mmol), L-phenylalanine benzyl ester p-toluenesulfonate 705.7 m
g (1.65 mmol) and 1-hydroxybenzotriazole
Dissolve 254.5 mg (1.66 mmol) of monohydrate in 6.5 ml of DMF,
Under ice-cooling, add 0.235 ml (1.65 mmol) of triethylamine
After stirring for 15 minutes, water-soluble carbodiimide monohydrochloride 318.4 mg
(1.65 mmol) was added and the mixture was stirred for 2 hours. In addition, 16
Stirred for hours. After completion of the reaction, 50 ml of a 10% aqueous citric acid solution was added, and the mixture was extracted twice with 50 ml of ethyl acetate. The organic layer was washed once with 50 ml of a 10% aqueous citric acid solution, twice with 50 ml of a 4% aqueous sodium hydrogen carbonate solution and once with 50 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and evaporated to remove crude Boc.
-L-histidyl (Nπ-benzyloxymethyl)-
937.1 mg of L-phenylalanine benzyl ester was obtained. Ethyl acetate was added to the crude to dissolve it, and hexane was added to the crude to crystallize at low temperature. The crystals were collected by filtration, washed with hexane, and dried to give Boc-L-
838.8 mg of histidyl (Nπ-benzyloxymethyl) -L-phenylalanine benzyl ester was obtained. Yield 9
1.3%. FAB-MS m / Z 613 (M + H) +.

Example 11 Boc-AHPA-L-histidyl (Nπ-benzylo
Xymethyl) -L-phenylalanine benzyl ester
Synthesis of Boc-L-histidyl (Nπ-benzyloxymethyl) -L-phenylalanine benzyl ester 805.1 mg
(1.31 mmol) was added with 4N hydrochloric acid-dioxane (8 ml), and the mixture was stirred at room temperature for 1 hour.
877.8 mg of -histidyl (Nπ-benzyloxymethyl) -L-phenylalanine benzyl ester dihydrochloride / 1 dioxane was obtained. To 335.8 mg (0.50 mmol) of these, 164.8 mg (0.56 mmol) of Boc-AHPA, 86.0 mg (0.56 mmol) of 1-hydroxybenzotriazole monohydrate and 3.5 ml of DMF were added and dissolved. After adding 0.154 ml (1.1 mmol) of triethylamine and stirring for 15 minutes, 108.3 mg (0.56 mmol) of water-soluble carbodiimide monohydrochloride was added, and the mixture was stirred for 2 hours. Further, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, 50 ml of a 10% aqueous citric acid solution was added, and the mixture was extracted twice with 50 ml of ethyl acetate. Wash the organic layer with 50 ml of 10% aqueous citric acid solution.
The mixture was washed twice with 50 ml of a 4% aqueous sodium hydrogen carbonate solution and once with 50 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and evaporated to remove crude Boc-AHPA-L-histidyl (Nπ-benzyloxyl). 425.4 mg of methyl) -L-phenylalanine benzyl ester were obtained. This crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 3)) to give 348.1 mg of Boc-AHPA-L-histidyl (Nπ-benzyloxymethyl) -L-phenylalanine benzyl ester as white powder ( 0.44 mmol). Yield 88.0%. FAB-MS m / Z 790 (M + H) +.

Example 12 Synthesis of AHPA-L-histidyl-L-phenylalanine
Formed Boc-AHPA-L- histidyl (Enupai- benzyloxymethyl) -L- phenylalanine benzyl ester
To 242.0 mg (0.31 mmol), 50 mg of palladium black, 2 m of acetic acid and 2 ml of methanol were added, and the system was replaced with hydrogen and subjected to catalytic reduction at room temperature for 27 hours. Further 50 mg of palladium black and 3 ml of acetic acid
Was added and the mixture was catalytically reduced at room temperature for 20 hours. After filtering off the catalyst,
By evaporating the solvent of the filtrate, white powder Boc-AHPA was obtained.
Thus, 206.0 mg of -L-histidyl-L-phenylalanine was obtained. To this, 1 ml of trifluoroacetic acid was added, and after stirring at room temperature for 1 hour, the solvent was distilled off, and crude AHPA-L-histidyl-
L-phenylalanine / 1 trifluoroacetate 266.7mg
I got This crude product was purified by HPLC (column: CAPCE).
LL PAK C18, SHISEIDO, 20 × 250 mm, eluent: acetonitrile: 0.1% trifluoroacetic acid = 9: 4
1, flow rate: 8 ml / min., Detection wavelength: 210 nm), and AHPA
The fraction containing -L-histidyl-L-phenylalanine was concentrated to dryness. This was dissolved in 2 ml of methanol, and Sephadex LH-20 swollen with methanol.
(550 ml) and developed with methanol. AH
The fractions containing PA-L-histidyl-L-phenylalanine / 1 trifluoroacetate were concentrated under reduced pressure, and 151.
8 mg (0.256 mmol) of a white powder were obtained. 83.7% yield. FAB-MS m / Z 480 (M + H) +.

Example 13 Synthesis of Boc-AHCA To 2.38 g (10.0 mmol) of AHCA, 2.42 g (11.1 mmol) of di-t-butyl dicarbonate, 20 ml of water and 40 ml of dioxane were added and dissolved, and triethylamine was added under ice cooling. 2.94ml (21.0m
mol) was added and stirred for 30 minutes. After stirring at room temperature for further 16 hours, the solvent was distilled off. Then, the reaction mixture was diluted with 10 ml of water, adjusted to pH 2 with a 5% aqueous solution of potassium hydrogen sulfate, and extracted three times with 30 ml of ethyl acetate. The oil layer was washed with 30 ml of a saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain 3.00 g of Boc-AHCA. 99.4% yield. Rf 0.52 (chloroform: methanol: acetic acid = 9
0: 10: 5). FAB-MS m / z 324 (M + Na) +.

Example 14 Boc-AHCA-L-valyl-L-phenylalanine
Synthesis of benzyl ester L-valyl-L-phenylalanine benzyl ester monohydrochloride (100.4 mg, 0.26 mmol) obtained in Example 2 was added to Bo
86.8 mg (0.29 mmol) of c-AHCA, 44.2 mg (0.29 mmol) of 1-hydroxybenzotriazole monohydrate and DMF
Add 1 ml to dissolve, and add 0.04 ml of triethylamine under ice-cooling.
(0.29 mmol) and 55.3 mg of water-soluble carbodiimide monohydrochloride
(0.767 mmol) was added, and the mixture was stirred for 2 hours and further stirred at room temperature for 16 hours. After completion of the reaction, 50 ml of a 10% aqueous citric acid solution was added, and the mixture was extracted twice with 50 ml of ethyl acetate. The organic layer is once with 50 ml of 10% citric acid aqueous solution, and 4% aqueous sodium hydrogen carbonate solution
After washing twice with 50 ml and once with 50 ml of saturated saline, drying over anhydrous sodium sulfate and distilling off the solvent, crude Boc-
165.1 mg of AHCA-L-valyl-L-phenylalanine benzyl ester was obtained. Chloroform was added to the crude product to dissolve it, and hexane was added to the crude product to crystallize at a low temperature. The crystals were collected by filtration, washed with hexane, and dried to obtain 148.2 mg of Boc-AHCA-L-valyl-L-phenylalanine benzyl ester as white crystals. Yield 89.4
%. FAB-MS m / Z 638 (M + H) +.

Example 15 Synthesis of AHCA-L-valyl L-phenylalanine Boc-AHCA-L-valyl-L-phenylalanine benzyl ester (109.4 mg, 0.17 mmol) was added to palladium black 15
mg, 1 ml of acetic acid and 1 ml of methanol were added, and the system was replaced with hydrogen and catalytically reduced at room temperature for 1 hour. After the catalyst was separated by filtration, the filtrate was evaporated to remove Boc-AHCA- as a white powder.
106.1 mg of L-valyl-L-phenylalanine were obtained. To this was added 1 ml of trifluoroacetic acid, and the mixture was stirred at room temperature for 1 hour, and the solvent was distilled off to obtain 72.9 mg of crude AHCA-L-valyl-L-phenylalanine. Methanol was added to the crude product to dissolve it, and isopropyl ether was added thereto to crystallize. The crystals were collected by filtration, washed with isopropyl ether, and dried. It was further dissolved in 1 ml of methanol, applied to a column of Sephadex LH-20 (550 ml) swollen with methanol, and developed with methanol. The fraction containing AHCA-L-valyl-L-phenylalanine was concentrated under reduced pressure to obtain 59.6 mg of a white powder. Yield 78.3%. FAB-MS m / Z 448 (M + H) +.

Example 16 Boc-AHCA-L-histidyl (Nπ-benzylo
Xymethyl) -L-phenylalanine benzyl ester
Obtained in Example 14 of L- histidyl (Enupai- benzyloxymethyl) -L- phenylalanine benzyl ester · 2 Bo to hydrochloride 1 dioxane 236.4mg (0.35mmol)
117.4 mg (0.39 mmol) of c-AHCA, 61.7 mg (0.39 mmol) of 1-hydroxybenzotriazole monohydrate and DM
F 2.5 ml was added and dissolved.
After adding 108 ml (0.77 mmol) and stirring for 15 minutes, 74.6 mg (0.39 mmol) of water-soluble carbodiimide monohydrochloride was added and stirred for 2 hours. The mixture was further stirred at room temperature for 16 hours. 10% after reaction
An aqueous citric acid solution (50 ml) was added, and the mixture was extracted twice with ethyl acetate (50 ml). The organic layer is once with 50 ml of 10% aqueous citric acid solution, twice with 50 ml of 4% aqueous sodium hydrogen carbonate solution and 50 ml of saturated saline solution.
And dried over anhydrous sodium sulfate, and then the solvent was distilled off to obtain crude Boc-AHCA-L-histidyl (N
272.0 mg of π-benzyloxymethyl) -L-phenylalanine benzyl ester were obtained. The crude product was purified by column chromatography (silica gel, chloroform: methanol (100: 2)) to obtain a white powder Boc.
-AHCA-L-histidyl (Nπ-benzyloxymethyl) -L-phenylalanine benzyl ester 240.1 m
g (0.30 mmol) were obtained. Yield 86.3%. FAB-MS m / Z 796 (M + H) +.

Example 17 Synthesis of AHCA-L-histidyl-L-phenylalanine
Formed Boc-AHCA-L- histidyl (Enupai- benzyloxymethyl) -L- phenylalanine benzyl ester
To 240.1 mg (0.30 mmol), 60 mg of palladium black, 2.5 ml of acetic acid and 2.5 ml of methanol were added, and the system was replaced with hydrogen and subjected to catalytic reduction at room temperature for 25 hours. Further, 60 mg of palladium black and 1 ml of acetic acid were added, and the mixture was subjected to catalytic reduction at room temperature for 28 hours. After the catalyst was separated by filtration, the filtrate was evaporated to remove Boc as a white powder.
-AHCA-L-histidyl-L-phenyalanine 223.
5 mg were obtained. 1.5 ml of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 1 hour, and the solvent was distilled off to obtain crude AHCA-L-histidyl-L-phenylalanine / 1 trifluoroacetate.
271.3 mg were obtained. This crude product was purified by HPLC (column: C
APCELL PAK C18, SHISEIDO, 20 × 250m
m, eluent: acetonitrile: 0.1% trifluoroacetic acid
(11:39, flow rate: 8 ml / min., Detection wavelength: 210 nm)
Then, the fraction containing AHCA-L-histidyl-L-phenylalanine was concentrated to dryness. This was dissolved in 2 ml of methanol, applied to a column of Sephadex LH-20 (550 ml) swollen with methanol, and developed with methanol. The fraction containing AHCA-L-histidyl-L-phenylalanine / 1 trifluoroacetate was concentrated under reduced pressure to obtain 125.4 mg (0.21 mmol) of a white powder. Yield 59.7
%. FAB-MS m / Z 486 (M + H) +.

Example 18 Synthesis of Z-AHCA To 2.38 g (10.0 mmol) of AHCA was added 3.30 g (10.0 g) of S-benzyloxycarbonyl-4,6-dimethyl-2-thiopyrimidine.
2.0 mmol), 9.0 ml of water, 9.0 ml of dioxane and 3.50 ml (25.0 mmol) of triethylamine, and the mixture was stirred at room temperature for 20 hours. After completion of the reaction, 45 ml of water was added, and the mixture was washed twice with 40 ml of ethyl acetate, and the aqueous layer was cooled with an ice bath. 3.5 ml of 6N hydrochloric acid in the aqueous layer
After adjusting the pH to 2, the mixture was extracted three times with 30 ml of ethyl acetate. The oil layer was washed three times with 30 ml of 1N hydrochloric acid and twice with 30 ml of saturated saline.
After washing twice and drying over anhydrous sodium sulfate, the solvent was distilled off to obtain 1.48 g of Z-AHCA. On the other hand, the crude Z-AHCA recovered from the previous ethyl acetate wash was purified by column chromatography (silica gel, chloroform: methanol: acetic acid = 200: 2: 1 to 50: 2: 1) to give Z-AH.
1.68 g of CA was obtained. 94.1% yield.

Melting point 124-125 ° C [α] 24 / D + 51.3 ° (c1.0, methanol) FAB-MS m / z 334 (M-1) -1H-NMR (CD3 OD) δ: 0.75 -1.37 (m, 6H) 1.38-1.90 (m, 7H) 4.15 (d, 1H, J = 1.5 Hz) 4.27 (m, 1H) 5.06, 5.21 (ABq, 2H, J = 12.2 Hz) 5.15 (d, 1H, J = 9.8 Hz) 6.09 (br d) 7.23-7.40 (m, 5H)

Example 19 Boc-L-arginyl (Z2) -L-phenylalani
Synthesis of benzyl ester Boc-L-arginine (Z2) 352.7 mg (0.650 mmol),
292.2 mg (0.683 mmol) of L-phenylalanine benzyl ester / p-toluenesulfonate and 175.9 mg (1.302 mmol) of 1-hydroxybenzotriazole were dissolved in DMF (3.0 ml), and triethylamine 0.096 ml (0.686 mmo) was cooled on ice.
l) and 162.0 mg (0.845 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride were added, and the mixture was stirred for 2 hours and further stirred at room temperature for 6 hours. After completion of the reaction, the reaction solution was diluted with 30 ml of ethyl acetate, washed successively with a saturated aqueous solution of sodium hydrogencarbonate, a 10% aqueous solution of citric acid and 15 ml of saturated saline, dried over anhydrous sodium sulfate, and evaporated to remove the solvent. Thus, a crude white solid was obtained. Then, the white solid is purified by column chromatography (silica gel, chloroform: methanol (100: 1)).
By doing, Boc-L-arginyl (Z
2) 492.9 mg of -L-phenylalanine benzyl ester
I got 97.2% yield.

Melting point 138.5-139.5 ° C. [α] 26 / D + 6.6 ° (c1.0, chloroform) FAB-MS m / z 780 (M + H) + 1H-NMR (CDCl 3) δ: 1. 40 (s, 9H) 1.45-1.78 (m, 4H) 2.81 (dd, 1H, J = 6.8, 14.0 Hz) 3.00 (dd, 1H, J = 5.9, 14.0 Hz) 3.70-3.94 (m, 2H) 4.19 (m, 1H) 4.77 (ddd, 1H, J = 5.9, 6.8, 7.8 Hz) 5.05, 5.09 (ABq, 2H, J = 12.2 Hz) 5.05, 5.14 (ABq, 2H, J = 12.7 Hz) 5.21, 5.23 (ABq, 2H, J = 12.2 Hz) 5.55 (br d, 1H, J = 6.8 Hz) 6.74 (d, 1H, J = 7.8 Hz) 6.94 ( , 2H) 7.02-7.47 (m, 18H) 9.24 (br s, 1H) 9.42 (br s, 1H)

Example 20 Z-AHCA-L-arginyl (Z2) -L-phenyl
Synthesis of alanine benzyl ester To 253.4 mg (0.325 mmol) of Boc-L-arginyl (Z2) -L-phenylalanine benzyl ester, 2 ml of trifluoroacetic acid was added, and after stirring at room temperature for 40 minutes, the solvent was distilled off. The solvent was distilled off twice to obtain a syrupy L-arginyl (Z2) -L-phenylalanine benzyl ester trifluoroacetate. 118.3 mg (0.353 mmol) of Z-AHCA, 1-
88.5 mg (0.655 mmol) of hydroxybenzotriazole and 2.5 ml of DMF were added and dissolved, and under ice-cooling 0.050 ml (0.357 mmol) of triethylamine and 87.2 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
(0.455 mmol), and the mixture was stirred for 2 hours, and further stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was diluted with 25 ml of ethyl acetate, and washed successively with a saturated aqueous solution of sodium bicarbonate, a 10% aqueous solution of citric acid and 10 ml of saturated saline solution. After the oil layer was dried over anhydrous sodium sulfate, the solvent was distilled off to obtain an oily crude product. The crude product was purified by column chromatography (silica gel, hexane: ethyl acetate: methanol (70: 30: 1 to 60: 40: 2)) to give Z-AHCA-L-arginyl (Z2) -L as a white solid. -85.7 mg of phenylalanine benzyl ester were obtained. Yield 57.3
%. Next, this white solid was crystallized with a chloroform-hexane system.

Melting point 131-133 ° C. [α] 26 / D + 3.4 ° (c1.1, chloroform) FAB-MS m / z 997 (M + H) +1 H-NMR (CDCl 3) δ: 0.72-0. 99 (m, 2H) 1.00-1.88 (m, 15H) 2.95 (dd, 1H, J = 7.3, 13.8 Hz) 3.06 (dd, 1H, J = 6.1, (13.8 Hz) 3.85 (m, 3H) 3.95 (m, 1H) 4.10 (d, 1H, J = 5.9 Hz) 4.45 (br ddd, 1H) 4.76 (br ddd, 1H) 4.96, 5.03 (ABq, 2H, J = 12.7 Hz) 5.04, 5.10 (ABq, 2H, J = 12.2 Hz) 5.05, 5.10 (ABq, 2H, J = 11.7 Hz) 5.19, 5.22 (ABq, 2H, J = 12.2 Hz) 32 (d, 1H, J = 8.8 Hz) 6.94-7.05 (m, 3H) 7.06-7.19 (m, 3H) 7.20-7.43 (m, 21H) 9. 27 (br s, 1H) 9.39 (br s, 1H)

Example 21 Synthesis of AHCA-L-arginyl-L-phenylalanine
Forming Z-AHCA-L- arginyl (Z2)-L-phenylalanine benzyl ester 183.5Mg (0.184 mmol) in methanol - acetic acid - water (7: 2: 3) was dissolved in a solution 6 ml, Pd
-Stirred for 2 days under a hydrogen atmosphere in the presence of a black catalyst. After filtering off the catalyst, the crude product obtained by evaporating the filtrate was purified by column chromatography (Sephadex LH-20, developed with 1% acetic acid-methanol, then OD
S, water-methanol-acetic acid (50: 50: 1)) gave 92.4 mg of AHCA-L-arginyl-L-phenylalanine / 3/2 acetate as a white powder.

Melting point 141-148 ° C. [α] 26 / D + 8.3 ° (c 0.72, methanol) FAB-MS m / z 505 (M-acetic acid + 1) + 1H-NMR (CD3 OD) δ: 0. 86-1.06 (m, 2H) 1.14-1.92 (m, 15H) 1.93 (s, 4.5H) 2.94 (dd, 1H, J = 8.3, 13.9 Hz) 3.13 (t, 2H, J = 6.8 Hz) 3.22 (dd, 1H, J = 4.4, 13.9 Hz) 3.54 (m, 1H) 4.15 (d, 1H, J = (3.4 Hz) 4.33 (t, 1H, J = 6.6 Hz) 4.49 (dd, 1H, J = 4.4, 8.3 Hz) 7.10-7.30 (m, 5H)

Example 22 Boc-L-phenylalanyl-L-phenylalanine
Synthesis of benzyl ester Boc-L-phenylalanine 132.9 mg (0.501 mmol), L
-224.6 mg (0.525 mmol) of phenylalanine benzyl ester / p-toluenesulfonate and 135.4 mg (1.002 mmol) of 1-hydroxybenzotriazole were dissolved in 1.5 ml of DMF, and 0.074 ml (0.529 mmol) of triethylamine was cooled on ice.
And 1-ethyl-3- (3-dimethylaminopropyl)
124.6 mg (0.650 mmol) of carbodiimide hydrochloride was added, and the mixture was stirred for 2 hours and further stirred at room temperature for 19 hours. After the reaction,
The reaction solution was diluted with 15 ml of ethyl acetate, washed successively with a 4% aqueous sodium hydrogen carbonate solution, a 10% aqueous citric acid solution and 10 ml of saturated saline, and dried over anhydrous sodium sulfate.
The solvent was distilled off to obtain a crude crystal. Then, the white solid was purified by column chromatography (Sephadex LH-20, developed with methanol) to obtain Boc-
251.2 mg of L-phenylalanyl-L-phenylalanine benzyl ester were obtained. 99.8% yield.

Melting point 127 ° C [α] 23 / D + 8.8 ° (c1.0, chloroform) FAB-MS m / z 503 (M + H) + 1H-NMR (CDCl3) δ: 1.39 (s, 9H) 2.94-3.10 (m, 4H) 4.31 (m, 1H) 4.81 (dt, 1H, J = 6.4, 6.8 Hz) 4.93 (br s, 1H) 5.09 (S, 2H) 6.27 (d, 1H, J = 6.8 Hz) 6.89 (m, 2H) 7.10-7.40 (m, 13H)

Example 23 Z-AHCA-L-phenylalanyl-L-phenyla
Synthesis of lanine benzyl ester To 250.2 mg (0.498 mmol) of Boc-L-phenylalanyl-L-phenylalanine benzyl ester was added 2 ml of trifluoroacetic acid, and the mixture was stirred at room temperature for 40 minutes, and the solvent was distilled off. By performing the operation of distilling off the solvent twice, a white solid L-phenylalanyl-L-phenylalanine benzyl ester / trifluoroacetate was obtained.
175.4 mg (0.523 mmol) of Z-AHCAL, 135.1 mg (1.000 mmol) of 1-hydroxybenzotriazole and DM
Then, 2.0 ml of F was added and dissolved.
074 ml (0.529 mmol) and 124.1 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.64 mmol)
7 mmol), and the mixture was stirred for 2 hours and further stirred at room temperature for 17 hours. After completion of the reaction, the reaction mixture was diluted with 20 ml of ethyl acetate, and washed sequentially with a 4% aqueous sodium hydrogen carbonate solution, a 10% aqueous citric acid solution 10 ml, and a saturated saline solution 15 ml each. After the oil layer was dried over anhydrous sodium sulfate, the solvent was distilled off to obtain a crude white solid. This crude product was purified by column chromatography (silica gel, hexane: ethyl acetate: methanol (60: 40: 1 to 60: 40: 2)) to give Z-AHCA-L-phenylalanyl-L- as a white solid. 316.9 mg of phenylalanine benzyl ester were obtained. Yield 88.4
%. Next, this white solid was crystallized with a chloroform-hexane system.

Melting point 127-128 ° C [α] 28 / D-3.7 ° (c1.0, chloroform) FAB-MS m / z 720 (M + H) + 1H-NMR (CDCl3) δ: 0.73- 1.00 (m, 2H) 1.02-1.48 (m, 5H) 1.52-1.84 (m, 6H) 2.85-3.12 (m, 4H) 3.93 (m, 2H) 1H) 3.98 (dd, 1H, J = 2.9, 5.9 Hz) 4.53 (br d, 1H) 4.61 (br ddd, 1H) 4.78 (br ddd, 1H) 4.94 −5.13 (m, 4H) 5.35 (d, 1H, J = 8.3 Hz) 6.61 (br d, 1H) 6.92 (m, 2H) 7.05-7.39 (m, 19H)

Example 24 AHCA-L-phenylalanyl-L-phenylalani
Synthesis of Z-AHCA-L-phenylalanyl-L-phenylalanine benzyl ester 316.5 mg (0.440 mmol) was dissolved in methanol-acetic acid-water (6: 2: 1) solution (6 ml), and Pd-
The mixture was stirred under a hydrogen atmosphere in the presence of a black catalyst for 24 hours. After the catalyst was separated by filtration, the crude product obtained by distilling off the solvent of the filtrate was purified by column chromatography (Sephadex LH-20, developed with 5% acetic acid-methanol to obtain AH).
CA-L-phenylalanyl-L-phenylalanine 21
7.0 mg were obtained. 99.6% yield.

FAB-MS m / z 496 (M + H) + 1H-NMR (CD3 OD) δ: 0.77-1.00 (m, 2H) 1.13-1.40 (m, 5H) 1.48 (M, 1H) 1.60-1.79 (m, 5H) 2.96 (dd, 1H, J = 9.6, 13.4 Hz) 3.03 (dd, 1H, J = 6.4, 13) 3.4 Hz) 3.16 (dd, 1H, J = 4.8, 13.4 Hz) 3.21 (dd, 1H, J = 4.8, 13.4 Hz) 4.42 (ddd, 1H, J = 3) .4, 7.0, 7.0 Hz) 4.10 (d, 1H, J = 3.4 Hz) 4.45 (dd, 1H, J = 4.8, 6.4 Hz) 4.53 (dd, 1H) , J = 4.8, 9.6 Hz) 7.10-7.32 (m, 10H)

Example 25 Synthesis of Z-EtAhp 1.97 g (7.18 mmol) of S-benzyloxycarbonyl-4,6-dimethyl-2-thiopyrimidine corresponding to 5.99 mmol of EtAhp.trifluoroacetate, 6.0 ml of water, dioxane
6.0 ml and triethylamine 2.10 ml (15.0 mmol) were added,
Stirred at room temperature for 24 hours. Subsequently, the reaction mixture was diluted by adding 40 ml of water, adjusted to pH 2 by adding 14 ml of 1N hydrochloric acid, and extracted three times with 20 ml of ethyl acetate. 1N hydrochloric acid 30
After washing twice with water and once with 30 ml of saturated saline, drying over anhydrous sodium sulfate and distilling off the solvent, crude Z-EtA was obtained.
hp. This crude product was purified by column chromatography (silica gel, chloroform: methanol: acetic acid = 96:
3: 1) to obtain 1.18 g of Z-EtAhp. Yield 73.7%. FAB-MS m / z 268 (M + H) +.

Example 26 Z-EtAhp-L-arginyl (Z2) -L-phenyl
Synthesis of lualanine benzyl ester To 239.1 mg (0.307 mmol) of Boc-L-arginyl (Z2) -L-phenylalanine benzyl ester, 2 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour, and the solvent was distilled off. In addition, the operation of distilling off the solvent was performed three times to obtain L-arginyl (Z2) -L-phenylalanine benzyl ester trifluoroacetate. 86.2 mg (0.323 mmol) of Z-EtAhp was added to this salt, and Bop reagent 142.
7 mg (0.323 mmol) and 3.0 ml of chloroform were added and dissolved, and 0.129 ml (0.921 mmol) of triethylamine was added, followed by stirring at room temperature for 24 hours. After distilling off the solvent from the reaction mixture, the mixture was diluted with 15 ml of ethyl acetate, and washed successively with a 4% aqueous sodium hydrogen carbonate solution, a 10% aqueous citric acid solution and 10 ml each of saturated saline. After the oil layer was dried over anhydrous sodium sulfate, the solvent was distilled off to obtain an oily crude product. The crude product was purified by column chromatography (silica gel, hexane: ethyl acetate: methanol (50: 50: 1)) to obtain 192.9 mg of an amorphous solid. Yield 67.7%. A part of this solid was further purified by column chromatography (silica gel, chloroform: acetonitrile (100
: 1 to 10: 1)) to obtain Z-EtAhp-L-
Arginyl (Z2) -L-phenylalanine benzyl ester was obtained.

FAB-MS m / z 929 (M + H) + 1H-NMR (CDCl 3) δ: 0.88 (t, 3H, J = 7.3 Hz) 1.30-1.95 (m, 6H) 95 (dd, 1H, J = 7.3, 13.9 Hz) 3.07 (dd, 1H, J = 5.9, 13.9 Hz) 3.74 (m, 1H) 3.79-3.98 ( m, 3H) 4.09 (br, 1H) 4.45 (br ddd, 1H) 4.76 (br ddd, 1H) 4.88-5.16 (m, 6H) 5.19,5.21 (m, 3H) ABq, 2H, J = 12.2 Hz) 5.37 (d, 1H, J = 9.3 Hz) 6.91-ca. 7.41 (m, 26H, overlapping) 7.42 (d, 1H, J = 8.3 Hz) 9.28 (brs, 1H) 9.40 (brs, 1H)

Example 27 EtAhp-L-arginyl-L-phenylalanine
84.9 mg (0.440 mmol) of synthetic Z-EtAhp-L-arginyl (Z2) -L-phenylalanine benzyl ester was dissolved in 10 ml of a methanol-acetic acid-water (7: 2: 1) solution,
Stirred in a hydrogen atmosphere for 24 hours in the presence of d-black catalyst. After removing the catalyst by filtration, the filtrate was evaporated to remove E.
49.5 mg of tAhp-L-arginyl-L-phenylalanine acetate was obtained. Yield 100% (but as 1.75 acetate).

FAB-MS m / z 437 (M-acetic acid + 1) + 1H-NMR (CD3 OD) δ: 1.04 (t, 3H, J = 7.3 Hz) 1.46-ca. 1.87 (m, 6H, overlapping) 2.95 (dd, 1H, J = 8.3, 13.7 Hz) 3.03-ca. 3.17 (m, 2H, overlapping) 3.22 (dd, 1H, J = 4.0, 13.7 Hz) 3.37 (m, 1H) 4.22 (br s) 4.32 (t, 1H) , J = 6.4 Hz) 4.48 (m, 1H) 7.07-7.30 (m, 5H) However, there is a singlet peak derived from acetic acid at δ 1.95.

Test Examples Anti-aminopeptidase N activity was determined according to J. Antibiotics, 38,
1629-1630 (1985). That is, 0.05 ml of 2 mM L-leucine-β-naphthylamide,
A mixed solution containing 0.1 ml of 0.1 M Tris-HCl buffer (pH 7.0) and 0.035 ml of a solution containing a specimen was heated at 37 ° C. for 3 minutes, and then aminopeptidase N (Boehringer Mannheim) solution 0.015 ml was added and reacted at 37 ° C. for 1 hour. 0.5M containing 10% polyoxyethylene (20) sorbitan monolaurate (manufactured by Wako Pure Chemical Industries) and 0.2% fast garnet GBC salt (Sigma Chemical Company)
The reaction was stopped by adding a sodium citrate buffer (pH 3.78), and the absorbance (a) at 525 nm was measured. At the same time, the absorbance (b) of a blind test using only a buffer solution containing no sample was measured, and the inhibition rate of aminopeptidase N (AP-N) was calculated by the formula [(ba) / b] × 100. The concentration of the sample showing 50% inhibition was defined as the IC50 value. The anti-leucine aminopeptidase (Leu-AP) and anti-aminopeptidase A (AP-A) inhibitory activities were determined by the method of Aoyagi et al. [J. Antibio
tics, 31, 636 (1978)], anti-aminopeptidase B
(AP-B) inhibitory activity was determined by the method of Umezawa et al. [J.
tics, 29, 97 (1976)]. Table 4 shows the results. As is evident from the results in Table 4, the compounds of the present invention show excellent inhibitory activity, especially for aminopeptidase N.

[0066]

Table 4 ──────────────────────────────────── Enzyme inhibitory activity IC50 (μg / ml) ) Compound {AP-N Leu-AP AP-A AP-B} ─────────────────────────── Reference Example 3 0.18 0.030 9.0 9.0 (Phefestin) Example 30. 18 0.031 11 100 Example 6 0.60 0.070 40 55 Example 9 1.1 0.29> 100> 100 Example 12 0.10 0.38 19 2.3 Example 15 0.120 .16 15 11 Example 17 0.0070 12.5 100 40 Example 21 0.022 17 135 3.5 Example 24 0.060 3.0 − −─────────── ─────────────────────────

Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 AED A61K 37/02 AED (72) Inventor Makoto Tsuda 40-3-908 Iwabuchi-cho, Kita-ku, Tokyo

Claims (7)

[Claims] (1) Formula (1) [In the formula, R represents a saturated or unsaturated lower hydrocarbon group which may have a substituent, and X and Y each represent an amino acid residue. However, the case where R is a phenyl group, X is a valine residue, and Y is a phenylalanine residue is excluded. Or a pharmacologically acceptable salt thereof. 2. R is a phenyl group which may have a substituent, a cycloalkyl group having 3 to 9 carbon atoms which may have a substituent, or an alkyl group having 1 to 8 carbon atoms which may have a substituent. Or the alkenyl group having 2 to 8 carbon atoms which may have a substituent, or a pharmacologically acceptable salt thereof. 3. R is a phenyl group, a cyclohexyl group,
Or the compound of claim 1 which is a lower alkyl group having 1 to 4 carbon atoms, or a pharmacologically acceptable salt thereof. 4. The compound according to claim 2, wherein X is an amino acid residue having a basic side chain, or a pharmaceutically acceptable salt thereof. 5. R is a cyclohexyl group, X is a residue of valine, histidine, arginine or phenylalanine,
2. The compound according to claim 1, wherein Y is a residue of phenylalanine, histidine or proline, or a pharmaceutically acceptable salt thereof. 6. A medicament comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient. 7. An aminopeptidase N inhibitor comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.