JPH0971533A - Freeze-dried preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a pharmaceutical preparation containing an unstable physiologically active substance in stable state. SOLUTION: This freeze-dried preparation contains 1 pt.wt. of a physiologically active substance having a (substituted) amidino group and expressed by formula (R is H or an alkyl; X is H or an alkyl; Y is a cyclic hydrocarbon group; Z is carboxyl) or its salt and 1-1,000 pts.wt., preferably 5-500 pts.wt. of a disaccharide such as galactose, lactose or sucrose. It can be produced by dissolving both components in water or an aqueous solvent (e.g. an alcohol) and freeze-drying the aqueous solution by conventional method. An example of the compound of formula is (S)-4-(4-amidinobenzoylglycyl)-3- methoxycarbonylmethyl-2-oxopiperazine-1-acetic acid. It can be administered by oral or parenteral administration as an agent for the treatment of cerebral thrombosis (acute stage), cerebral ischemic syndrome, unstable angina, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition containing an unstable physiologically active substance in a stable manner, and is used in the field of pharmaceuticals and other pharmaceutical preparations.

[0002]

BACKGROUND OF THE INVENTION Some of the physiologically active substances known so far are relatively stable over time, and some are unstable. For example, amidino in the molecule. Compounds having a group are unstable over time, and it is desired to keep these compounds stable.

[0003]

Means for Solving the Problems As a result of intensive studies to solve this point, the present inventors have found that the incorporation of a disaccharide improves the stability of a compound having an amidino group in the molecule. I found it. That is, the present invention relates to a pharmaceutical composition comprising a physiologically active substance having an optionally substituted amidino group and a disaccharide. The physiologically active substance having an optionally substituted amidino group (hereinafter simply referred to as a physiologically active substance) may be one having at least one amidino group optionally substituted in the molecule. The optionally substituted amidino group is preferably an amidino group substituted with an alkyl group, and examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and a tertiary butyl group. Examples include groups having 1 to 4 carbon atoms. The present invention can be more effectively applied to a compound having an unsubstituted amidino group.

Specific examples of the physiologically active substance in the present invention include (1) (S) -4- (trans-4-guanidinomethylcyclohexylcarbonylglycyl) -2-
Oxopiperazine-1,3-diacetic acid, (2) (S) -4-
(4-guanidinomethylbenzoylglycyl) -2-oxopiperazine-1,3-diacetic acid, (3) (S) -4- (4
-Guanidinobenzoylglycyl) -2-oxopiperazine-1,3-diacetic acid, (4) (S) -4- (4-guanidinobenzoylsarcosyl) -2-oxopiperazine-
1,3-diacetic acid, (5) (S) -4- (4-guanidinomethylbenzoylsarcosyl) -2-oxopiperazine-
1,3-diacetic acid, (6) (S) -1-carboxymethyl-4
-(4-guanidinobenzoylsarcosyl) -2-oxopiperazine-3-propionic acid, (7) (S) -4- (3
-Guanidinophenylacetylglycyl) -2-oxopiperazine-1,3-diacetic acid, (8) (S) -4- (4-amidinobenzoylglycyl) -2-oxopiperazine-
1,3-Diacetic acid, (9) 4- (4-amidinobenzoylglycyl) -2-oxopiperazine-1-acetic acid, (10)
(S) -4- (4-amidinobenzoylglycyl) -3-
Methoxycarbonylmethyl-2-oxopiperazine-1
-Acetic acid, (11) (S) -4- (4-amidinobenzoylglycyl) -3-benzyl-2-oxopiperazine-1
-Acetic acid,

(12) (S) -4- (4-amidinobenzoylglycyl) -3-carbamoylmethyl-2-oxopiperazine-1-acetic acid, (13) (S) -4- (4-amidinobenzoylglycyl) Sil) -1-carboxymethyl-2
-Oxopiperazine-3-propionic acid, (14) 4-
(4-Amidinobenzoyl) aminoacetyl-3- [3
-(4-Amidinobenzoyl) aminopropyl] -2-
Oxopiperazine-1-acetic acid, (15) 4- (4-amidinobenzoyl) aminoacetyl-3- [4- (4-amidinobenzoyl) aminobutyl] -2-oxopiperazine-1-acetic acid, (16) 4- (4-amidinobenzoyl)
Aminoacetyl-3- [2- (4-amidinobenzoyl) aminoethyl] -2-oxopiperazine-1-acetic acid, (17) 4- (4-amidinobenzoyl) aminoacetyl-3- [2- (4-amidino) Phenylaminocarbonyl) ethyl] -2-oxopiperazine-1-acetic acid, (1
8) 4- (4-amidinobenzoyl) aminoacetyl-
3- [3- (4-amidinophenylaminocarbonyl)
Propyl] -2-oxopiperazine-1-acetic acid, (19) 4
-(4-amidinobenzoyl) aminoacetyl-3-
[4- (4-Amidinophenylaminocarbonyl) butyl] -2-oxopiperazine-1-acetic acid, (20) (S)-
4- (4-guanidinobenzoylamino) acetyl-3
-[3- (4-guanidinobenzoylamino) propyl] -2-oxopiperazine-1-acetic acid, (21) (S)
-4- (4-amidinobenzoylamino) acetyl-3
-[2- (4-guanidinobenzoylamino) ethyl]
-2-oxopiperazine-1-acetic acid, (22) (S) -4
-[4- (2-Aminoethyl) benzoylamino] acetyl-3- [3- (4-amidinobenzoylamino)]
Propyl-2-oxopiperazine-1-acetic acid, (23)
(S, S) -3-3- (4-guanidinobenzoylamino) propyl] -4- [3- (4-methoxyphenyl)
-2- [4- (5-trifluoromethyl- [1,2,4]
Oxadiazol-3-yl-amino) benzoylamino] propionyl] -2-oxopiperazine-1-acetic acid, (24) (S, S) -4- [2- (4-guanidinobenzoyl) amino-3- ( 4-Methoxyphenyl) propionyl] -3- [3- (4-guanidinobenzoyl)
Aminopropyl] -2-oxopiperazine-1-acetic acid and pharmacologically acceptable esters or salts thereof and the like can be mentioned. Further, these compounds may be in an optically active form or a racemic form.

In addition to the above compounds, SB-20744
8, GR-200976, SC-58052, G-74
53, G-7461, RO-43-5054, RO-4
3-8857, SC-52012, Fradafiban (Fr
adafiban), SC-5684-A, SC-54701
GR-144053, DX-9065-A, FR-14
Compounds having antithrombotic activity such as 4633 and SB-208651, Coumamidine Gamma-1
a-1), Coumamidine Gamma-
2) and other compounds having antibacterial action, FCE-2521
7, CP-79328, CGP-48664, B-62
3, FCE-24561, Tallimustin
e), a compound having an antitumor effect such as distamycin A (Distamycin-A), a compound having an antirheumatic effect and an antinephritis effect such as TO-195, a compound having an antiinflammatory effect such as BBE, CGS-25019- Compounds having anti-rheumatic and anti-asthmatic actions such as C, compounds having anti-anginal action and anti-myocardial infarction action such as Lamifiban, anti-anginal action and anti-myocardial infarction action such as SC-54684-A And antithrombotic compounds, compounds with anti-pancreatitis, anti-emphysema and anti-esophagitis effects, such as sepimostat, Pentamidine Isethi
Nitrafudam Hydrochlorid, a compound with antithrombotic and antiprotozoal activity such as onate)
Compounds having antidepressant action such as e), compounds having antipancreatitis action, antianginal action, antimyocardial infarction action such as Nafamostat, ribamidine (Ribamidin)
In addition to compounds having an antiviral action such as e), for example, CGP-40215-A, Diminazen (Diminazen)
e), Olmidine, Propamidin
dine), isometamamidium, TAN
-868-A, Acetamide Hydrochl
oride), Amidinomycin, Tapam, Amicarbalide, Dibrompropamidine, Renyfoline Hydrochloride, Hexamidine, Hydroxylstilbamidine Isethionate (Hydroxystilbamidine) ), Isometamamidium chloride, and stilbamidine isethionate.

The above-mentioned pharmacologically acceptable salts are usually salts with acids such as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, such as acetic acid, trifluoroacetic acid,
Examples thereof include salts with organic acids such as tartaric acid, citric acid, fumaric acid, maleic acid, toluenesulfonic acid and methanesulfonic acid. Depending on the compound, salts with bases, for example salts with alkali metals such as sodium and potassium, for example, It may be a salt with an alkaline earth metal such as calcium or magnesium. As a target of the preparation of the present invention, a non-peptidic compound is preferable. The compounds (1) to (2
4) Alternatively, a piperazine derivative having a side chain containing an amidino group at the 4- or 3-position similar thereto or a pharmacologically acceptable salt thereof, etc. is a preferable object, and among such piperazine derivatives, Formula (I)

Embedded image [Wherein R is hydrogen or an alkyl group, X is hydrogen or an optionally substituted alkyl group, and Y is 1 to 2].
A cyclic hydrocarbon which may be bonded via individual atoms,
Z represents a carboxyl group which may be amidated or esterified, respectively] and a pharmacologically acceptable salt thereof are particularly preferred targets. In the above general formula, the alkyl group represented by R is a C _1-6 (C _1-6 ) alkyl (eg, methyl, ethyl, propyl, isopropyl, butyl, sec-
(Butyl, tert-butyl, pentyl, hexyl), and preferably a C _1-4 alkyl group.

The optionally substituted alkyl group represented by X includes linear C _1-4 alkyl (eg, methyl, ethyl, propyl, butyl), for example, esterified or amidated. Optionally a carboxyl group (for example, a C _1-4 alkyl ester such as methyl ester or ethyl ester), phenyl, or a formula:
Substituted with a group represented by A-B-D [A represents an amino, amidino or guanidino group, B represents phenylene and D represents -CONH- (any bond may be bonded to B)] May be. The cyclic hydrocarbon which may be bonded via 1 or 2 atoms represented by Y is a saturated or unsaturated 5- or 6-membered cyclic carbon which may be bonded via a carbon atom or a nitrogen atom. Hydrogen groups (eg, cyclopentane, cyclohexyl, phenyl groups, etc., which may be bonded via an imino group, a methylene group, etc.) are included, and any or both bonds may be bonded via an atom. Good. Phenyl is preferred as the cyclic hydrocarbon group. Examples of the optionally esterified carboxyl group represented by Z include C _1-4 alkyl ester (eg, methyl ester, ethyl ester, etc.). When these compounds have an asymmetric carbon in the molecule, they may have either R configuration or S configuration. The compounds (1) to (13) are described in JP-A-6-25285, and the compounds (14) to (24) can be produced, for example, according to the method described in the reference example described below. Further, the compound of the above general formula can be produced according to these methods. Examples of the disaccharide used in the present invention include maltose-type disaccharides such as maltose, cellobiose, gentiobiose, melibiose, lactose, turanose and solohorse, and trehalose-type disaccharides such as trehalose, isotrehalose, sucrose and isosaccharose. But it's okay. Of these, sucrose, lactose and maltose are preferred, and sucrose is most preferred. These disaccharides can be used alone or in admixture of two or more.

The pharmaceutical composition of the present invention contains both a physiologically active substance and a disaccharide, and the mixing ratio of these is about 1 to 1,000, preferably 5 to 1 disaccharide to 1 physiologically active substance. 500. Next, a method for producing the freeze-dried preparation of the present invention will be described. In the freeze-dried preparation of the present invention, for example, an aqueous solution obtained by dissolving both a physiologically active substance and a disaccharide in water or a suitable aqueous solvent (for example, a mixture of water and alcohol) is filtered with a filter of 0.22 μm, if desired. As a result, it is preferable to easily prepare a sterile preparation and then freeze-dry it to give a solid form. The freeze-dried preparation of the present invention can suppress the decomposition and deterioration of the physiologically active substance for a long period of time and keep it stable. When preparing an aqueous liquid, both the physiologically active substance and the disaccharide may be dissolved in water or an aqueous solvent (for example, a mixture of water and alcohol) according to a method known per se. Either may be dissolved first. An isotonicity agent may be added to the aqueous solution of the above physiologically active substance and disaccharide to adjust the osmotic pressure. Examples of the isotonicity agent include those known as isotonicity agents such as monosaccharides such as glucose, sugar alcohols such as mannitol and salts such as salt.
The freeze-dried preparation of the present invention can be usually obtained by first dissolving both the physiologically active substance and the disaccharide in water or an aqueous solvent to give an aqueous solution, and freeze-drying the aqueous solution by a method known per se. More specifically, the above-mentioned aqueous liquid is preferably frozen below the eutectic point, and then the shelf temperature is gradually raised to the primary drying temperature while keeping the inside of the drying chamber in vacuum, and the primary drying is performed at the same temperature. After the completion of the primary drying, the shelf temperature is further raised until the secondary drying temperature is reached, and the secondary drying is performed at the same temperature. The shelf temperatures of the primary drying and the secondary drying may be the same.
At this time, the concentration of the physiologically active substance in the aqueous liquid is usually 0.01 m.
g / ml-500 mg / ml, disaccharide concentration is usually 0.0
It is 1 mg / ml to 1,000 mg / ml. The freeze-dried pharmaceutical composition of the present invention thus obtained can keep a physiologically active substance stable for a long period of time.

The lyophilized preparation of the present invention is usually used alone or as a pharmaceutical composition prepared by mixing with a pharmaceutically acceptable carrier or excipient, and can be used orally or parenterally. The freeze-dried preparation of the present invention can be tableted by compressing it, or filled in a capsule to give a capsule, and can be injected with water for injection or infusion (eg, physiological saline, glucose, etc.) before use. It can also be dissolved and used as an injection such as an intravenous injection, a subcutaneous injection, an intramuscular injection, a drip injection or an eye drop. The liquid dissolved in this water for injection or infusion is sufficiently stable. In this case, the concentration of the physiologically active substance in the solution is, for example, about 0.01 mg / ml.
It is 10 mg / ml. The concentration of disaccharide is about 0.01 mg / ml
It is 1,000 mg / ml. In the case of a ready-to-use preparation for injection, the above aqueous solution is preferably prepared by a known aseptic preparation method such as filtration sterilization. Further, before preparing the freeze-dried preparation, the disaccharide or a mixture of the disaccharide and other additives may be previously pyrolyzed and used. The freeze-dried preparation of the present invention is used for these target diseases based on the pharmacological effect of physiologically active substances.

In the freeze-dried preparation of the present invention, the compounds (1) to (24) used as physiologically active substances are mammals (eg, mouse, rat, rabbit, cat, dog, cow, goat, monkey, It has anti-platelet activity (such as humans), low toxicity, safe for humans and animals, cerebral thrombosis (acute phase), transient cerebral ischemic attack, unstable angina in mammals including humans. Can be used orally or parenterally. The dose in this case varies depending on the dosage form, administration method, type of medicinal component to be used, etc., but when administered intravenously as an injection, for example, about 0 physiologically active substance having an antithrombotic action is taken per day for a human adult. Administer 1 to 10 mg. The composition is once or twice a day
It may be administered in three to three divided doses. Moreover, you may use it as a drip injection. As the infusion solution used for the infusion, physiological saline, 5% glucose solution or the like can be used.
In this case, it can also be used as a kit preparation that is combined with an infusion solution. It can also be used as a high-value-added preparation such as a prefilled syringe preparation or a DPS preparation (dual chamber prefilled syringe). When an aqueous solution such as an injection is prepared, it is preferable that the solution is weakly acidic or weakly basic. For example hydrochloric acid,
Inorganic acids such as hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, such as acetic acid, tartaric acid, citric acid, fumaric acid, maleic acid,
The pH can be adjusted to weakly acidic or weakly basic by using an organic acid such as toluenesulfonic acid or methanesulfonic acid, or a sodium salt thereof.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in more detail below with reference to Examples and Reference Examples, but the present invention is not limited to these exemplifications. Example 1 (S) -4- (4-amidinobenzoylglycyl) -3
-Methoxycarbonylmethyl-2-oxopiperazine-
1-Acetic acid hydrochloride (Compound A) and saccharides (mannitol, sucrose, lactose or maltose) were mixed at a ratio of [Table 1] to prepare a frozen preparation, and an accelerated stability test was carried out. It was Lyophilization was carried out by adding Compound A to 0.2
%, And 2% saccharides were prepared and frozen at about -40 ° C, and the shelf temperature was about 7.5 ° C / hour while keeping the vacuum degree in the drying chamber at about 0.1 torr or less. A method of raising the temperature until reaching 30 ° C. and drying at the same temperature for 10 hours or more was adopted.

[Table 1]: Composition list of formulations A to D

[Table 2]: Stability results of formulations A to D Formulations B, C and D, which are the pharmaceutical compositions of the present invention, have better stability at each temperature for up to 1 month than formulation A, which is a comparative composition.

Example 2 (S) -4- (4-amidinobenzoylglycyl) -3-
Methoxycarbonylmethyl-2-oxopiperazine-1
-A freeze-dried preparation was prepared by adding 0.5 mg of acetic acid hydrochloride (Compound A) and 5 mg of sucrose. For the freeze-dried preparation, an aqueous solution containing 0.05% of compound A and 0.5% of sucrose was prepared, and this was prepared in the same manner as in Example 1. The appearance of the obtained preparation was a white lump, and the solubility when dissolved in distilled water was colorless and clear.

Example 3 (S) -4- (4-guanidinobenzoyl) aminoacetyl-3- [3- (4-guanidinobenzoyl) amino] propyl-2-oxopiperazine-1-acetic acid hydrochloride (Compound B) A freeze-dried preparation containing 2 mg and 20 mg of sucrose was prepared. For freeze-drying, an aqueous solution containing 0.2% of compound B and 2% of sucrose was prepared and prepared in the same manner as in Example 1. The appearance of the obtained preparation was a white lump, and the solubility when dissolved in distilled water was colorless and clear.

Reference Example 1 (S) -3- (3- ^t -Butoxycarbonylaminopropyl) -2-
Oxopiperazine-1-acetic acid ^t- butyl ester oxalate (2,2-dimethoxyethyl) aminoacetic acid ^t- butyl ester (6.0 g 27.7 mmol), NZ-Orn (Boc) -OH (10.0 g
(27.7 mmol) was dissolved in 54.6 ml of acetone, and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (5.6 g, 29.2 mmol) was added with stirring at 15 ° C.
After stirring at room temperature for 1 hour, the reaction system was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, and washed with 5% aqueous potassium hydrogen sulfate and saturated aqueous sodium hydrogen carbonate. The organic layer was concentrated under reduced pressure to obtain a pale yellow oily substance. This oil and p-toluenesulfonic acid 1.0
The hydrate (1.04 g 5.46 mmol) was dissolved in 137 ml of toluene, and the mixture was stirred at 70 ° C. for 2 hours. The reaction system was poured into saturated aqueous sodium hydrogen carbonate, and the layers were separated and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (hexane / ethyl acetate = 3/2) to obtain 8.3 g of a pale yellow oily substance. This oil (8.3 g, 16.5 mmol) was dissolved in 166 ml of ethyl acetate, and 10% palladium-carbon (P
1.7 g of dC) was added, and the mixture was stirred under a hydrogen atmosphere for 2 hours. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in 16.6 ml of methanol and oxalic acid 2.0 hydrate (2.1
g (16.5 mmol) was added, and the mixture was concentrated under reduced pressure. The precipitated crystals were washed with ethyl acetate to give the title compound (5.1 g, 66.8%).
Was obtained as white crystals. Specific rotation: [α] _D -29.3 ° (C = 0.73, H ₂ O) Melting point: 181 ° C Elemental analysis: C ₁₈ H ₃₃ N ₃ O ₅・ (CO ₂ H) ₂ (461.511) Calculated: C, 52.05; H, 7.64; N, 9.10 Found: C, 51.98; H, 7.61; N, 9.20

Reference Example 2 (S) -4-benzyloxycarbonylaminoacetyl-3-
(3- ^t -Butoxycarbonylaminopropyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester (S) -3- (3- ^t -butoxycarbonylaminopropyl) -2-
Oxopiperazine-1-acetic acid ^t- butyl ester oxalate (1.6 g, 3.47 mmol) was dissolved in saturated aqueous sodium hydrogen carbonate, and the mixture was extracted with ethyl acetate and concentrated under reduced pressure. Residue and NZ-Gl
y-OH (0.87 g 4.16 mmol) was dissolved in 16.0 ml of acetone and stirred at 15 ° C. 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.87 g 4.51 mm).
ol). After stirring at room temperature for 1 hour, the reaction system was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, and washed with 5% aqueous potassium hydrogen sulfate and saturated aqueous sodium hydrogen carbonate. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to give the title compound (1.95 g, 100%) as a colorless amorphous powder. IR ν max cm ^-1 : 3360, 2970, 2930, 1713, 1650, 151
3, 1448, 1363, 1246, 1158, 1045, 964, 848, 744, 69
5 NMR (CDCl ₃ ) δ: 1.43 (9H, s), 1.46 (9H, s), 1.50-2.20 (4
H, m), 3.02-4.28 (10H, m), 4.52-4.80 (1H, m), 5.01 (1H, d
d, J = 8.8Hz, J = 4.6Hz), 5.13 (2H, s), 5.64-5.86 (1H, m),
7.37 (5H, s)

Reference Example 3 (S) -4- (4-amidinobenzoyl) aminoacetyl-3-
(3-Aminopropyl) -2-oxopiperazine-1-acetic acid trifluoroacetate (S) -4-benzyloxycarbonylaminoacetyl-3-
(3- ^t -Butoxycarbonylaminopropyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester (1.34g 2.38mm
ol) was dissolved in 13.4 ml of methanol and 10% Pd-C was added.
0.54 g was added, and the mixture was stirred under a hydrogen atmosphere for 30 minutes. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. Residue and baking soda
(0.4 g 4.76 mmol) was dissolved in 26.8 ml of H ₂ O and 13.4 ml of 1,4-dioxane, and 4-amidinobenzoyl chloride hydrochloride (0.68 g 3.09 mmol) was added with stirring at room temperature. After stirring for 3 hours, the reaction system was adjusted to pH 4 with 1N hydrochloric acid water and concentrated to dryness. The residue is trifluoroacetic acid 3.75 m
It was dissolved in 1 and stirred at room temperature for 2 hours. The reaction system was concentrated under reduced pressure and subjected to CHP-20 (Mitsubishi Kasei) column chromatography (H ₂
O) to give the title compound (1.0 g, 63.3%) as a colorless amorphous powder. Specific rotation: [α] _D + 35.4 ° (C = 0.75, MeOH) Elemental analysis: C ₁₉ H ₂₆ N ₆ O ₅・ 2CF ₃ CO ₂ H ・ H ₂ O (664.515) Calculated: C, 41.57; H, 4.55; N, 12.65 Found: C, 41.86; H, 4.50; N, 12.60

Reference Example 4 (S) -4- (4-amidinobenzoyl) aminoacetyl-3-
[3- (4-amidinobenzoyl) amino] propyl-2-oxopiperazine-1-acetic acid trifluoroacetate (S) -4- (4-amidinobenzoylamino) acetyl-3-
(3-Aminopropyl) -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt (0.5 g 0.94 mmol) and sodium bicarbonate (0.3
2 g (3.76 mmol) was dissolved in 5.0 ml of H ₂ O and 2.5 ml of 1,4-dioxane, and 4-amidinobenzoyl chloride hydrochloride (0.22 g, 0.99 mmol) was added with stirring at room temperature. Two
After stirring for an hour, the reaction system was adjusted to pH 4 with 1N HCl water and then concentrated under reduced pressure. The residue was purified by CHP-20 column chromatography (H ₂ O → 5% CH ₃ CN) to give the title compound (0.1%).
34 g (50.7%) was obtained as a colorless amorphous powder. Specific rotation: [α] _D + 41.9 ° (C = 0.73, MeO
H) Elemental _{analysis: C 27 H 32 N 8 O} 6 · CF 3 CO 2 H
· _2H 2 O (714.653) Calculated: C, 48.74; H, 5.22 ; N, 15.68 Found: C, 48.52; H, 5.22 ; N, 15.57

Reference Example 5 (S) -3- (4- ^t -Butoxycarbonylaminobutyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester oxalate By the same method as in Reference Example 1, NZ-Lys (Boc) Synthesized using -OH. Specific rotation: [α] _D -29.0 ° (C = 1.02, DMSO) Melting point: 170-172 ° C Elemental analysis: C ₁₉ H ₃₅ N ₃ O ₅・ (CO ₂ H) ₂ (475.540) Calculated: C, 53.04; H, 7.84; N, 8.84 Found: C, 52.75; H, 7.65; N, 8.66 Reference Example 6 (S) -4-benzyloxycarbonylaminoacetyl-3-
(4- ^t -Butoxycarbonylaminobutyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester By the same method as in Reference Example 2, (S) -3- (4- ^t -butoxycarbonylaminobutyl) -2-oxopiperazine- 1-acetic acid
Synthesized using ^t- butyl ester oxalate. IR ν max cm ^-1 : 3400, 2990, 2945, 1713, 1657, 152
0, 1458, 1368, 1253, 1166, 1070, 745, 700 NMR (CDCl ₃ ) δ: 1.42 (9H, s), 1.46 (9H, s), 1.18-2.12 (6
H, m), 2.92-4.28 (10H, m), 4.48-4.84 (1H, m), 5.02 (1H, d
d, J = 8.6Hz, J = 4.8Hz), 5.13 (2H, s), 5.60-5.88 (1H, m),
7.36 (5H, s)

Reference Example 7 (S) -4- (4-amidinobenzoylamino) acetyl-3-
(4-Aminobutyl) -2-oxopiperazine-1-acetic acid trifluoroacetate salt In the same manner as in Reference Example 3, (S)-[4-benzyloxycarbonylaminoacetyl-3- (4-t-butoxycarbonylamino- Butyl) -2-oxopiperazin-1-yl] -acetic acid
Synthesized using ^t- butyl ester. Specific rotation: [α] _D + 46.8 ° (C = 1.01, H ₂ O) Elemental analysis value: Calculated as C ₂₀ H ₂₈ N ₆ O ₅・ 1.7CF ₃ CO ₂ H ・ 2H ₂ O (662.394) : C, 42.43; H, 5.13; N, 12.69 Actual value: C, 42.53; H, 4.88; N, 12.78 Reference Example 8 (S) -4- (4-amidinobenzoylamino) acetyl-3-
[4- (4-amidinobenzoylamino) butyl] -2-oxopiperazine-1-acetic acid 1-trifluoroacetic acid salt 1-hydrochloride By the same method as in Reference Example 4, (S) -4- (4-amidinobenzoylamino) Acetyl-3- (4-aminobutyl) -2-oxopiperazine-1-acetic acid was synthesized using trifluoroacetic acid salt. Specific rotation: [α] _D ＋ 44.3 ° (C = 1.01, H ₂ O) Elemental analysis value: C ₂₈ H ₃₄ N ₈ O ₆・ CF ₃ CO ₂ H ・ HCl ・ 3H ₂ O (783.157)
Calculated as: C, 46.01; H, 5.41; N, 14.31 Found: C, 46.23; H, 5.22; N, 14.54

Reference Example 9 (S, S) -4- [2-benzyloxycarbonylamino-3-
(4-Methoxyphenyl) propionyl] -3- (3- ^t -butoxycarbonylaminopropyl) -2-oxopiperazine-1
-Acetic acid ^t- butyl ester It was synthesized using NZ-Tyr (OMe) -OH in the same manner as in Reference Example 2. IR ν max cm ^-1 : 3360, 2975, 2925, 1710, 1643, 151
2, 1448, 1360, 1245, 1152, 1033, 743, 696 NMR (CDCl ₃ ) δ: 1.41 (9H, s), 1.44
(9H, s), 1.30-2.10 (3H, m),
2.20-2.44 (1H, m), 2.80-3.84
(10H, m), 3.77 (3H, s), 4.23
(1H, d, J = 17.2 Hz), 4.50-4.8
5 (1H, m), 4.93 (1H, dd, J = 6.2)
Hz, J = 7.0 Hz), 5.09 (2H, dd, J
= 12.0 Hz, J = 16.4 Hz), 5.67 (1
H, d, J = 8.8 Hz), 6.80 (2H, d, J
= 8.8 Hz), 7.09 (2H, d, J = 8.8H
z), 7.35 (5H, s) Reference Example 10 (S, S) -4- [2- (4-amidinobenzoylamino) -3-
(4-Methoxyphenyl) -propionyl] -3- (3-aminopropyl) -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt By the same method as in Reference Example 3, (S, S) -4- [2- It was synthesized using benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- (3- ^t -butoxycarbonylaminopropyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester. Specific rotation: [α] _D ＋ 78.2 ° (C = 0.62, H ₂ O) Elemental analysis: C ₂₇ H ₃₄ N ₆ O ₆・ CF ₃ CO ₂ H ・ 3H ₂ O (706.672) Calculated value: C, 49.29; H, 5.85; N, 11.89 Found: C, 49.53; H, 5.68; N, 11.90

Reference Example 11 (S, S) -4- [2- (4-amidinobenzoylamino) -3- (4
-Methoxyphenyl) -propionyl] -3- [3- (4-amidinobenzoylamino) propyl] -2-oxopiperazine-
1-Acetic acid trifluoroacetic acid salt By the same method as in Reference Example 4, (S, S) -4- [2- (4-amidinobenzoylamino) -3- (4-methoxyphenyl) propionyl] -3- (3- Aminopropyl) -2-oxopiperazine-1-acetic acid was synthesized using trifluoroacetic acid salt. Specific rotation: [α] _D ＋ 52.8 ° (C = 0.76, MeOH) Elemental analysis: C ₃₅ H ₄₀ N ₈ O ₇・ CF ₃ CO ₂ H ・ 3H ₂ O (852.820) Calculated: C, 52.11; H, 5.55; N, 13.14 Found: C, 52.27; H, 5.50; N, 13.26 Reference Example 12 (S, S) -4- [2-benzyloxycarbonylamino-3--
(4-Methoxyphenyl) -propionyl] -3- (4- ^t -butoxycarbonylaminobutyl) -2-oxopiperazine-1-
Acetic acid ^t- butyl ester (S) -3- (4- ^t -butoxycarbonylaminobutyl) -2-oxopiperazine-1-acetic acid was prepared in the same manner as in Reference Example 2.
Synthesized using ^t- butyl ester oxalate and NZ-Tyr (OMe) -OH. IR ν max cm ^-1 : 3345, 2975, 2930, 1712, 1646, 151
2, 1447, 1364, 1244, 1155, 1034, 743, 696 NMR (CDCl ₃ ) δ: 1.43 (9H, s), 1.44 (9H, s), 1.00-2.45 (6
H, m), 2.80-3.90 (10H, m), 3.78 (3H, s), 4.23 (1H, d, J = 1
7.4Hz), 4.70-5.10 (2H, m), 5.10 (2H, d, J = 2.4Hz), 5.74 (1
H, d, J = 8.8Hz), 6.81 (2H, d, J = 8.6Hz), 7.10 (2H, d, J = 8.6H
z), 7.35 (5H, s)

Reference Example 13 (S, S) -4- [2- (4-amidinobenzoylamino) -3-
(4-Methoxyphenyl) propionyl] -3- (4-aminobutyl) -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt By the same method as in Reference Example 3, (S, S)-[4- [2-- Synthesis was performed using benzyloxycarbonylamino-3- (4-methoxyphenyl) -propionyl] -3- (4- ^t -butoxycarbonylaminobutyl) -2-oxopiperazin-1-yl] -acetic acid ^t- butyl ester. Specific rotation: [α] _D + 53.1 ° (C = 0.64, MeOH) Elemental analysis: C ₂₈ H ₃₆ N ₆ O ₆・ CF ₃ CO ₂ H ・ 3H ₂ O (720.699) Calculated: C, 50.00; H, 6.01; N, 11.66 Found: C, 49.87; H, 5.77; N, 11.45 Reference Example 14 (S, S) -4- [2- (4-amidinobenzoylamino) -3-
(4-Methoxyphenyl) propionyl] -3- [4- (4-amidinobenzoylamino) butyl] -2-oxopiperazine-
1-Acetic acid hydrochloride In the same manner as in Reference Example 4, (S, S) -4- [2- (4-amidinobenzoylamino) -3- (4-methoxyphenyl) propionyl] -3- (4-aminobutyl) ) -2-Oxopiperazine
-1-Acetic acid Synthesized using trifluoroacetic acid salt. Specific rotation: [α] _D ＋ 54.5 ° (C = 0.88, H ₂ O) Elemental analysis value: Calculated as C ₃₆ H ₄₂ N ₈ O ₇・ HCl ・ 6H ₂ O (843.329): C, 51.27; H, 6.57; N, 13.29 Found: C, 51.24; H, 6.37; N, 13.26

Reference Example 15 (S) -4-benzyloxycarbonylaminoacetyl-3-
[3- (6- ^t -Butoxycarbonylaminohexanoylamino) propyl] -2-oxopiperazine-1-acetic acid (S) -4-benzyloxycarbonylaminoacetyl-3- (3- ^t -butoxy) obtained in Reference Example 2 Carbonylaminopropyl) -2-oxopiperazine-1-acetic acid ^t -butyl ester
(0.6 g, 1.07 mmol) was dissolved in 3.0 ml of trifluoroacetic acid, stirred at room temperature for 30 minutes, and the reaction system was concentrated under reduced pressure. 6- ^t butoxyaminocaproic acid (0.26g 1.12m
mol), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.22 g 1.14 mmol) and 1-
Hydroxybenzotriazole (0.15g 1.12mmol)
Was dissolved in 2.1 ml of DMF and stirred for 1 hour. To this solution was added the above residue and triethylamine (0.3 ml 2.14 mmol).
2.1 ml of a DMF solution in which was dissolved was added, and the mixture was stirred at room temperature for 4 hours. The reaction system was diluted with ethyl acetate and washed with 5% aqueous potassium hydrogen sulfate solution and saturated aqueous sodium hydrogen carbonate solution. The organic layer was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography.
Purification by (ethyl acetate / methanol / acetic acid = 20/10 / 0.6) gave 0.42 g (y.63.3%) of the title compound as a colorless amorphous powder. IR ν max cm ^-1 : 3320, 2930, 1643, 1533, 1448, 120
3, 1173, 1046 NMR (CDCl ₃ ) δ: 1.42 (9H, s), 1.20-2.09 (10H, m), 2.17
(2H, t, J = 7.3Hz), 2.92-4.20 (12H, m), 4.80-4.98 (1H, m),
5.11 (2H, s), 7.22-7.44 (5H, m)

Reference Example 16 (S) -4- (4-amidinobenzoylamino) acetyl-3-
[3- (6-Aminohexanoylamino) propyl] -2-oxopiperazine-1-acetic acid trifluoroacetate (S) -4-benzyloxycarbonylaminoacetyl-3-
[3- (6- ^t -Butoxycarbonylaminohexanoylamino) propyl] -2-oxopiperazine-1-acetic acid (0.42 g
0.68 mmol) was dissolved in 8.4 ml of methanol and 10%
0.17 g of Pd-C was added, and the mixture was stirred under a hydrogen atmosphere for 1 hour.
The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue and sodium bicarbonate (0.18 g 2.14 mmol) were dissolved in 8.4 ml of H ₂ O and 4.2 ml of 1,4-dioxane, and 4-amidinobenzoyl chloride hydrochloride (0.20 g 0.93 mmol) was added with stirring at room temperature. It was After stirring for 1 hour, the reaction system was adjusted to pH 4 with 1N HCl water and then concentrated to dryness. The residue was dissolved in 4.3 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. The reaction system was concentrated under reduced pressure, and the residue was subjected to CHP-20 column chromatography.
Purified with (H ₂ O → 5% CH ₃ CN), 0.26 g of the title compound
(y.55%) was obtained as a colorless amorphous powder. Specific rotation: [α] _D + 42.7 ° (C = 0.99, MeOH) Elemental analysis value: Calculated as C ₂₅ H ₃₇ N ₇ O ₆・ 1.1CF ₃ CO ₂ H ・ 2H ₂ O (693.067) C: 47.14 H: 6.12 N: 14.15 Measured value C: 47.30 H: 5.82 N: 14.40

Reference Example 17 (S) -4-benzyloxycarbonylaminoacetyl-3-
[3- (5- ^t -Butoxycarbonylaminopentanoylamino) propyl] -2-oxopiperazine-1-acetic acid It was synthesized in the same manner as in Reference Example 15 using 5- ^t -butoxyaminovaleric acid. IR ν max cm ^-1 : 3370, 2940, 1650, 1533, 1455, 125
4, 1170, 1050 NMR (CDCl ₃ ) δ: 1.42 (9H, s), 1.28-2.08 (8H, m), 2.18 (2
H, t, J = 7.0Hz), 3.03 (2H, t, J = 6.8Hz), 3.10-4.20 (10H,
m), 4.82-5.00 (1H, m), 5.11 (2H, s), 7.22-7.52 (5H, m) Reference Example 18 (S) -4- (4-amidinobenzoylamino) acetyl-3-
[3- (5-Aminopentanoylamino) propyl] -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt By the same method as in Reference Example 16, (S) -4-benzyloxycarbonylaminoacetyl-3- [3 It was synthesized using-(5- ^t -butoxycarbonylaminopentanoylamino) propyl] -2-oxopiperazine-1-acetic acid. Specific rotation: [α] _D ＋ 46.0 ° (C = 1.01, MeOH) Elemental analysis: C ₂₄ H ₃₅ N ₇ O ₆・ CF ₃ CO ₂ H ・ 2.5H ₂ O (676.646) Calculated: C , 46.15; H, 6.11; N, 14.49 Found: C, 46.43; H, 6.1.
5; N, 14.20

Reference Example 19 (S) -4-benzyloxycarbonylaminoacetyl-3
-[3- (4- ^t -Butoxycarbonylaminobutanoylamino) propyl] -2-oxopiperazine-1-acetic acid It was synthesized in the same manner as in Reference Example 15 using 4- ^t -butoxyaminobutyric acid. IR ν max cm ^-1 : 3350, 2930, 1642, 1530, 1452, 125
2, 1170, 1050 NMR (CDCl ₃ ) δ: 1.42 (9H, s), 1.30-2.10 (4H, m), 1.73 (2
H, t, J = 7.2Hz), 2.18 (2H, t, J = 7.5Hz), 3.04 (2H, t, J = 6.8H
z), 3.10-4.20 (10H, m), 4.83-4.97 (1H, m), 5.11 (2H, s),
7.22-7.50 (5H, m) Reference Example 20 (S) -4- (4-amidinobenzoylamino) acetyl-3-
[3- (4-Aminobutanoylamino) propyl] -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt By the same method as in Reference Example 16, (S) -4-benzyloxycarbonylaminoacetyl-3- [3 It was synthesized using-(4- ^t -butoxycarbonylaminobutanoylamino) propyl] -2-oxopiperazine-1-acetic acid. Specific rotation: [α] _D + 47.9 ° (C = 1.00, H ₂ O) Elemental analysis value: Calculated as C ₂₃ H ₃₃ N ₇ O ₆・ 1.5CF ₃ CO ₂ H ・ 2H ₂ O (710.623) : C, 43.95; H, 5.46; N, 13.80 Found: C, 44.23; H, 5.63; N, 13.52

Reference Example 21 (S, S) -4- [2- (4-amidinobenzoylamino) -3-
(4-Methoxyphenyl) propionyl] -3- (4-guanidinobutyl) -2-oxopiperazine-1-acetic acid hydrochloride (S, S) -4- [2-benzyloxycarbonylamino-obtained in Reference Example 12 3- (4-methoxyphenyl) propionyl]
-3- (4- ^t -butoxycarbonylaminobutyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester (0.6g 0.86m
mol) was dissolved in 2.0 ml of trifluoroacetic acid. After stirring at room temperature for 1 hour, the reaction system was concentrated under reduced pressure. 5.6 ml of an aqueous solution of the residue and sodium bicarbonate (0.22 g 2.57 mmol) was added to 5.6 ml of an aqueous solution of S-methylisothiourea sulfate (0.48 g 1.71 mmol) and 2N aqueous NaOH (0.86 ml 1.71 mmol).
In addition, the mixture was stirred at room temperature for 14 hours. The precipitated solid was collected by filtration, washed with water and dried. This solid is methanol 5.
It was dissolved in 8 ml, 10% Pd-C (0.12 g) was added, and the mixture was stirred under a hydrogen atmosphere for 1 hour. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue was subjected to CHP-20 column chromatography (H
₂ O → 5% CH ₃ CN → 10% CH ₃ CN), then (S, S)-
4- [2-amino-3- (4-methoxyphenyl) propionyl] -3- (4-guanidinobutyl) -2-oxopiperazine-
1-acetic acid was obtained. This intermediate (0.16 g 0.36 mmol) and sodium bicarbonate (0.09 g 1.07 mmol) were added to H ₂ O 3.2 ml, 1,4-
It was dissolved in 1.6 ml of dioxane, and 4-amidinobenzoyl chloride hydrochloride (0.10 g 0.46 mmol) was added thereto with stirring at room temperature. After stirring for 1.5 hours, pour the reaction system into 1N HCl water.
It was adjusted to H4 and concentrated under reduced pressure. The residue was purified by CHP-20 column chromatography (H ₂ O → 5% CH ₃ CN) to give the title compound (0.16 g, y.27.3%) as a colorless amorphous powder. Specific rotation: [α] _D + 62.7 ° (C = 0.99, MeOH) Elemental analysis value: Calculated as C ₂₉ H ₃₈ N ₈ O ₆ · HCl · 3H ₂ O (685.176) C, 50.84; H, 6.62 ; N, 16.35 Found C, 50.76; H, 6.47; N, 16.11

Reference Example 22 (S) -4- (4-amidinobenzoylamino) acetyl-3-
[3- (4-guanidinobutanoylamino) propyl] -2-oxopiperazine-1-acetic acid hydrochloride (S) -4-benzyloxycarbonylaminoacetyl-3- [3- (4-- ^t -Butoxycarbonylaminobutanoylamino) propyl] -2-oxopiperazine-1-acetic acid (0.33 g, 0.56 mmol) was dissolved in 6.6 ml of trifluoroacetic acid, and the mixture was stirred at room temperature for 1 hour, and then the reaction system was mixed. It was concentrated under reduced pressure. Residue and baking soda (0.14g 1.68mmo
3.3 ml of an aqueous solution of l) is added to S-methylisothiourea sulfate
(0.93 g 3.35 mmol) and 2N aqueous NaOH (1.68 ml 3.35).
(35 mmol) in 3.3 ml of an aqueous solution, and the mixture was stirred at room temperature for 14 hours. The reaction system was concentrated under reduced pressure, and the residue was subjected to CHP-20 column chromatography (H ₂ O → 5% CH ₃ CN → 10% CH ₃ CN → 1
5% CH ₃ CN) to give (S)-[4- (benzyloxycarbonylamino) -acetyl-3- [3- (4-guanidinobutyrylamino) -propyl] -2-oxopiperazine-1- Ill] -acetic acid was obtained. This intermediate was dissolved in 6.0 ml of methanol, added with 0.30 g of 10% Pd-C, and added with hydrogen under an atmosphere of 1
Stirred for hours. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue and sodium bicarbonate (0.19 g 2.23 mmol) were added to H ₂ O 6.0.
ml, dissolved in 3.0 ml of 1,4-dioxane, and stirred at room temperature with 4-amidinobenzoyl chloride hydrochloride (0.16 g, 0.16 g).
73 mmol) was added. After stirring for 1.0 hour, the reaction system was 1NH
The pH was adjusted to 4 with Cl water, and concentrated under reduced pressure. CH residue
Purification by P-20 column chromatography (H ₂ O) gave the title compound (0.09 g, y.24.7%) as a colorless amorphous powder. Specific rotation: [α] _D + 48.4 ° (C = 0.96, H ₂ O) Elemental analysis: Calculated as C ₂₄ H ₃₅ N ₉ O ₆・ 2HCl ・ 3.5H ₂ O (681.572) C, 42.29; H, 6.51; N, 18.50 Found C, 42.34; H, 6.59; N, 18.28

Reference Example 23 4- (N-benzyloxycarbonyl) glycyl-1- ^t -butoxycarbonylmethyl-2-oxopiperazine-3-acetic acid 4- (N-benzyloxycarbonyl) glycyl-1- ^t -butoxycarbonylmethyl- 1.46 g of 2-oxopiperazine-3-acetic acid methyl ester was dissolved in a mixture of 5 ml of water and 5 ml of methanol, and 190 mg of lithium hydroxide monohydrate was added at 0 ° C.
Was added in 5 minutes. After stirring at the same temperature for 1 hour, the mixture was stirred at room temperature for 1 hour. The reaction solution was adjusted to pH 7 with 5% aqueous potassium hydrogen sulfate, and methanol was removed by concentration under reduced pressure.
Further, the pH was adjusted to 3 with 5% potassium hydrogen sulfate and then extracted with ethyl acetate. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the title compound (1.1 g) as a colorless oil. NMR: (CDCl ₃ ) δ: 1.452 (9H, s), 2.80-4.65 (10H, m), 5.
10 (2H, s), 5.82 (1H, m), 6.03 (1H, m), 7.33 (5H, s). IRν _max , cm ⁻¹ : 3000, 1730, 1
660, 1465, 1370, 1230, 11
60.

Reference Example 24 3- (4-amidinophenyl) aminocarbonylmethyl-
4- (N-benzyloxycarbonyl) glycyl-2-oxopiperazine-1-acetic acid ^t- butyl ester 4- (N-benzyloxycarbonyl) glycyl-1- ^t -butoxycarbonylmethyl-2-oxopiperazine obtained in Reference Example 9 820 mg of 3-acetic acid and 370 mg of 4-aminobenzamidine dihydrochloride were dissolved in 5 ml of pyridine, 370 mg of dicyclohexylcarbodiimide and 10 mg of 4-dimethylaminopyridine were added at room temperature, and the mixture was stirred at the same temperature for 24 hours.
The crude product obtained by filtering the insoluble matter and concentrating the filtrate under reduced pressure was 1
It was dissolved in a hydrochloric acid aqueous solution and subjected to column chromatography using CHP-20. The fraction eluted with 5% acetonitrile-water was freeze-dried to obtain 550 mg of the title compound as a colorless powder. NMR (DMSO _d-6 ) δ: 1.42 (9H, s), 2.83-4.44 (13H, m), 5.0
2 (2H, s), 7.34 (5H, s), 7.78-7.82 (4H, m), 9.03-9.25 (3
H, m) IR ν _max , cm ^-1 : 3325, 1730, 1680, 1640, 1480, 1365,
1260, 1155.

Reference Example 25 (S) -4- [N- (4-amidinobenzoyl) glycyl] -3- (4
-Amidinophenyl) aminocarbonylmethyl-2-oxopiperazine-1-acetic acid 3- (4-amidinophenyl) aminocarbonylmethyl-4- (N-benzyloxycarbonyl) glycyl-2-oxopiperazine-obtained in Reference Example 12 1-acetic acid ^t- butyl ester 93
0 mg was dissolved in 15 ml of methanol and 10% palladium-
100 mg of carbon was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1 hour. The reduction catalyst was filtered off, and the oily substance obtained by concentrating the filtrate under reduced pressure was combined with 350 mg of sodium hydrogen carbonate and water 2
To a solution dissolved in a mixture of 5 ml and 25 ml of dioxane, 307 mg of 4-amidinobenzoic acid with vigorous stirring at room temperature.
Was added in 5 minutes. The crude product obtained by concentrating the reaction solution was dissolved in 5 ml of dichloromethane and mixed with 5 ml of trifluoroacetic acid.
Was added at room temperature and stirred for 1 hour. The crude product obtained by concentrating the reaction solution under reduced pressure was purified by column chromatography using CHP-20 to obtain 490 mg of the title compound as a colorless powder. Specific rotation: [α] _D ²³ ＋ 57.5 ° (C = 0.9, H ₂ O) Elemental analysis: C ₂₅ H ₂₈ N ₈ O ₆・ CF ₃ CO ₂ H ・ 2.7H ₂ O Calculated: C , 46.41; H, 4.96; N, 16.04. Found: C, 46.56; H, 4.80; N, 15.84.

Reference Example 26 (S) -4- (4-guanidinobenzoylamino) acetyl-3- [3- (4-guanidinobenzoylamino)] propyl-2-oxopiperazine-1-acetic acid hydrochloride Reference Example 2 The obtained (S) -4-benzyloxycarbonylaminoacetyl-3- (3- ^t -butoxycarbonylamino) propyl-2-oxopiperazine-1-acetic acid ^t- butyl ester 0.7 g was dissolved in trifluoroacetic acid 4.9 ml. The mixture was stirred at room temperature for 1 hour. The reaction solution is concentrated under reduced pressure,
It was azeotropically distilled several times with toluene. The residue was subjected to column chromatography using CHP-20 (Mitsubishi Kasei) and 20%
Concentrate the fraction eluted with acetonitrile / water (S)
-4-benzyloxycarbonylaminoacetyl-3-
(3-Amino) propyl-2-oxopiperazine-1-
Acetic acid was obtained as a crude product. This product is methanol 12.0m
Dissolve in l, add 10% palladium-carbon 250mg,
The mixture was stirred under a hydrogen atmosphere for 1 hour. Filter off the catalyst,
The filtrate was concentrated under reduced pressure. Residue and overlay 836 mg 1,4-
Dissolve in a mixed solvent of 7.0 ml of dioxane and 14.0 ml of water,
4-Guanidinobenzoic acid N-hydroxy-under stirring at room temperature
1.27 g of 5-norbornene-2,3-dicarboxylic acid imide ester hydrochloride was added. After stirring for 1 hour, the reaction system was adjusted to pH 3-4 with 1N hydrochloric acid and concentrated under reduced pressure. The residue was subjected to CHP-20 column chromatography (5% CH
₍ Elution with ₃ CN / H ₂ O), and the equal fraction was lyophilized to give 0.48 g of the title compound as a colorless amorphous powder. Specific rotation: [α] _D ²⁰ + 56.3 ° (C = 1.017, H ₂ O) Elemental analysis value: Calculated as C ₂₇ H ₃₄ N ₁₀ O ₆・ 1.0HCl ・ 3.5H ₂ O C, 46.72; H , 6.10; N, 20.18 Found C, 46.56; H, 6.17;
N, 20.05

Reference Example 27 (S) -3- (2- ^t -butoxycarbonylamino) ethyl-2-oxopiperazine-
1-acetic acid ^t-butyl ester oxalate (S) -N ² - benzyloxycarbonyl -N ⁴ - ^{t-butoxycarbonyl-2,4-diaminobutane} acid 26 g, N
15.5 g of-(2,2-dimethoxyethyl) glycine ^t- butyl ester was dissolved in 200 ml of acetonitrile, and 19 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added with stirring at room temperature. After stirring at the same temperature for a further 2 hours, the reaction mixture was concentrated, the obtained oily substance was dissolved in ethyl acetate, washed with 5% aqueous potassium hydrogensulfate and saturated aqueous sodium hydrogencarbonate, and dried over anhydrous magnesium sulfate, It was concentrated under reduced pressure. The residue was dissolved in 500 ml of toluene and p-toluenesulfonic acid was added.
4 g was added and the mixture was stirred at 70 ° C. for 3 hours. The reaction solution cooled to room temperature was washed with saturated aqueous sodium hydrogen carbonate solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was dissolved in 500 ml of methanol and 10% Pd-C was added to 10 ml.
g was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 10 hours. The catalyst was removed by filtration, 6.4 g of oxalic acid was added to the filtrate, and the mixture was concentrated under reduced pressure. The obtained crude crystals were recrystallized from methanol / ethyl acetate to give the title compound (9.5 g) as colorless crystals. Mp: 165 - 169 ° C. Elemental analysis _{C 17 H 31 N 3 O 5} · (CO 2 H) 2 Calculated: C, 51.00; H, 7.43 ; N, 9.39 Found: C, 50.78; H, 7.59 ; N , 9.14

Reference Example 28 (S) -4- (benzyloxycarbonylamino) acetyl-3- (2- ^t -butoxycarbonylamino) ethyl-2-oxopiperazine-1
-Acetic acid ^t- butyl ester (S) -3- (2- ^t -butoxycarbonylaminoethyl) -2-oxopiperazine-1-acetic acid obtained in Reference Example 27
^T- butyl ester oxalate (900 mg) was suspended in dichloromethane (20 ml), saturated aqueous sodium hydrogencarbonate (20 ml) was added, and the mixture was vigorously stirred for 10 minutes. The organic layer was separated, dried over anhydrous magnesium sulfate, and then N-benzyloxycarbonylglycine (420 mg) and 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide hydrochloride 50
0 mg was added and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 5% potassium hydrogen sulfate and saturated aqueous sodium hydrogen carbonate solution, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (eluent: ethyl acetate-hexane = 3: 1) to give 1.05 g of the title compound as a colorless oil. IR ν max cm ^-1 : 3450, 1705, 1655, 1640, 1500, 145
0, 1360, 1240, 1160. NMR (CDCl ₃ ) δ: 1.43 (9H, s), 1.46 (9H, s), 2.05-2.3
3 (1H, m), 2.73-2.95 (1H, m), 3.15-4.20 (10H, m), 5.0
5 (1H, dd, J = 3Hz), 5.13 (2H, s), 5.30 (1H, brs), 5.8
3 (1H, brs), 7.36 (5H, s).

Reference Example 29 (S) -4- (4-Amidinobenzoylamino) acetyl-3- [2- (4-guanidinobenzoylamino)] ethyl-2-oxopiperazine-1-acetic acid hydrochloride In Reference Example 28 550 mg of the obtained (S) -4- (N-benzyloxycarbonylamino) acetyl-3- (2- ^t -butoxycarbonylamino) ethyl-2-oxopiperazine-1-acetic acid ^t- butyl ester was added to 5 ml of trifluoroacetic acid.
And was stirred at room temperature for 1 hour. The oily substance obtained by concentrating the reaction solution and 400 mg of sodium hydrogen carbonate were dissolved in a mixed solution of 25 ml of water and 25 ml of dioxane, and the mixture was stirred at room temperature.
250 mg of guanidinobenzoyl chloride hydrochloride were added. The reaction mixture was adjusted to pH 7 with 1N hydrochloric acid, 10% palladium-carbon (100 mg) was added, and the mixture was stirred under hydrogen atmosphere for 1 hr. The catalyst was filtered off and 30 ml of dioxane was added to the filtrate.
And 400 mg of sodium hydrogen carbonate were added thereto, and 230 mg of 4-amidinobenzoic acid hydrochloride was added with vigorous stirring. The reaction solution was adjusted to pH 3 with 1N hydrochloric acid and then concentrated under reduced pressure to a half volume. The concentrate was purified by CHP-20 column (5% acetonitrile / water) to obtain 250 mg of the title compound as a colorless amorphous solid. Non-optical rotation: [α] _D ²⁰ + 26.112 ° (C = 0.450, MeOH) Elemental analysis Calculated as C ₂₆ H ₃₁ N ₉ O ₆ HCl.5H ₂ O: C, 45.12; H, 6.12; N, 18.21 Found: C, 45.61; H, 6.06; N, 18.22

Reference Example 30 (S) -3- [3- (4-Amidinobenzoylamino)] propyl-4-benzyloxycarbonylaminoacetyl-2-oxopiperazine-1-acetic acid Obtained in Reference Example 2 (S). 1.35 g of -4-benzyloxycarbonylaminoacetyl-3- (3- ^t -butoxycarbonylaminopropyl) -2-oxopiperazine-1-acetic acid ^t- butyl ester was dissolved in 6.8 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. After that, it was concentrated under reduced pressure. The residue was dissolved in a mixed solvent of 20 ml of water and 10 ml of dioxane,
At room temperature, 806 mg of sodium hydrogen carbonate and then 683 mg of 4-amidinobenzoyl chloride hydrochloride were added, and the mixture was vigorously stirred for 30 minutes. The crude product obtained by concentrating the reaction solution is CH
Purification by P-20 column (eluted with 20% acetonitrile / H ₂ O) gave 1.0 g of the title compound as a colorless amorphous powder. Specific rotation: [α] _D ²⁰ + 106.6 ° (C = 0.478, 0.1N HCl) Elemental analysis Calculated as C ₂₇ H ₃₂ N ₆ O ₇・ 2H ₂ O: C, 55.09; H, 6.16; N, 14.28 Found: C, 55.36; H, 6.10; N, 14.35

Reference Example 31 (S) -4- [4- (2-aminoethyl) benzoylamino] acetyl-3- [3-
(4-Amidinobenzoylamino)] propyl-2-oxopiperazine-1-acetic acid trifluoroacetate (S) -3- [3- (4-amidinobenzoylamino)] propyl-4-benzyl obtained in Reference Example 30. Oxycarbonylaminoacetyl-2-oxopiperazine-1-acetic acid (300 mg) was dissolved in methanol (20 ml), 10% palladium-carbon (120 mg) was added, and the mixture was stirred under hydrogen atmosphere at room temperature for 1 hr. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to give an oily substance, which was dissolved in 5 ml of dimethylformamide, and 4- (2-
^t -butoxycarbonylamino) benzoic acid 173 mg and N
5 ml of DMF solution of active ester synthesized from 94 mg of hydroxysuccinimide and 167 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
Was added and the mixture was stirred at room temperature for 2 hours. The oily substance obtained by concentrating the reaction solution was dissolved in 7 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. The crude product obtained by concentrating the reaction solution under reduced pressure was used as CHP.
Purification by -20 column (eluted with 10% acetonitrile / water) gave 110 mg of the title compound as a colorless amorphous powder. Specific rotation [α] _D ²⁰ = ＋ 41.7 ° (C = 1.018, MeOH) Elemental analysis C ₂₈ H ₃₅ N ₇ O ₆・ 1.1CF ₃ CO ₂ H ・ 4H ₂ O Calculated value: C, 47.53; H , 5.82; N, 12.85 Found: C, 47.64; H, 5.60; N, 12.72

Reference Example 32 (S, S) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- (3-tert-butoxycarbonylaminopropyl) -2-oxopiperazine -1-
Synthesis of acetic acid tert-butyl ester (S) -3- (3-tert-butoxycarbonylamino) propyl-2-oxopiperazine-1-obtained in Reference Example 5
4.2 g of acetic acid tert-butyl ester oxalate was added to 5 parts of water.
It was dissolved in 0 ml, added with 2.3 g of sodium hydrogen carbonate, extracted with 50 ml of dichloromethane twice, dried (Na ₂ SO ₄ ), and concentrated under reduced pressure. To the residue was added 3 g of Z-Tyr (OMe) -OH and dissolved in 150 ml of dichloromethane, then WSC 1.92 g.
Was added and the mixture was stirred at room temperature for 2 hours. After dichloromethane was distilled off under reduced pressure, it was extracted with ethyl acetate. The ethyl acetate phase was washed with 3% aqueous potassium hydrogen sulfate and saturated aqueous hydrogen carbonate, dried (Na ₂ SO ₄ ), and concentrated under reduced pressure. The residue was chromatographed on silica gel (hexane / ethyl acetate = 1: 2).
~ Ethyl acetate) to give 5.88 g of the title compound. ¹ H-NMR (CDCl ₃ ) δ 1.35-2.10 (4H, m), 1.41 (9H, s), 1.46
(9H, s), 2.30 (1H, m), 2.80-3.85 (7H, m), 3.41 (1H, d, J = 1
7.4Hz), 3.78 (3H, s), 4.24 (1H, d, J = 17.4Hz), 4.75 (2H,
m), 4.94 (1H, t, J = 6.5Hz), 5.10 (2H, q, J = 12.4Hz), 5.69
(1H, d, J = 8.2Hz), 6.80 (2H, d, J = 8.6Hz), 7.09 (2H, d, J = 8.
6Hz), 7.35 (5H, s).

Reference Example 33 (S, S) -3- (3-aminopropyl) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl]-
Synthesis of 2-oxopiperazine-1-acetic acid (S, S) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- (3-tert- obtained in Reference Example 32. Butoxycarbonylaminopropyl) -2-oxopiperazine-1-acetic acid ter
5.7 g of t-butyl ester was suspended in 20 ml of toluene and stirred under ice cooling, and then 20 ml of trifluoroacetic acid was added.
After stirring at room temperature for 2 hours, toluene was added and the mixture was concentrated under reduced pressure. The residue was dissolved in 30 ml of water, and the pH was adjusted to 5 with concentrated aqueous ammonia.
After that, XAD-2 column chromatography (H ₂ O
→ eluted with 50% CH ₃ CN water) to obtain 4.3 g of the title compound. ¹ H-NMR (CD ₃ OD) δ 1.40-2.10 (4H, m), 2.32 (1H, m), 2.80-
4.00 (7H, m), 3.16 (1H, d, J = 16.5Hz), 3.77 (3H, s), 4.61-
4.85 (2H, m), 4.72 (1H, d, J = 16.5Hz), 5.05 (2H, q, J = 12.3H
z), 6.82 (2H, d, J = 8.4Hz), 7.11 (2H, d, J = 8.4Hz), 7.32 (5
H, s).

Reference Example 34 (S, S) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- (3-benzyloxycarbonylaminopropyl) -2-oxopiperazine- 1-
Synthesis of acetic acid (S, S) -3- (3-aminopropyl) -4- [2-benzyloxycarbonylamino-3 obtained in Reference Example 33
3.8 g of-(4-methoxyphenyl) propionyl] -2-oxopiperazine-1-acetic acid was dissolved in 100 ml of 50% aqueous dioxane, 1.52 g of sodium hydrogen carbonate was added, and Z-chloride 1 was cooled with ice. .24 ml was added dropwise, and the mixture was further stirred at room temperature for 1.5 hours. Distilled off dioxane,
The mixture was adjusted to pH 2 with 3% aqueous potassium hydrogen sulfate, extracted with ethyl acetate, washed with saturated aqueous sodium hydrogen carbonate, dried (sodium sulfate), and concentrated under reduced pressure. Ether was added to the residue and decantation was performed twice to obtain 4 g of the title compound. ¹ H-NMR (CDCl ₃ ) δ 1.40-2.05 (4 H.m),
2.22 (1H, m), 2.75 (9H, m),
3.74 (3H, s), 4.65-5.20 (6H,
m), 5.52 (1H, t, J = 5.5Hz),
5.94 (1H, d, J = 8.6Hz), 6.78
(2H, d, J = 8.6Hz), 7.05 (2H,
d, J = 8.6 Hz), 7.31 (10H, s).

Reference Example 35 (S, S) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- (3-benzyloxycarbonylaminopropyl) -2-oxopiperazine- 1-
Synthesis of acetic acid tert-butyl ester (S, S) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- (3-benzyloxycarbonylaminopropyl) obtained in Reference Example 34 ) -2-Oxopiperazine-1-acetic acid 1.
After dissolving 7 g, 2 ml of tert-butanol and 1.6 g of 4-dimethylaminopyridine in 50 ml of dichloromethane, W
SC 0.6g was added and it stirred at room temperature for 24 hours. Dichloromethane was distilled off, and the mixture was extracted with ethyl acetate. The ethyl acetate phase was washed with water and saturated aqueous NaCl, dried (Na ₂ SO ₄ ), and concentrated under reduced pressure. The residue was purified by silica gel chromatography (AcOEt) and crystallized from ether / hexane to give 1.02 g of the title compound as colorless crystals.
mp 138-140 ° C. Specific rotation: [α] _D ²⁰ + 49.7 ° (C
= 0.431, MeOH). Elemental analysis value: C ₃₉ H ₄₈ N ₄ O ₉ (716.832), calculated value: C, 65.35; H, 6.75; N, 7.82 Analysis value: C, 65.17; H, 6.69; N, 7.91

Reference Example 36 (S, S) -4- [2-benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- [3- (4-guanidinobenzoylamino) propyl] -2- Oxopiperazine-
Synthesis of 1-acetic acid hydrochloride (S, S) -3- (3-aminopropyl) -4- [2-benzyloxycarbonylamino-3 obtained in Reference Example 37.
0.5 g of-(4-methoxyphenyl) propionyl] -2-oxopiperazine-1-acetic acid was dissolved in 50 ml of 50% dioxane water, 0.24 g of sodium hydrogen carbonate was added, and 4-guanidinobenzoic acid 3,5- 0.3377 g of dioxo-4-aza-tricyclo [5,2,1,0 2,6] dec-8-en-4-yl ester hydrochloride was added, and the mixture was stirred at room temperature for 2 hours. The pH of the reaction solution is adjusted to 3 with 1N hydrochloric acid, and dioxane is distilled off. The residue was subjected to XAD-2 column chromatography (H ₂ O → 10% CH ₃ CN water → 20%
CH ₃ CN water → 50% CH ₃ CN water was used for elution) to obtain 0.43 g of the title compound. ¹ H-NMR (CD ₃ OD) δ: 1.50-2.10 (4H, m), 2.45 (1H, m), 2.8
0-4.25 (9H, m), 3.77 (3H, s), 4.60-5.00 (2H, m), 5.00 (2
H, s), 6.83 (2H, d, J = 8.5Hz), 7.13 (2H, d, J = 8.5Hz), 7.30
(5H, s), 7.35 (2H, d, J = 8.5Hz), 7.92 (2H, d, J = 8.5Hz).

Reference Example 37 (S, S) -3- [3- (4
-Guanidinobenzoylamino) propyl] -4- [3
-(4-Methoxyphenyl) -2- [4- (5-trifluoromethyl- [1,2,4] oxadiazol-3-ylamino) benzoylamino] propionyl] -2-oxopiperazine-1-acetic acid. Synthesis of Hydrochloride (S, S) -4- [2-Benzyloxycarbonylamino-3- (4-methoxyphenyl) propionyl] -3- [3- (4-guanidinobenzoylamino) propyl obtained in Reference Example 36 ] -2-Oxopiperazine-1-acetic acid hydrochloride was dissolved in 40 ml of methanol to obtain 10% Pd-C.
0.2 g was added and catalytic reduction was performed at room temperature for 2 hours. The catalyst was removed by filtration, the filtrate was concentrated under reduced pressure and then dissolved in 50 ml of 50% aqueous dioxane. While keeping alkaline with sodium hydrogen carbonate, 4- (5-trifluoromethyl- [1,2,
4] Oxadiazol-3-ylamino) benzoic acid and a dioxane solution of acid chloride prepared from oxalyl chloride were added dropwise, and the mixture was stirred at room temperature for 30 minutes. The pH of the reaction solution is 1N
-After adjusting to 3 with hydrochloric acid, the mixture was concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate: acetic acid: water = 8: 1: 1), and ether was added to give 0.17 g of the title compound as a colorless powder. Specific rotation: [α] _D ²⁰
+ 42.8 ° (C = 0.94, DMSO). Elemental analysis: C ₃₇ as _{H 39 N 10 O 8 F 3} · HCl · 0.1Et 2 O (852.649), Calcd: C, 52.68; H, 5.08 ; N, 16.43 analysis: C, 52.62; H, 5.01 ; N, 16.58

Reference Example 38 (S, S) -4- [2- (4
-Guanidinobenzoyl) amino-3- (4-methoxyphenyl) propionyl] -3- [3- (4-guanidinobenzoyl) aminopropyl] -2-oxopiperazine-1-acetic acid hydrochloride obtained in Reference Example 32 (S , S) -3- (3-Aminopropyl) -4- [2-benzyloxycarbonylamino-3
250 mg of-(4-methoxyphenyl) propionyl] -2-oxopiperazine-1-acetic acid was dissolved in 5 ml of methanol, 100 mg of 10% palladium-carbon was added, and the mixture was stirred under hydrogen at room temperature for 1 hour. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure to give an oily substance, 10 ml of dioxane and 10 ml of water.
210 ml of a mixed solvent of sodium hydrogencarbonate and 4-guanidinobenzoic acid 3,5-dioxo-4-azatricyclo [5,2,1,0 2,6] dec-8-ene-4
450 mg of yl ester was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was adjusted to 1N. After adjusting the pH to 3 with hydrochloric acid, dioxane was distilled off under reduced pressure. The obtained aqueous solution is CHP-20.
The mixture was applied to a column, and the fraction eluted with 10% acetonitrile / water was freeze-dried to obtain 130 mg of the title compound as an amorphous powder. Elemental analysis: Calculated as C ₃₅ H ₄₂ N ₁₀ O ₇ · HCl · 2H ₂ O: C, 53.40; H, 6.02, N, 17.79 Found: C, 53.11; H, 5.86, N, 18.06

Reference Example 39 (S) -4- (4-amidinobenzoylamino) acetyl-3- [2- (4-amidinobenzoylamino)] ethyl-2-oxopiperazine-1-acetic acid trifluoroacetic acid salt Reference Example (S) -4- (Benzyloxycarbonylamino) acetyl-3- (2- ^t -butoxycarbonylamino) ethyl-2-oxopiperazine-1-acetic acid obtained in 28.
^{The t-} butyl ester was subjected to the same operations as in Reference Examples 3 and 4 to obtain the title compound as a colorless amorphous powder. Specific rotation: [α] _D ²⁰ + 30.299 ° (C = 0.470, H ₂ O) Elemental analysis Calculated as C ₂₆ H ₃₀ N ₈ O ₆・ CF ₃ CO ₂ H ・ 3H ₂ O: C, 46.80; H, 5.19; N, 15.59. Found: C, 46.67; H, 4.99; N, 15.39.

Reference Example 40 (S) -4- (4-amidinobenzoylamino) acetyl-3- [2- (4-amidinophenylaminocarbonyl) ethyl] -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt ( S) -4-Benzyloxycarbonylaminoacetyl-1- ^t -butoxycarbonylmethyl-2-oxopiperazine-3-propanoic acid methyl ester was used in Reference Examples 23 and 2.
This was subjected to the same operations as in Nos. 4 and 25 to obtain the title compound as a colorless amorphous powder. Specific rotation: [α] _D ²⁰ + 59.625 ° (C = 0.360, H ₂ O) Elemental analysis Calculated as C ₂₆ H ₃₀ N ₈ O ₆・ CF ₃ CO ₂ H ・ 4H ₂ O: C, 45.65; H, 5.34; N, 15.21 Found: C, 45.70, H, 5.10; N, 14.91

Reference Example 41 (S) -4- (4-amidinobenzoylamino) acetyl-3- [3- (4-amidinophenylaminocarbonyl) propyl] -2-oxopiperazine-1-acetic acid trifluoroacetic acid salt ( S) -4-Benzyloxycarbonylaminoacetyl-1- ^t -butoxycarbonylmethyl-2-oxopiperazine-3-butanoic acid methyl ester was used, and Reference Example 40 was used.
The title compound is obtained by performing the same operation as. Reference Example 42 (S) -4- (4-amidinobenzoylamino) acetyl-3- [4- (4-amidinophenylaminocarbonyl) butyl] -2-oxopiperazine-1
-Acetic acid trifluoroacetic acid salt (S) -4-benzyloxycarbonylaminoacetyl-1- ^t -butoxycarbonylmethyl-2-oxopiperazine-3-pentanoic acid methyl ester was used, and Reference Example 4 was used.
The title compound is obtained by the same procedure as for 0.

[0049]

According to the present invention, a physiologically active substance having an optionally substituted amidino group can be kept stable for a long period of time.

Claims (11)

[Claims] 1. A freeze-dried preparation containing a physiologically active substance having an optionally substituted amidino group and a disaccharide. 2. A physiologically active substance is represented by the formula: [Wherein R is hydrogen or an alkyl group, X is hydrogen or an optionally substituted alkyl group, and Y is 1 to 2].
A cyclic hydrocarbon which may be bonded via individual atoms,
Z represents a carboxyl group which may be amidated or esterified, respectively] or a pharmaceutically acceptable salt thereof, wherein the lyophilized preparation according to claim 1. 3. R is a C _1-6 alkyl group, X is a carboxyl group which may be amidated or esterified, a phenyl group or the formula: —A—B—D— [wherein A is amino or amidino]. Or a guanidino group, B a phenylene group, D
Represents -CONH-], a C _1-4 alkyl group which may be substituted with a group represented by the formula, Y is a 5- to 6-membered cyclic hydrocarbon group which may be bonded via an imino group, Z The lyophilized preparation according to claim 2, wherein is a carboxyl group. 4. A physiologically active substance is (S) -4- (4-amidinobenzoylglycyl) -3-methoxycarbonylmethyl-2-oxopiperazine-1-acetic acid or a pharmaceutically acceptable salt thereof. The freeze-dried preparation according to claim 1. 5. The physiologically active substance is (S) -4- (4-amidinobenzoyl) aminoacetyl-3- [3- (4-amidinobenzoyl) amino] propyl-2-oxopiperazine-1-acetic acid or a drug thereof. The freeze-dried preparation according to claim 1, which is a physically acceptable salt. 6. The physiologically active substance is (S) -4- (4-guanidinobenzoyl) aminoacetyl-3- [3- (4-
The freeze-dried preparation according to claim 1, which is guanidinobenzoyl) amino] propyl-2-oxopiperazine-1-acetic acid or a pharmaceutically acceptable salt thereof. 7. The freeze-dried preparation according to claim 1, wherein the disaccharide is maltose, cellobiose, gentiobiose, melibiose, lactose, turanose, solohorse, trehalose, isotrehalose, isosaccharose or sucrose. 8. The freeze-dried preparation according to claim 7, wherein the disaccharide is maltose, lactose or sucrose. 9. The freeze-dried preparation according to claim 1, wherein the content of the disaccharide is 1 to 1000 by weight with respect to 1 of the physiologically active substance. 10. The freeze-dried preparation according to claim 9, wherein the content of the disaccharide is 5 to 500 by weight with respect to 1 of the physiologically active substance. 11. An in-use dissolution preparation for injection, which comprises the freeze-dried preparation according to claim 1.