JP2003026698A - New compound, immobilized product and method for screening protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound useful for diabetic medicines, to provide an immobilized product, and to provide a method for screening the protein. SOLUTION: This compound of formula (I) [R<1> is an aryl group or a heterocyclic group; X is a binding bond, a lower alkylene or a lower alkenylene; R<2> is a lower alkyl, or may form a heterocyclic group together with the adjacent nitrogen atom; R<3> is H or a halogen atom; R<4> is H or a lower alkyl, or R<3> and R<4> may be bound to each other to form a heterocyclic group; R<5> is H, a lower alkyl or a lower alkenyl; R<6> is H, hydroxyl, a lower alkyl, a hydroxy(lower)alkyl or an amino(lower)alkylamino; (n) is an integer of 0 to 2] and its salt are provided.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel compound, a salt thereof, an immobilization product and a protein screening method.

[0002]

2. Description of the Related Art Improvement and treatment of lifestyle-related diseases are indispensable in modern society in an aging society. Among them, the number of patients with diabetes continues to increase, but many of the therapeutic agents have side effects such as promoting liver damage and obesity, and it is possible to obtain satisfactory drug efficacy and side effects. The current situation is that it has not been done. On the other hand, as one technology for drug development, we identified bioreceptors that catch drugs,
A method has been proposed in which the receptor is utilized for screening of drugs and structural analysis for drug design by separation and purification. Therefore, with diabetes as a target disease, by utilizing the above-mentioned drug development technology, the pharmacologically active substance-biological receptor interaction is analyzed, and a new pharmacologically active substance is created based on combinatorial chemistry. There is a need for a method of early development of an ideal drug for treating diabetes.

[0003]

According to the invention, the formula (I):

[Chemical 4] [Wherein R ¹ is an aryl group or a heterocyclic group, and the aryl group and the heterocyclic group are selected from the group consisting of a heterocyclic group, an aryl group, a hydroxy group and a lower alkyl group. It may be substituted with one or more substituents, and among these substituents, the heterocyclic group and the aryl group may be further substituted with a lower alkenyl group or a halogen atom, and the hydroxy group may be further substituted. Optionally, X is a bond, a lower alkylene group or a lower alkenylene group, and R ² represents a lower alkyl group,
Alternatively, it may form a heterocycle with an adjacent nitrogen atom, and the lower alkyl group is an amino group, a hydroxy group,
It may be substituted with one or more substituents selected from the group consisting of a halogen atom, a carboxy group, a carbamoyl group, a heterocyclic group, an aryl group and a lower alkylthio group, and among these substituents, an amino group and a hydroxy group. The group may be further substituted, R ³ is a hydrogen atom or a halogen atom, R ⁴ represents a hydrogen atom or a lower alkyl group optionally substituted with a hydroxy group, or R ³ and R ⁴ And may combine with each other to form a heterocycle, which may be substituted with a hydroxy group and / or a lower alkyl group. R ⁵ is a hydrogen atom, a lower alkyl group or a lower alkenyl group. There, R ⁶ is hydrogen atom, hydroxy group, lower alkyl group, hydroxy (lower) alkyl or amino (lower) alkylamino group, hydrin of these Alkoxy group, hydroxy (lower)
The alkyl group and the amino (lower) alkylamino group may be further substituted, and n represents an integer of 0 to 2.

However, R ¹ is a quinolin-4-yl group substituted with a lower alkenyl group and a phenyl group substituted with a hydroxy group, X is a bond, and R is
² is an amino group substituted with an amidino group, a hydroxy group and a lower alkyl group substituted with a halogen atom, and R ³ and R ⁴ are bonded to each other and are pyrrolidine substituted with a hydroxy group and a lower alkyl group. Forming a ring, R ⁵
Is a lower alkenyl group and R ⁶ is a hydroxy group, n is not 0. ] And its salts are provided.

According to the invention, the formula (Ia):

[Chemical 5] [Wherein R ^1a represents an aryl group or a heterocyclic group, and these aryl group and heterocyclic group are selected from the group consisting of a heterocyclic group, an aryl group, a hydroxy group and a lower alkyl group. It may be substituted with one or more substituents, and among these substituents, the heterocyclic group and the aryl group may be further substituted with a lower alkenyl group or a halogen atom, and the hydroxy group may be further substituted. Optionally Xa is a bond, a lower alkylene group or a lower alkenylene group, R ^2a represents a lower alkyl group, or may form a heterocycle with an adjacent nitrogen atom, a lower alkyl group Is 1 or more selected from the group consisting of an amino group, a hydroxy group, a halogen atom, a carboxy group, a carbamoyl group, a heterocyclic group, an aryl group and a lower alkylthio group. R ^3a is a hydrogen atom or a halogen atom, R ^4a is a hydrogen atom or a hydroxy group, and the amino group and the hydroxy group of these substituents may be further substituted. Represents a lower alkyl group which may be substituted with a group, or R ^3a and R ^4a may combine with each other to form a heterocycle, which is a hydroxy group and / or a lower alkyl group. R ^5a , which may be substituted with, is a hydrogen atom,
A lower alkyl group or a lower alkenyl group, R ^6a represents a hydrogen atom, a hydroxy group, a lower alkyl group, a hydroxy (lower) alkyl group or an amino (lower) alkylamino group, and among these, a hydroxy group, a hydroxy (lower) group The alkyl group and the amino (lower) alkylamino group may be further substituted, and n represents an integer of 0 to 2. ] The immobilization product obtained by bonding the compound of the above] to the fine particles made of a styrene-glycidyl methacrylate polymer through a spacer is provided.

Further, according to the present invention, there is provided a method for screening a protein, which comprises screening a protein capable of binding to the compound of formula (Ia) using the above-mentioned immobilized product.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The novel compound of the present invention is represented by the above formula (I). Unless otherwise specified, the term "lower" in the definition of the substituent of the formula (I) and the following description means a carbon number of 1 to 6, but a "lower alkenyl group".
The term "lower" in the "lower alkenylene group" means 2 to 6 carbon atoms. Moreover, the number of the substituents is not particularly limited, and examples thereof include 1 to 4, 1 to 3, and 1 to 2.

In the formula (I), R ¹ is an optionally substituted aryl group or heterocyclic group. Examples of the aryl group include phenyl, naphthyl, tolyl, xylyl and biphenyl, with phenyl being particularly preferred.
Examples of the heterocyclic group include a 5- to 10-membered monocyclic or condensed heterocyclic group having 1 to 3 nitrogen, oxygen and / or sulfur atoms, and specifically, furyl, thienyl, pyrrolyl,
Examples thereof include imidazolyl, oxazolyl, thiazolyl, benzofuryl, indolyl, quinolyl, isoquinolyl, imidazopyridyl, triazolyl, oxadiazolyl and thiadiazolyl.

Examples of the substituents on the aryl group and the heterocyclic group include a heterocyclic group, an aryl group, a hydroxy group and a lower alkyl group. Examples of the heterocyclic group and aryl group here include the same ones as described above,
Further, it may be substituted with a lower alkenyl group or a halogen atom. Examples of the lower alkenyl group include linear or branched ones, and specifically, vinyl, allyl,
Propenyl, isopropenyl, etc. are mentioned. Examples of the halogen atom include fluorine, chlorine, bromine and iodine atoms.

Examples of the lower alkyl group include linear or branched ones, specifically, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, t-pentyl. , Hexyl and the like. The hydroxy group which is a substituent in the aryl group and the heterocyclic group of R ¹ may be further substituted, and examples of such a substituent include lower alkyl and optionally substituted hydroxy (lower) alkyl group. And optionally substituted amino (lower) alkyl groups.

Examples of the above-mentioned hydroxy (lower) alkyl group include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl,
Hydroxyhexyl and the like can be mentioned. Examples of the amino (lower) alkyl group include aminomethyl, aminoethyl, aminopropyl, aminobutyl, aminopentyl, aminohexyl and the like. When these hydroxy (lower) alkyl group and amino (lower) alkyl group are substituted, examples of the substituent include acyl, cyclo (lower) alkenyl, ar (lower) alkyl, tri (lower) alkylsilyl, lower alkyl. , Lower alkoxy (lower) alkyl, heterocyclic (lower) alkyl, acyl (lower) alkyl, lower alkenyl and the like.

As the acyl, lower alkanoyl (eg, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl etc.), lower alkoxycarbonyl (eg methoxycarbonyl, ethoxycarbonyl etc.), lower alkanesulfonyl (eg mesyl, ethanesulfonyl). Etc.) aliphatic acyl groups such as arenesulfonyl (eg, benzenesulfonyl, tosyl, etc.), aroyl (eg, benzoyl, toluoyl, xyloyl, naphthoyl, phthaloyl, etc.), ar (lower) alkanoyl (eg, phenylacetyl, phenyl, etc. Propionyl etc.), ar (lower) alkoxycarbonyl (for example, benzyloxycarbonyl, phenethyloxycarbonyl,
Such as acyl containing an aromatic or heterocyclic ring (such as indanecarbonyl).

Cyclo (lower) alkenyl includes cyclobutenyl, cyclopentenyl, cyclohexenyl and the like. Examples of ar (lower) alkyl include benzyl and phenethyl. Examples of tri (lower) alkylsilyl include trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, tert-butyldimethylsilyl and the like.
Examples of lower alkoxy (lower) alkyl include methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, tert-butoxymethyl, tert-butoxyethyl and the like.

Examples of the heterocyclic (lower) alkyl include furfuryl, thienylmethyl, pyrrolylmethyl and the like. Examples of acyl (lower) alkyl include formylmethyl, acelylmethyl, benzoylethyl and the like. Examples of the lower alkenyl include the same ones as described above. Examples of the lower alkyl group include the same ones as described above. As the lower alkylene group represented by X,
Methylene, ethylene, propylene and the like can be mentioned. Examples of the lower alkenylene group include arylene and isopropenylene.

R ² is an optionally substituted lower alkyl group, and when the lower alkyl group is substituted, the substituent is, for example, an optionally substituted amino group or an optionally substituted hydroxy group. One or more substituents selected from a group, a halogen atom, a carboxy group, a carbamoyl group, a heterocyclic group, an aryl group and a lower alkylthio group can be mentioned.

As the amino group which may be substituted,
Unsubstituted amino group, amino-protecting group (for example, t-Bo
c etc.), carbamoyl group or formula

[Chemical 6] [In the formula,-. Means a bond on the amino group side. ] An amino group substituted with a group represented by Examples of the optionally substituted hydroxy group include an unsubstituted hydroxyl group and a lower alkyl-substituted hydroxy group.

As the heterocyclic group and the aryl group, the same ones as mentioned above can be mentioned. Examples of the lower alkylthio group include methylthio, ethylthio, propylthio, isopropylthio and the like. Examples of the heterocycle formed by R ² together with the adjacent nitrogen atom include a 5 to 10-membered heterocycle having 1 to 3 nitrogen atoms, and specific examples thereof include pyrrolidine, piperidine, piperazine, morpholine and pyrroline. Can be mentioned. As the halogen atom in R ³ ,
The same as the above is mentioned.

As the lower alkyl group for R ⁴ , the same as mentioned above can be mentioned. When R ³ and R ⁴ are bonded to each other to form a heterocycle, the same heterocycles as mentioned above can be mentioned. Examples of the lower alkyl group and lower alkenyl group for R ⁵ include the same ones as described above. Examples of the optionally substituted hydroxy group and lower alkyl group for R ⁶ include the same ones as described above.

As the hydroxy (lower) alkyl group,
The same as the above is mentioned. Examples of the amino (lower) alkylamino group include aminomethylamino, aminoethylamino, aminopropylamino, amino-n-butylamino and the like. When the hydroxy (lower) alkyl group and the amino (lower) alkylamino group are substituted, the substituent is acyl, cyclo (lower) alkenyl, ar (lower) alkyl, tri (lower) alkylsilyl, lower alkyl, lower. Alkoxy (lower) alkyl,
Heterocyclic (lower) alkyl, acyl (lower) alkyl, lower alkenyl and the like can be mentioned. Specific examples of these are:
The same as the above is mentioned.

In the formula (I), R ¹ is a quinolin-4-yl group substituted with a lower alkenyl group and a phenyl group substituted with a hydroxy group, X is a bond, and R ² Is an amino group substituted with an amidino group, a hydroxy group and a lower alkyl group substituted with a halogen atom, and R ³ and R ⁴ are bonded to each other to form a pyrrolidine ring substituted with a hydroxy group or a lower alkyl group. When R ⁵ is a lower alkenyl group and R ⁶ is a hydroxy group, compounds in which n is 0 are excluded.

[Chemical 7] It is preferred that only the compound represented by In addition, R ^1a , R ^2a , R ^3a , R ^4a in the formula (Ia),
Specific examples of R ^5a , R ^6a, and Xa are R ¹ , R ² , and R, respectively.
Specific examples of ³ , R ⁴ , R ⁵ , R ⁶ and X are the same.

The salts of compound (I) and compound (Ia) include metal salts such as alkali metal salts (eg sodium or potassium salt) and alkaline earth metal salts (eg calcium or magnesium salt), ammonium salts. ,
Salts with organic bases (for example, trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine or salts with N, N'-dibenzylethylenediamine), salts with organic acids (for example, acetic acid, maleic acid, tartaric acid, methanesulfonic acid) , Benzenesulfonic acid, formic acid, toluenesulfonic acid or salts with trifluoroacetic acid), salts with inorganic acids (for example, salts with hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid), salts with amino acids (for example, arginine) , A salt with aspartic acid or glutamic acid) and the like.

The compound (I) may contain one or more stereoisomers due to an asymmetric carbon atom and a double bond, and such isomers and mixtures thereof are also included in the scope of the present invention. Compound (I) and a salt thereof can be produced, for example, by the following method.

Method 1

[Chemical 8] [In the formula, R ^{1 to} R ⁶ , X and n have the same meanings as described above. ] It can manufacture by the method represented. In this amidation reaction, compound (IV) and compound (V) are usually combined with a suitable condensing agent (for example, 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide, 1,3-dicyclohexylcarbodiimide, etc. ) And optionally adjuvants (eg N
-Hydroxybenzotriazole, N-hydroxysuccinimide, etc.) in a solvent that does not adversely affect the reaction (eg, acetonitrile, benzene, N, N-dimethylformamide, tetrahydrofuran, methylene chloride, ethylene chloride, chloroform, diethyl ether, etc.) It can be carried out. The reaction temperature is not particularly limited and is usually under cooling to heating.

When the substituent of the resulting compound (I) is protected, this protecting group may be removed by a conventional method. Compound (IV) and compound (V), which are the starting materials of this reaction, can be prepared by subjecting, for example, to the method described in Reference Examples to be described later or the compounds described in WO99 / 63102 or analogs of this compound. It can be produced by a similar method.

Method 2 In compound (I), compound (Ib) in which R ⁶ is an optionally substituted amino lower alkylamino group is

[Chemical 9] [Wherein R ¹ , R ³ , R ⁴ and R ⁵ have the same meanings as defined above, and C
OR ⁶ ″ is a carboxy group or an activated carboxy group, and R ⁶ ′ is an optionally substituted amino lower alkyl group].

The compound (II) as a starting material is, for example,
The compounds described in WO 99/63102 or analogues of this compound can be used. The above-mentioned amidation reaction can be usually carried out by compound (II) and compound (III) together with a suitable condensing agent and any auxiliary agent in a solvent that does not adversely influence the reaction. The reaction temperature is
The method is not particularly limited, and as in Method 1 above,
Usually, it is carried out under cooling to heating, and examples of the condensing agent, the auxiliary agent, and the solvent include the same ones as exemplified in the above method 1. In addition, when the amino group of the obtained compound (Ib) is protected, this protecting group may be removed by a usual method.

Method 3 Of Compound (I) and its salt, Compound (VI) to Compound (IX) are produced by a library synthesis method for producing chemically diverse compounds, for example, using combinatorial chemistry. can do. More specifically,
It is as shown below. (1)

[Chemical 10]

(2)

[Chemical 11]

(3)

[Chemical 12]

(4)

[Chemical 13]

[In the formula, the asterisk represents a carrier, and AA ₁ , AA ₂ , A
A ₂ ', AA ₃ , Ar, Ar' and R have the same definitions as described later. This carrier is a carrier usually used in synthesis, and a desirable carrier includes, for example, crown. These reactions can be carried out by using a known compound or a compound derived from a known compound, by the method described in Examples below or a method analogous thereto.

The compound (I) of the present invention and a salt thereof have an inhibitory activity on gluconeogenesis and are useful as a therapeutic and prophylactic agent for diabetes, obesity and the like. When the compound (I) is used as a therapeutic or preventive agent for diabetes, obesity, etc., it varies depending on the age and symptoms of the person to be treated or prevented, but as a therapeutic agent, for example, in the case of systemic administration, usually, About 0.01-1000 mg per day, preferably about 0.1-
500 mg, more preferably about 0.5 to 100 mg of the compound (I) or an elementary salt is administered, specifically, an average of 0.2 to 0.5 mg, 1 mg, 5 mg, 10 m per day.
g, 50 mg, 100 mg, 250 mg and 500 mg
Is administered. In case of long-term administration, it is about 0.
It is administered in the range of 3 mg / body to 1000 mg / body.

The compound (I) is bonded to fine particles of a styrene-glycidyl methacrylate polymer (hereinafter referred to as "SG particles") through a spacer to form the immobilized product of the present invention. The SG particles are not particularly limited in terms of polymerization state, morphology and the like, but it is preferable that the SG particles can be used as a stationary phase in ordinary liquid chromatography. For example, a polymer obtained by polymerizing styrene and glycidyl methacrylate and having a glycidyl group on the surface of the polymer can be mentioned. The size of SG particles is, for example, 0.05 to 0.
It is preferably about 5 μm.

The spacer has one end bound to the SG particles and the other end bound to the compound (I), as long as the SG particles and the compound (I) are bound via an appropriate distance. The structure, form, etc. are not particularly limited. For example, it is preferable that both ends of the spacer have the same or different functional groups such as an amino group, a carboxy group, and an epoxy group before being bonded to the SG particles and / or the compound (I).

As the spacer bonded to the SG particles, for example,

[Chemical 14] Etc. These are usually converted to an active ester, a halogen compound (for example, an iodo compound, etc.) or the like, and then bound to the compound (I).

The protein screening method of the present invention comprises:
The immobilization product described above is used to screen a protein capable of binding to compound (I). Specifically, it is carried out by mixing the above-mentioned immobilized product with a cell extract containing a protein, stirring the mixture as necessary, separating the immobilized product, eluting the protein from the immobilized product, and dissociating the protein. . In this way, a specific protein (eg, receptor) containing a site acting on compound (I) can be screened. The specific bond between the immobilized product and the protein may be a chemical bond such as hydrogen bond, chemical or physical adsorption, or the like.

The obtained protein can be optionally purified by a known method such as chromatography, a gene encoding the protein can be cloned, and its amino acid sequence and the base sequence of the gene can be determined. By producing a large amount of a peptide having a peptide or a partial sequence thereof with a genetically modified microorganism, it can be used for drug screening, structural analysis for drug design, and the like. That is, a compound having a stronger inhibitory activity against gluconeogenesis can be found by searching for a compound having an affinity for the protein as the receptor obtained as described above.

[0038]

EXAMPLES Examples of the compounds and immobilizates of the present invention will be specifically described below. Reference Example 1: N- (4-triphenylmethylaminobutyl) -2-
([[1- (5-[[amino (imino) methyl] amino] -3-chloro-
4-hydroxy-2-[[4-hydroxy-3- (3-isopropenyl-
4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenamide 2-([[1- (5-[[amino (imino ) Methyl] amino] -3-chloro-4-hydroxy-2-[[4-hydroxy-3- (3-isopropyl
-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoic acid (50mg, 0.068mmol), 1-triphenylmethylamino -4-aminobutane (22.4 mg, 0.68
mmol), WSCD.HCl (13 mg, 0.068 mmol) and 1-hydroxybenzotriazole (9.18 mg, 0.068 mmol) in N, N-dimethylformamide (5 ml) overnight,
Stir at room temperature. Dilute the reaction mixture with ethyl acetate,
Wash sequentially with water and brine. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure to give the title compound
(35 mg) was obtained as an amorphous solid. MS: 1047 (M-1)

Reference Example 2: 2-[[(3-hydroxy-3-methyl-
2-Pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoic acid 2-([[1- (5-[[amino (imino) methyl] amino] -2-[[4-hydroxy-3- (3 -Isopropenyl-4-quinolinyl) benzoyl] amino] -3-chloro-4-hydroxypentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoic acid ( 2.0g, 2.72mmol) was dissolved in 1N aqueous ammonia (25mL), and 1N aqueous sodium hydroxide solution was added.
(25 mL) was added, and the mixture was stirred at room temperature for 48 hours. The reaction mixture
It was adjusted to acidic (pH = 2) with 1N hydrochloric acid.

ODS-AM (ODS-AM120-S50, made by YMC) resin
(180 mL) was equilibrated with 0.05% TFA water, and the above acidic solution was passed therethrough. C-terminal dipeptide 2-when flushed with 0.05% TFA water
[[(3-Hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoic acid eluted as the TFA salt. Further, when 20% acetonitrile-0.05% TFA water was flown, the N-terminal acylarginine moiety was eluted as a TFA salt. These were collected and concentrated under reduced pressure.

For the C-terminal dipeptide, the residue was first dissolved in 50 mL of water. ODS-AM equilibrated with water (ODS-AM120-S
50, made by YMC) and passed through a resin (180 mL). After elution by flushing with water, the mixture was concentrated, the residue was dissolved in methanol, and triturated with ethyl acetate to give the desired compound (417 mg, 63
%) Was obtained. 1H-NMR (300MHz, DMSO-d6) δ1.50 (3H, s), 1.80 (3H, s),
1.88-1.98 (2H, m), 2.06 (3H, s), 3.13-3.241 (1H, m), 3.2
5-3.37 (1H, m), 3.88 (1H, s),

With respect to the N-terminal acylarginine portion, 2.65 g was obtained as a concentrated residue and used as a starting material for the next step without being produced. Reference Example 3: Methyl 2-[[(3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoate 2-[[(3-hydroxy-3-methyl-2-pyrrolidinyl ] Carbonyl] amino) -3-methyl-2-butenoic acid (100 mg, 0.413 mmol) is dissolved in methanol (2.0 mL) and chloroform (2.0 mL), and 2M trimethylsilyldiazomethanehexane solution is prepared.
(0.45 mL) was added dropwise at 0 ° C. over 5 minutes and the mixture was stirred at room temperature for 15 minutes. Furthermore, to the mixture, 2M
Trimethylsilyl diazomethane hexane solution (0.3 mL)
Was added dropwise at 0 ° C. over 5 minutes and the mixture was stirred at room temperature for 15 minutes. The reaction was quenched with acetic acid (3 drops) and the resulting mixture was diluted with chloroform (10 mL). The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution (10 mL), and then the aqueous layer was extracted with chloroform (10 mLx5). The combined layers were dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent; 10% methanol in chloroform),
The title compound (53 mg, 50%) was obtained as a colorless oil. ESI-MS; 257.2 (M + H)

Reference Example 4: Methyl 2-([[1- (5-([[(tert-butoxycarbonyl) amino] [(tert-butoxycarbonyl) imino] methyl] amino) -2-[[(9H- Fluoren-9-ylmethoxy) carbonyl] amino] pentanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino)-
3-Methyl-2-butenoate methyl 2-[[(3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoate (50 mg, 0.
195 mmol), 5-([[(tert-butoxycarbonyl) amino] [(t
ert-butoxycarbonyl) imino] methyl] amino) -2-
[[(9H-Fluoren-9-ylmethoxy) carbonyl] amino]
Valeric acid (116 mg, 1.0 eq.) And 1-hydroxybenzotriazole (29 mg, 1.1 eq.) Were added to N, N-dimethylformamide.
Dissolved in (2.0 mL), 1-ethyl-3- (3-dimethylethylaminopropyl) carbodiimide hydrochloride (41 mg, 1.1
eq.) was added portionwise at 0 ° C. and the mixture was stirred at room temperature for 4 days. The resulting mixture was diluted with water (10 mL) and ethyl acetate (10 mL). The organic layer was collected, washed successively with saturated aqueous sodium hydrogen carbonate solution (10 mL) and brine (10 mL), dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent; 0.5-5% methanol in chloroform) to give the title compound (137 mg, 84%) as a colorless syrup. ESI-MS; 835.5 (M + H)

Reference Example 5: 4-Hydroxy-3- (3-isopropenyl-4-quinolinyl) benzoic acid The concentrated residue (2.65 g) of the N-terminal acylarginine moiety obtained in Reference Example 2 was added to water (50 mL). After dissolution, solid sodium hydroxide (3.26 g, 30 eq.) Was added and the mixture was heated under reflux for 48 hours.
The reaction mixture was naturally cooled to room temperature, diluted with water (50 mL), and then adjusted to acidic (pH = 2) with 6N hydrochloric acid.

After removing insoluble matter by filtration, ODS-AM (ODS
-AM120-S50, YMC) resin (180 mL) was equilibrated with water,
The filtrate was passed there. Flowing through with 20% acetonitrile-0.05% TFA water, 4-hydroxy-3- (3-isopropenyl-4-quinolinyl) benzoic acid elutes as a TFA salt. This was collected and concentrated under reduced pressure. The residue was dissolved in 100 mL of water, the pH was adjusted to 5 with a 1N aqueous sodium hydroxide solution, and the precipitated solid was collected by filtration. By drying at 30 ° C. under reduced pressure for 3 hours, 4-hydroxy-3- (3-isopropenyl-4-quinolinyl) benzoic acid was obtained as a brown solid in an amount of 490 mg (58.6%). 1H-NMR (300MHz, DMSO-d6) δ1.81 (3H, s), 4.96 (1H, s),
5.16 (1H, s), 7.08 (1H, d, J = 8Hz), 7.37 (1H, d, J = 8Hz), 7.
52 (1H, t, J = 8Hz), 7.61 (1H, s), 7.73 (1H, t, J = 8Hz), 7.92
(1H, d, J = 8Hz), 8.06 (1H, d, J = 8Hz), 8.86 (1H, s). Reference Example 6: 3-[(tert-butoxycarbonyl) amino] propyl 4- [3- [ (tert-Butoxycarbonyl) amino] propoxy] -3- (3-isopropenyl-4-quinolinyl) benzoate

4-Hydroxy-3- (3-isopropenyl-4-quinolinyl) benzoic acid and tert-butyl 3-bromopropylcarbamate were dissolved in N, N-dimethylformamide (2.5 mL) and potassium carbonate (136 mg) was added. Was added and the mixture was stirred at room temperature for 4.5 hours. The temperature was raised to 50 ° C. and the mixture was stirred at that temperature for 15 hours. The resulting mixture is water (10 mL)
And diluted with ethyl acetate (20 mL), then the layers were separated. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate solution and brine, dried over magnesium sulfate and concentrated under reduced pressure. Silica gel column chromatography of the residue
(Eluent; n-hexane: ethyl acetate = 3: 2-1: 1)
The title compound (128 mg, 63
%) ESI-MS; 620.4 (M + H)

Reference Example 7: 4- [3-[(tert-butoxycarbonyl) amino] propoxy] -3- (3-isopropenyl-4-quinolinyl) benzoic acid 3-[(tert-butoxycarbonyl) amino] propyl 4- [3-
[(tert-Butoxycarbonyl) amino] propoxy] -3- (3-
Isopropenyl-4-quinolyl) benzoate in methanol
Dissolve in (2.0 mL), 1N aqueous sodium hydroxide solution (1.0 mL)
Was added and the mixture was stirred at room temperature for 8 hours. The resulting mixture was adjusted to acidic with 1N hydrochloric acid (pH = ca.6) and extracted with ethyl acetate.
Dilute (10 mL), then separate layers. The organic layer was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residual solid was triturated with ether-n-hexane (1: 2) to give the title compound (85 mg, 91%) as a white solid. 1H-NMR (300MHz, DMSO-d6) δ1.33 (9H, s), 1.45-1.57 (2
H, m), 1.76 (3H, s), 2.54-2.68 (2H, m), 3.98-4.07 (2H,
m), 4.94 (1H, s), 5.16 (1H, s), 6.66-6.73 (1H, m), 7.28
(1H, d, J = 8Hz), 7.33 (1H, d, J = 8Hz), 7.51 (1H, t, J = 8Hz),
7.64 (1H, d, J = 2Hz), 7.73 (1H, t, J = 8Hz), 8.05 (1H, d, J = 8H
z), 8.07 (1H, dd, J = 2,8Hz), 8.86 (1H, s). ESI-MS; 463.3 (M + H)

Reference Example 8: Methyl 2-[([1- [2-amino-5-
([[(tert-Butoxycarbonyl) amino] [(tert-butoxycarbonyl) imino] methyl] amino) pentanoyl-3-
Hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoate methyl 2-([[1- (5-([((tert-butoxycarbonyl) amino] [(tert- Butoxycarbonyl) imino] methyl] amino) -2-[[(9H-fluoren-9-ylmethoxy) carbonyl]
Amino] pentanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoate (130 mg, 0.156 mmol) in N, N-dimethylformamide
Dissolve in (3.4 mL) and piperazinomethyl polystyrene
(680 mg, 0.69 mmol / g Loading, Nova) was added, and the suspension was shaken by a multi-shaker at room temperature for 23.5 hours. The resulting mixture was filtered, then the residue was washed with N, N-dimethylformamide (3.4 mL x 5). Combined solvent
It was diluted with 1N hydrochloric acid (48 mL) and washed with ether (20 mL). The aqueous layer was adjusted to a base with saturated aqueous sodium hydrogen carbonate solution, and extracted with chloroform (25 mLx5). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give the title compound (45
(47 mg, 47%) was obtained as a pale yellow oily substance. ESI-MS; 613.4 (M + H)

Reference Example 9: Methyl 2-([[1- (5-([[(tert-butoxycarbonyl) amino] [(tert-butoxycarbonyl))
(Imino] methyl] amino) -2-[[4- [3-[(tert-butoxycarbonyl) amino] propoxy] -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy -3-Methyl-2-pyrrolidinyl] carbonyl] amino) -3-
Methyl-2-butenoate methyl 2-[([1- [2-amino-5-([[(tert-butoxycarbonyl) amino] [(tert-butoxycarbonyl) imino] methyl]
Amino) pentanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl) amino] -3-methyl-2-butenoate (45 mg, 0.073 mmol), 4- [3-[(tert-butoxycarbonyl) amino] Propoxy] -3- (3-isopropenyl-4-quinolinyl) benzoic acid (36mg, 1.05eq.) And 1-hydroxybenzotriazole (11mg, 1.1eq.) Were dissolved in N, N-dimethylformamide (3.0mL). 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (16 mg, 1.
1 eq.) Was added portionwise at 0 ° C. and the mixture was stirred at room temperature for 16 hours. The resulting mixture was ethyl acetate (12 mL)
And the organic layer was diluted with saturated aqueous sodium hydrogen carbonate solution (10 m
L) and brine (10 mL), washed successively, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (eluent; 2% methanol in chloroform) to give the title compound (59 mg, 76%) as a colorless syrup. ESI-MS; 1058.7 (M + H)

Reference Example 10: 2-([[1- (5-([[(tert-butoxycarbonyl) amino] imino] methyl] amino) -2-[[4- [3
-[(tert-Butoxycarbonyl) amino] propoxy] -3- (3
-Isopropenyl-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl]
Carbonyl] amino) -3-methyl-2-butenoic acid methyl 2-[([1- (5-([[(tert-butoxycarbonyl) amino] [(tert-butoxycarbonyl) imino] methyl] amino) -2 -[[4- [3-[(tert-Butoxycarbonyl) amino] propoxy] -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl ] Carbonyl] amino) -3-methyl-2-butenoate (57 mg, 0.054 mmol) was dissolved in methanol (2.0 mL), 1N aqueous sodium hydroxide solution (1.0 mL) was added, and the mixture was stirred at room temperature for 6.5 hr. did. The resulting mixture was acidified with 1N hydrochloric acid (pH = ca.4) and extracted with chloroform (10 mL). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue (62 mg) was purified by preparative thin layer chromatography (chloroform: methanol: acetic acid = 8: 1: 1) to obtain the title compound (30 mg, 59%) as a colorless oily substance. ESI-MS; 943.7 (M + H)

Reference Example 11: 2-[([1-[(9H-fluorene-9-
Ilmethoxy) carbonyl] -3-hydroxy-3-methyl-2-
Pyrrolidinyl] carbonyl) amino] -3-methyl-2-butenoic acid 2-[[(3-Hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoic acid (208mg, 0.86mmol )
With 10% aqueous sodium carbonate solution (5.0 ml) and dioxane.
(7.5 ml), and under ice cooling, a solution of 9-fluorenylmethyl chloroformate (222 mg, 1.0 eq.) In dioxane (5.
(0 ml) was added dropwise over 5 minutes, the temperature was raised to room temperature, and the mixture was stirred for 23 hours. The reaction mixture was distilled water (100 ml)
Poured in and washed twice with ether (50 ml). The aqueous layer was adjusted to pH = 2 with concentrated hydrochloric acid and extracted with ethyl acetate (50 ml). The organic layer was dried over magnesium sulfate, and the solvent was evaporated to give the title compound (405 mg, 102%) as an amorphous powder. 1H-NMR (300MHz, DMSO-d6) δ1.32 (1.5H, s), 1.42 (1.5H,
s), 1.69-1.83 (4H, m), 1.92-2.04 (4H, m), 3.28-3.40 (1
H, m), 3.49-3.62 (2H, m), 3.92-4.00 (1H, m), 4.15-4.33
(3H, m), 7.26-7.46 (4H, m), 7.59-7.72 (2H, m), 7.85-7.9
3 (2H, m), 9.03 (0.5H, s), 9.13 (0.5H, s). ESI-MS; 465.1 (M + H)

Reference Example 12: [2-[([1-[(9H-fluorene-9-
Ilmethoxy) carbonyl] -3-hydroxy-3-methyl-2-
Pyrrolidinyl] carbonyl) amino] -3-methyl-2-butenoyloxy] -2-chlorotrityl polystyrene The resin used for the reaction was 2-chlorotrityl polystyrene (240 m) in 8 microcan reactors (IRORI).
g, Loading: 1.20 mmol / g) was introduced in an amount of 30 mg each. 2-[([1-[(9H-Fluoren-9-ylmethoxy) carbonyl] -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl) amino] -3-methyl-2-butenoic acid (187.3mg, 0.403 mm
ol) in methylene chloride solution (8.0 ml), add diisopropylethylamine (144 μl) and let it stand for 5 minutes, then add the 8 microcan reactors prepared previously and stir at room temperature for 45 hours and a half hours. did. The reaction solution was removed by filtration, and the resin was washed twice with methylene chloride (10 ml), four times with N, N-dimethylformamide (10 ml) and methanol (10 ml).
After washing once with methylene chloride and 4 times with methylene chloride (10 ml), the title compound was obtained by drying under reduced pressure for 30 minutes. Reference Example 13: (2-[[(3-Hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene [2-[([1- [ (9H-Fluoren-9-ylmethoxy) carbonyl]
-3-Hydroxy-3-methyl-2-pyrrolidinyl] carbonyl)
Amino] -3-methyl-2-butenoyloxy] -2-chlorotrityl polystyrene (8 microcan reactors obtained in the previous step) was added to 20% piperidine-methylene chloride solution (10.0 ml), and the mixture was stirred at room temperature for 1 hour. Stir for hours. The reaction solution was removed by filtration and the resin was washed with N-methylpyrrolidone.
(10 ml) twice, N, N-dimethylformamide-methylene chloride (1: 1, 10 ml) four times, and methylene chloride (10 ml) four times.
After washing twice, it was dried under reduced pressure for 30 minutes to obtain the title compound.

Reference Example 14: [2-([[1-((2S) -6-[(tert-butoxycarbonyl) amino] -2-[[9H-fluoren-9-ylmethoxy) carbonyl] amino] hexanoyl ) -3-Hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-
Methyl-2-butenoyloxy] -2-chlorotrityl polystyrene (2S) -6-[(tert-butoxycarbonyl) amino] -2-[[(9H-
Fluorene-9-ylmethoxy) carbonyl] amino] caproic acid (378 mg, 0.807 mmol) and 1-hydroxybenzotriazole (109 mg) in N-methylpyrrolidone (8.0 ml) was added with N, N'-diisopropylcarbodiimide (126 μl). After standing still for 5 minutes, (2-[[(3-hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (obtained in the previous step 8 Micro Can Reactors)
Was added and the mixture was stirred at room temperature for 21 hours. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (10 ml).
Times, N, N-dimethylformamide-methylene chloride (1: 1, 10
ml) 4 times and methylene chloride (10 ml) 4 times,
The title compound was obtained by drying under reduced pressure for 30 minutes.

Reference Example 15: (2-[[(1-[(2S) -6-[(tert-butoxycarbonyl) amino] -2-aminohexanoyl] -3-
Hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene [2-([[1-((2S) -6-[(tert-butoxycarbonyl )amino]-
2-[[(9H-Fluoren-9-ylmethoxy) carbonyl] amino] hexanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoyloxy] -2-chlorotrityl 20% of polystyrene (7 of the microcan reactors obtained in the previous step)
It was added to a piperidine-methylene chloride solution (10.0 ml), and the mixture was stirred at room temperature for 1 hour. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (10 ml) twice, N, N-dimethylformamide-methylene chloride (1: 1, 10 ml) four times, and methylene chloride (10 ml) four times. After that, the title compound was obtained by drying under reduced pressure for 30 minutes.

Reference Example 16: (2-[[(1-[(2S) -2-[[4- (3-
[[(9H-Fluoren-9-ylmethoxy) carbonyl] amino]
Propoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino]
Hexanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy)
2-Chlorotrityl polystyrene 4- (3-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] propoxy) -3- (3-isopropenyl-4-quinolinyl)
To a solution of benzoic acid (206 mg) and 1-hydroxybenzotriazole (48 mg) in N-methylpyrrolidone (7.0 ml) was added N,
Add N'-diisopropylcarbodiimide (55 μl) to add 5
After standing for a minute, (2-[[(1-[(2S) -6-[(tert-butoxycarbonyl) amino] -2-aminohexanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl) Carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (7 microcan reactors obtained in the previous step) was added, and the mixture was stirred at room temperature for 37 hours and a half. The reaction solution was removed by filtration and the resin was washed with N-methylpyrrolidone.
(10 ml) twice, N, N-dimethylformamide-methylene chloride (1: 1, 10 ml) four times, and methylene chloride (10 ml) four times.
After washing twice, it was dried under reduced pressure for 30 minutes to obtain the title compound.

Reference Example 17: 4- [3-[[(9H-Fluoren-9-ylmethoxy) carbonyl] amino] propoxy) -3- (3-isopropenyl-4-quinolinyl) benzoic acid 4- [3- [ (tert-Butoxycarbonyl) amino] propoxy]-
3- (3-isopropenyl-4-quinolinyl) benzoic acid (155mg,
0.335 mmol) of 10% trifluoroacetic acid-methylene chloride solution was allowed to stand at room temperature for 2.5 hours. The reaction solvent was evaporated under reduced pressure, and the residue was dissolved in 10% aqueous sodium carbonate solution (4.0 ml) and dioxane (2.0 ml). 9-Fluorenylmethyl chloroformate (87mg, 1.0eq.) In dioxane (4.
(0 ml) was added dropwise over 5 minutes, and the mixture was stirred for 24 hours.
Pour the reaction mixture into distilled water (80 ml) and add 1N hydrochloric acid to adjust the pH.
After adjusting to = 5, the precipitated solid was collected by filtration and washed with distilled water to give the title compound (197 mg, 101%) as a white solid. 1H-NMR (300MHz, DMSO-d6) δ1.48-1.59 (2H, m), 1.76 (3
H, s), 2.65-2.75 (2H, m), 3.97-4.06 (2H, m), 4.12-4.20 (1
H, m), 4.25 (2H, d, J = 8Hz), 4.94 (1H, s), 5.15 (1H, s), 7.
12-7.19 (1H, m), 7.23-7.43 (5H, m), 7.45-7.53 (1H, m),
7.60-7.75 (4H, m), 7.87 (2H, d, J = 8Hz), 8.02-8.10 (2H, m),
8.86 (1H, s) .ESI-MS; 585.4 (M + H)

Reference Example 18: ((2S) -2-[[(9H-fluorene-9
-Ylmethoxy) carbonyl] amino] -3-methylbutanoyloxy) -2-chlorotrityl polystyrene The resin used for the reaction was 2-chlorotrityl polystyrene (240 m) in 8 microcan reactors (IRORI).
g, Loading: 1.20 mmol / g) was introduced in an amount of 30 mg each. Diisopropylethylamine (286 μl) was added to a methylene chloride solution (8.0 ml) of (2S) -2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] -3-methylbutyric acid (272 mg, 0.801 mmol), and added. After leaving it for a minute, the eight microcan reactors prepared previously were added, and the mixture was stirred at room temperature for 17 hours and a half. The reaction solution was removed by filtration, and the resin was washed twice with methylene chloride (10 ml), four times with N, N-dimethylformamide (10 ml) and once with methanol (10 ml).
The extract was washed 4 times with methylene chloride (10 ml) and dried under reduced pressure for 30 minutes to give the title compound.

Reference Example 19: ((2S) -2-Amino-3-methylbutanoyloxy) -2-chlorotritylpolystyrene ((2S) -2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino ] -3-Methylbutanoyloxy) -2-chlorotrityl polystyrene (7 of the microcan reactors obtained in the previous step) was added to a 20% piperidine-methylene chloride solution (10.0 ml) and the mixture was allowed to stand at room temperature for 2 hours. Stir for half an hour. The reaction solution was removed by filtration, and the resin was washed twice with N-methylpyrrolidone (10 ml) and N, N-dimethylformamide-
4 times with methylene chloride (1: 1, 10 ml), methylene chloride (10 m
After washing 4 times with l), the title compound was obtained by drying under reduced pressure for 30 minutes.

Reference Example 20: [(2S) -2-[([(2S) -1-[(9H-fluoren-9-ylmethoxy) carbonyl] -2-pyrrolidinyl] carbonyl) amino] -3-methylbutanoyl Oxy] -2-
Chlorotrityl polystyrene (2S) -1-[(9H-fluoren-9-ylmethoxy) carbonyl-2
-Pyrrolidinecarboxylic acid (236.1 mg, 0.70 mmol) and bromo-tris-pyrrolidinophosphonium hexafluorophosphate (326 mg) in N-methylpyrrolidone (7.
Diisopropylethylamine (244 μl) was added to the solution, and the mixture was allowed to stand for 5 minutes, then ((2S) -2-amino-3-methylbutanoyloxy) -2-chlorotrityl polystyrene (7 obtained in the previous step Microcan Reactor) was added and the mixture was stirred at room temperature for 16 hours. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (10 ml) four times,
N, N-dimethylformamide-methylene chloride (1: 1, 10ml)
After washing 4 times with methylene chloride (4 times) and methylene chloride (10 ml) 4 times, the title compound was obtained by drying under reduced pressure for 30 minutes.

Reference Example 21: ((2S) -2-[[((2S) -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoyloxy)-
2-chlorotrityl polystyrene [(2S) -2-[([(2S) -1-[(9H-fluoren-9-ylmethoxy))
Carbonyl] -2-pyrrolidinyl] carbonyl) amino] -3-methylbutanoyloxy] -2-chlorotrityl polystyrene (4 of the microcan reactors obtained in the previous step) in 20% piperidine-methylene chloride solution (5.0
ml) and stirred at room temperature for 2 hours. The reaction solution was removed by filtration and the resin was washed with N-methylpyrrolidone (5 ml).
Times, N, N-dimethylformamide-methylene chloride (1: 1, 5m
It was washed 4 times with l) and 4 times with methylene chloride (5 ml), and dried under reduced pressure for 30 minutes to give the title compound.

Reference Example 22: [(2S) -2-([[(2S) -1-((2S) -6-
[(tert-Butoxycarbonyl) amino] -2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] hexanoyl)-
2-Pyrrolidinyl] carbonyl] amino) -3-methylbutanoyloxy] -2-chlorotritylpolystyrene (2S) -6-[(tert-butoxycarbonyl) amino] -2-[[(9H-
Fluorene-9-ylmethoxy) carbonyl] amino] caproic acid (375mg, 0.80mmol) and fluoro-N, N, N ', N'-tetramethylformamidinium hexafluorophosphate (211mg) in methylene chloride (8.0ml) ) In solution
After adding diisopropylethylamine (279 μl) and letting it stand for 5 minutes, ((2S) -2-[[((2S) -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoyloxy) -2-chlorotrityl was added. Polystyrene (4 microcan reactors obtained in the previous step) was added and stirred at room temperature for 16 hours and a half. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (10 ml) four times, N, N-dimethylformamide-methylene chloride (1: 1, 10 ml) four times, and methylene chloride.
After washing 4 times with (10 ml), the title compound was obtained by drying under reduced pressure for 30 minutes.

Reference Example 23: ((2S) -2-[[((2S) -1-[(2S) -6-
[(tert-Butoxycarbonyl) amino] -2-aminohexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoyloxy) -2-chlorotrityl polystyrene [(2S) -2-([[( 2S) -1-((2S) -6-[(tert-Butoxycarbonyl) amino] -2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] hexanoyl) -2-pyrrolidinyl] carbonyl] amino) Add -3-methylbutanoyloxy] -2-chlorotritylpolystyrene (2 of the microcan reactors obtained in the previous step) to a 20% piperidine-methylene chloride solution (5.0 ml) at room temperature for 2 hours. It was stirred. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (5 ml) twice, N, N-dimethylformamide-methylene chloride (1: 1, 5 ml) four times, and methylene chloride (5 ml).
After being washed 4 times with, the product was dried under reduced pressure for 30 minutes to obtain the title compound.

Reference Example 24: ((2S) -2-[[((2S) -1-[(2S) -2-
[[4- (3-[[(9H-fluoren-9-ylmethoxy) carbonyl]]
Amino] propoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl)
Amino] hexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoyloxy) -2-chlorotrityl polystyrene 4- (3-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] propoxy)- 3- (3-isopropenyl-4-quinolinyl)
Benzoic acid (105 mg) and 1-hydroxybenzotriazole (24 mg) N-methylpyrrolidone-methylene chloride (1: 1,
2.4 ml) solution with N, N'-diisopropylcarbodiimide (2
8 μl) and then left to stand for 5 minutes, then ((2S) -2-[[((2S) -1-
[(2S) -6-[(tert-Butoxycarbonyl) amino] -2-aminohexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-
Methylbutanoyloxy) -2-chlorotrityl polystyrene (two microcan reactors obtained in the previous step) was added and stirred at room temperature for 71 hours. The reaction solution was removed by filtration and the resin was washed with N-methylpyrrolidone (5m
l) 4 times with N, N-dimethylformamide-methylene chloride
The title compound was obtained by washing 4 times with (1: 1, 5 ml) and 4 times with methylene chloride (5 ml) and then drying for 30 minutes under reduced pressure.

Reference Example 25: ((2S) -2-[[((2S) -1-[(2S) -2-
[[4- (3-Aminopropoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-Methylbutanoyloxy) -2-chlorotrityl polystyrene ((2S) -2-[[((2S) -1-[(2S) -2-[[4- (3-[[(9H-fluorene -
9-ylmethoxy) carbonyl] amino] propoxy) -3- (3-
Isopropenyl-4-quinolinyl) benzoyl] amino] -6-
[(tert-Butoxycarbonyl) amino] hexanoyl] -2-
20% of pyrrolidinyl) carbonyl] amino] -3-methylbutanoyloxy) -2-chlorotritylpolystyrene (the two microcan reactors obtained in the previous step)
It was added to a piperidine-methylene chloride solution (5.0 ml), and the mixture was stirred at room temperature for 1 hour. The reaction solution was removed by filtration, and the resin was washed 4 times with N-methylpyrrolidone (5 ml), 4 times with N, N-dimethylformamide-methylene chloride (1: 1, 5 ml), and 4 times with methylene chloride (5 ml). After that, the title compound was obtained by drying under reduced pressure for 30 minutes.

Reference Example 26: (2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl Polystyrene The resin used in the reaction was 8 2-Chlorotrityl polystyrene (240m
g, Loading: 1.20 mmol / g) was introduced in an amount of 30 mg each. 2-[[(9H-Fluoren-9-ylmethoxy) carbonyl] amino] -3-methyl-2-butenoic acid (270mg, 0.80mmol)
Diisopropylethylamine (286 μl) was added to the methylene chloride solution (8.0 ml) of the above, and the mixture was allowed to stand for 5 minutes, then the eight microcan reactors prepared previously were added, and the mixture was stirred at room temperature for 17 hours. The reaction solution was removed by filtration, and the resin was washed twice with methylene chloride (10 ml), four times with N, N-dimethylformamide (10 ml) and once with methanol (10 ml).
The extract was washed 4 times with methylene chloride (10 ml) and dried under reduced pressure for 30 minutes to give the title compound.

Reference Example 27: (2-Amino-3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] -3-methyl-2 -Butenoyloxy) -2-chlorotrityl polystyrene (microcan obtained in the previous step.
Seven of the reactors) were added to a 20% piperidine-methylene chloride solution (10.0 ml) and stirred at room temperature for 2.5 hours.
The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (10 ml) twice, N, N-dimethylformamide-methylene chloride (1: 1, 10 ml) four times, and methylene chloride (10 ml) four times. After that, by drying under reduced pressure for 30 minutes,
The title compound was obtained.

Reference Example 28: [2-[([(2S, 3S) -1-[(9H-fluoren-9-ylmethoxy) carbonyl] -3-hydroxy-2-
Pyrrolidinyl] carbonyl) amino] -3-methyl-2-butenoyloxy] -2-chlorotrityl polystyrene (2S, 3S) -1-[(9H-fluoren-9-ylmethoxy) carbonyl-3-hydroxy-2-pyrrolidinecarboxylic acid (247.4 mg,
0.70 mmol) and bromo-tris-pyrrolidinophosphonium hexafluorophosphate (326 mg) in N-methylpyrrolidone (7.0 ml) was added diisopropylethylamine (244 μl), and the mixture was allowed to stand for 5 minutes, then (2-amino -3-Methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (7 microcan reactors obtained in the previous step) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was removed by filtration and the resin was washed with N-methylpyrrolidone
After washing with (10 ml) four times, N, N-dimethylformamide-methylene chloride (1: 1, 10 ml) four times, and methylene chloride (10 ml) four times, the product was dried under reduced pressure for 30 minutes to give the title compound. Got

Reference Example 29: (2-[[((2S, 3S) -3-Hydroxy-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene [2- [ ([(2S, 3S) -1-[(9H-fluoren-9-ylmethoxy) carbonyl] -3-hydroxy-2-pyrrolidinyl] carbonyl)
Amino] -3-methyl-2-butenoyloxy] -2-chlorotrityl polystyrene (4 of the microcan reactors obtained in the previous step) was added to 20% piperidine-methylene chloride solution (5.0 ml) at room temperature. And stirred for 2 hours. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (5 ml) twice, N, N-dimethylformamide-methylene chloride (1: 1, 5 ml) four times, and methylene chloride (5 ml).
After being washed 4 times with, the product was dried under reduced pressure for 30 minutes to obtain the title compound.

Reference Example 30: [2-([[(2S, 3S) -1-((2S) -6-
[(tert-Butoxycarbonyl) amino] -2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] hexanoyl)-
3-Hydroxy-2-pyrrolidinyl] carbonyl] amino) -3-
Methyl-2-butenoyloxy] -2-chlorotrityl polystyrene (2S) -6-[(tert-butoxycarbonyl) amino] -2-[[(9H-
Fluorene-9-ylmethoxy) carbonyl] amino] caproic acid (375mg, 0.80mmol) and fluoro-N, N, N ', N'-tetramethylformamidinium hexafluorophosphate (211mg) in methylene chloride (8.0ml) ) In solution
After adding diisopropylethylamine (279 μl) and allowing to stand for 5 minutes, (2-[[((2S, 3S) -3-hydroxy-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2 -Chlorotrityl polystyrene (4 microcan reactors obtained in the previous step) was added and stirred at room temperature for 16 hours and a half. The reaction solution was removed by filtration and the resin was washed 4 times with N-methylpyrrolidone (10 ml) and 4 times with N, N-dimethylformamide-methylene chloride (1: 1, 10 ml).
Wash 4 times with methylene chloride (10 ml), and then under reduced pressure 3
The title compound was obtained by drying for 0 minutes.

Reference Example 31: (2-[[((2S, 3S) -1-[(2S) -6-
[(tert-Butoxycarbonyl) amino] -2-aminohexanoyl] -3-hydroxy-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene [2-([[ (2S, 3S) -1-((2S) -6-[(tert-Butoxycarbonyl) amino] -2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] hexanoyl) -3-hydroxy-2 -Pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoyloxy] -2-chlorotrityl polystyrene (2 of the microcan reactors obtained in the previous step)
It was added to a 0% piperidine-methylene chloride solution (5.0 ml), and the mixture was stirred at room temperature for 2 hours. The reaction solution is removed by filtration,
The resin was washed twice with N-methylpyrrolidone (5 ml), N, N-dimethylformamide-methylene chloride (1: 1, 5 ml) four times, and methylene chloride (5 ml) four times, and then dried under reduced pressure for 30 minutes. By doing so, the title compound was obtained.

Reference Example 32: (2-[[((2S, 3S) -1-[(2S) -2-
[[4- (3-[[(9H-fluoren-9-ylmethoxy) carbonyl]]
Amino] propoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl)
Amino] hexanoyl] -3-hydroxy-2-pyrrolidinyl)
Carbonyl] amino] -3-methyl-2-butenoyloxy) -2-
Chlorotrityl polystyrene 4- (3-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] propoxy) -3- (3-isopropenyl-4-quinolinyl)
Benzoic acid (105 mg) and 1-hydroxybenzotriazole (24 mg) N-methylpyrrolidone-methylene chloride (1: 1,
2.4 ml) solution with N, N'-diisopropylcarbodiimide (2
8 μl) and leave it for 5 minutes, then (2-[[((2S, 3S) -1-
[(2S) -6-[(tert-Butoxycarbonyl) amino] -2-aminohexanoyl] -3-hydroxy-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl Polystyrene (2 microcan reactors obtained in the previous step) was added and stirred at room temperature for 71 hours. The reaction solution was removed by filtration, and the resin was washed with N-methylpyrrolidone (5 ml) 4 times, N, N-dimethylformamide-methylene chloride (1: 1, 5 ml) 4 times, and methylene chloride (5 ml).
(ml) four times and then dried under reduced pressure for 30 minutes to obtain the title compound.

Reference Example 33: (2-[[((2S, 3S) -1-[(2S) -2-
[[4- (3-Aminopropoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-2-pyrrolidinyl) Carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (2-[[((2S, 3S) -1-[(2S) -2-[[4- (3-[[( 9H-fluorene-9
-Ylmethoxy) carbonyl] amino] propoxy) -3- (3-
Isopropenyl-4-quinolinyl) benzoyl] amino] -6-
[(tert-Butoxycarbonyl) amino] hexanoyl] -3-
Hydroxy-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (2 microcan reactors obtained in the previous step) was treated with 20% piperidine-methylene chloride solution (5.0 m
l) and stirred at room temperature for 1 hour. The reaction solution was removed by filtration and the resin was washed with N-methylpyrrolidone (5 ml) 4 times.
Times, N, N-dimethylformamide-methylene chloride (1: 1, 5m
It was washed 4 times with l) and 4 times with methylene chloride (5 ml), and dried under reduced pressure for 30 minutes to give the title compound.

Example 1-1: N- (4-aminobutyl) -2-
([[1- (5-[[amino (imino) methyl] amino] -3-chloro-
4-hydroxy-2-[[4-hydroxy-3- (3-isopropenyl-
4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenamide N- (4-triphenylmethylaminobutyl) -2- ( [[1- (5-
[[Amino (imino) methyl] amino] -3-chloro-4-hydroxy-2-[[4-hydroxy-3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-
3-Methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenamide (30 mg) was dissolved in methanol (1 ml), chloroform (1 ml) and trifluoroacetic acid (1 ml) for 5 hours. , Stirred at room temperature and the solvent was evaporated. The residue was dissolved in water and the ODS column (acetonitrile: water = 1: 5) was used.
~ 1: 3). Lyophilize the target fraction to give the title compound
(10 mg, hereinafter "FR254149", molecular weight 806.4) was obtained. MS: 805 (M-1)

Example 2-1 2-([[1- (5-[[amino (imino) methyl] amino] -2-[[4- (3-aminopropoxy) -3- (3-
Isopropenyl-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoic acid penta (trifluoroacetate) 2-([[ 1- (5-[[[(tert-butoxycarbonyl) amino] (imino) methyl] amino] -2-[[4- [3-[(tert-butoxycarbonyl) amino] propoxy] -3- (3- Isopropenyl-4-quinolinyl) benzoyl] amino] pentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoic acid (30 mg, 0.032 mmol) in 10% trifluoro It was dissolved in an acetic acid-dichloromethane solution (2.0 mL) and left at room temperature for 1 hour. Dilute the mixture with toluene (2.0 mL),
Concentrated under reduced pressure. The residue was dissolved in 30% trifluoroacetic acid-dichloromethane solution (2.0 mL), and the mixture was left at room temperature for 1 hr. The mixture was diluted with toluene (2.0 mL) and concentrated under reduced pressure. The residue was converted to ether-n-hexane (1: 1, 2.
(0 mL) and the mixture was concentrated under reduced pressure to give the title compound (3
2 mg, 77%, "FR253761", molecular weight 742.9) was obtained as a pale yellow solid. ESI-MS; 743.6 (M + H)

Example 3-1 2-[[(1-[(2S) -2-[[4- (3-aminopropoxy) -3- (3-isopropenyl-4-quinolinyl))
Benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-methyl-2-butenoic acid diacetate (2- [[(1-[(2S) -2-[[4- (3-Aminopropoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(ter
t-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-
Methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (three of the microcan reactors obtained in the previous step) in acetic acid-trifluoroethanol-methylene chloride (1: 1: 8, 9.0 ml) In addition, the mixture was stirred at room temperature for 1 hour. Acetic acid-trifluoroethanol-methylene chloride (1: 1: 8, 21 ml) was added to the reaction mixture, the Microcan reactor was removed, and the solvent was evaporated under reduced pressure. The title compound was obtained by triturating the residue from ether-hexane.
(44 mg, hereinafter “FR259384”) was obtained. ESI-MS; 815.6 (M + H)

Example 4-1 2-[[(1-[(2S) -2-[[4- (3-aminopropoxy) -3- (3-isopropenyl-4-quinolinyl)
Benzoyl] amino] -6-aminohexanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-
Methyl-2-butenoic acid tritrifluoroacetate (2-[[(1-[(2S) -2-[[4- (3-aminopropoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl ] Amino] -6-[(ter
t-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-3-methyl-2-pyrrolidinyl) carbonyl] amino] -3-
Methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (one of the microcan reactors obtained in the previous step) was added to 10% trifluoroacetic acid-methylene chloride solution (2.5 ml) and the mixture was allowed to stand at room temperature for 1 hour. Stir for hours. After adding methylene chloride (7.5 ml) to the reaction mixture, the Microcan reactor was removed and the solvent was distilled off under reduced pressure to give the title compound (12.0 mg, hereinafter referred to as "FR259383").
Got ESI-MS; 715.6 (M + H)

Example 5-1: ((2S) -2-[[((2S) -1-[(2S)-
2-[[4- (3-aminopropoxy) -3- (3-isopropenyl-4-
(Quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoic acid diacetate ((2S) -2-[[((2S) -1-[(2S) -2-[[4- (3-aminopropoxy)
-3- (3-Isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoyloxy) -2 -Chlorotrityl polystyrene (2 microcan reactors obtained in the previous step) was mixed with acetic acid-
Trifluoroethanol-methylene chloride (1: 1: 8, 5.0ml)
The mixture was added to the mixture and stirred at room temperature for 1 hour. Acetic acid in the reaction mixture-
Trifluoroethanol-methylene chloride (1: 1: 8, 15ml)
Was added, the Microcan reactor was removed, and the solvent was evaporated under reduced pressure. The residue was triturated with ether-hexane to give the title compound (55.2 mg, hereinafter "FR2644").
I got 50). ESI-MS; 787.8 (M + H)

Example 6-1: ((2S) -2-[[((2S) -1-[(2S)-
2-[[4- (3-aminopropoxy) -3- (3-isopropenyl-4-
(Quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-methylbutanoic acid tritrifluoroacetate ((2S) -2-[[(( 2S) -1-[(2S) -2-[[4- (3-aminopropoxy)
-3- (3-Isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -2-pyrrolidinyl) carbonyl] amino] -3-methylbutyric acid diacetate (18.2 mg) of 10% trifluoroacetic acid-methylene chloride (1.0 ml) was allowed to stand at room temperature for 30 minutes. Toluene (1.0 ml) was added to the reaction mixture, and the solvent was evaporated under reduced pressure to give the title compound (20.2 mg, hereafter "FR26445").
1 ”). ESI-MS; 687.6 (M + H)

Example 7-1: (2-[[((2S, 3S) -1-[(2S) -2
-[[4- (3-Aminopropoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-2-pyrrolidinyl ) Carbonyl] amino] -3-methyl-2-butenoic acid diacetate (2-[[((2S, 3S) -1-[(2S) -2-[[4- (3-aminopropoxy)-
3- (3-Isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-2-pyrrolidinyl) carbonyl] amino]
-3-Methyl-2-butenoyloxy) -2-chlorotrityl polystyrene (2 microcan reactors obtained in the previous step) was added to acetic acid-trifluoroethanol-methylene chloride.
(1: 1: 8, 5.0 ml), and stirred at room temperature for 1 hour. Acetic acid-trifluoroethanol-methylene chloride in the reaction mixture
After adding (1: 1: 8, 15 ml), the Microcan reactor was removed, and the solvent was distilled off under reduced pressure. The residue is ether
By triturating from hexane, the title compound (67.9
mg, hereafter referred to as "FR264452"). ESI-MS; 801.7 (M + H)

Example 8-1: (2-[[((2S, 3S) -1-[(2S) -2
-[[4- (3-Aminopropoxy) -3- (3-isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-2-pyrrolidinyl ) Carbonyl] amino] -3-methyl-2-butenoic acid tritrifluoroacetate (2-[[((2S, 3S) -1-[(2S) -2-[[4- (3-aminopropoxy)) -
3- (3-Isopropenyl-4-quinolinyl) benzoyl] amino] -6-[(tert-butoxycarbonyl) amino] hexanoyl] -3-hydroxy-2-pyrrolidinyl) carbonyl] amino]
10% trifluoroacetic acid-methylene chloride (1.0 ml) of -3-methyl-2-butenoic acid diacetate (18.4 mg) was allowed to stand at room temperature for 30 minutes. Toluene (1.0 ml) was added to the reaction mixture, and the solvent was evaporated under reduced pressure to give the title compound (2
0.3 mg) was obtained. ESI-MS; 701.6 (M + H)

Example 9 Eighty kinds of compounds were synthesized according to the library synthesis method shown below.

[Chemical 15] [Where AA ₁ is

[Chemical 16] And AA ₂ is

[Chemical 17] Represents ]

1) Eight solid-phase supported reaction vessels were prepared, and 50% acetyl chloride-methylene chloride solution (10 ml) was introduced into each of them. Commercially available synphase crown (trityl alcohol linker, Mimoto
10 pes) were added to each reaction vessel, and the mixture was shaken with stirring at room temperature for 21 hours. The reaction solution was removed and washed with methylene chloride (10 ml) four times to obtain a chlorinated product (a).
Prepare 8 separate reaction vessels and use the Fmoc from A to H shown above.
Amino acid (AA ₁ , After 1.0 mmol) was added to each reaction vessel one by one, methylene chloride (10 ml) and diisopropylethylamine (2.05 mmol) were added to each vessel in this order and the mixture was allowed to stand for 10 minutes. Then, (a) was added 10 each. After stirring and shaking at room temperature for 16 hours, the reaction solution was removed and washed twice with methylene chloride (10 ml). All the FmocAA ₁ -crown derivatives (b) (80) produced were combined and washed with N, N-dimethylformamide (80 ml) three times, methanol (80 ml) once, and methylene chloride (80 ml) three times. Then, it was dried under reduced pressure at 40 ° C. for 30 minutes.

2) Fmoc group deprotection 1) in 20% piperidine-N-methylpyrrolidone solution (80 ml)
The crown derivative (b) (80 pieces) obtained in the above was added and the mixture was allowed to stand at room temperature for 1 hour. The reaction solution was removed by filtration and washed with N-methylpyrrolidone (80 ml) three times and methylene chloride.
After washing 3 times with (80 ml), it was dried under reduced pressure at 40 ° C. for 30 minutes.

3) Amidation reaction Ten reaction vessels were prepared, and the Fm of 1 to 10 shown above was prepared.
oc Amino acid (AA ₂ , 0.80 mmol) was introduced into each reaction vessel one by one, and 0.2M 1-hydroxybenzotriazole-N-methylpyrrolidone solution (4.0 ml) and 0.2MN, N'- were introduced into each vessel. A diisopropylcarbodiimide-N-methylpyrrolidone solution (4.0 ml) was added in this order, and the mixture was allowed to stand for 5 minutes. Crown derivative (c) obtained in the previous step (80)
Was divided into eight pieces according to the information on the attached tags and added to each reaction container. After standing at room temperature for 16 hours, the reaction solution was removed and N-methylpyrrolidone (8 ml) was added.
It was washed 3 times. All the obtained crown derivatives (d) (80 pieces) were combined to obtain N, N-dimethylformamide.
(80 ml) three times, methylene chloride (80 ml) three times, and N-methylpyrrolidone (80 ml) twice.

4) Fmoc group deprotection It carried out according to 2).

5) Amidation reaction (2S) -6-[(tert-butoxycarbonyl) amino] -2-[[(9H-
Fluoren-9-ylmethoxy) carbonyl] amino] caproic acid (3.75g, 8.0mmol) and 1-hydroxybenzotriazole (1.08g, 8.0mmol) N-methylpyrrolidone (8
0 ml) to a solution of N, N'-diisopropylcarbodiimide (1.2
(5 ml) was added and the mixture was allowed to stand for 5 minutes, and then the crown derivative (e) (80 pieces) obtained in the previous step was added. After standing at room temperature for 39 hours, the reaction solution was removed by filtration,
N-methylpyrrolidone (80 ml) 3 times, N, N-dimethylformamide (80 ml) 3 times, methylene chloride (80 ml) 3 times, N-
It was washed once with methylpyrrolidone (80 ml).

6) Deprotection of Fmoc group It carried out according to 2).

7) Amidation reaction 4-hydroxy-3- (3-isopropenyl-4-quinolinyl) benzoic acid (611 mg, 2.0 mmol) and 1-hydroxybenzotriazole (270 mg, 2.0 mmol) in N, N-dimethyl N, N′-diisopropylcarbodiimide (313 μl, 2.0 mmol) was added to the formamide (50 ml) solution and the mixture was allowed to stand for 5 minutes, and then the crown derivative (g) (80) obtained in the previous step was added. After standing at room temperature for 16 hours, the reaction solution was removed by filtration, and N, N-dimethylformamide (80 ml) three times, N, N-dimethylformamide-methylene chloride (1: 1, 8
It was washed 3 times with 0 ml) and 3 times with methylene chloride (80 ml). The amidated crown derivative (h) thus obtained was concentrated under reduced pressure at 40 ° C. for 3
Dry for 0 minutes.

8) Slicing reaction 10 of 80 wells out of 96 wells of deep well plate
% Trifluoroacetic acid-methylene chloride solution was continuously dispensed in 1.5 ml aliquots, and the crown derivative (h) obtained in the previous step was poured into each hole.
Were introduced one by one and left still for 45 minutes. After removing the crown, the solvent was distilled off. 9) MS data of 80 kinds of obtained compounds are shown below.

[0090]

[Table 1]

Example 10 Eighty kinds of compounds were synthesized according to the library synthesis method shown below.

[Chemical 18] [Wherein AA ₁ is as defined above and AA ₂ 'is

[Chemical 19] Is. ]

1) Solid phase support It was carried out according to 1) of Example 9.

2) Fmoc group deprotection It was carried out according to 2) of Example 9.

3) Amidation reaction Ten reaction vessels were prepared, and Fm of 1 to 10 shown above were prepared.
oc Amino acid (AA ₂ , 0.80mmol) was introduced into each reaction vessel one by one, and then 0.2M bromo-tris-pyrrolidinophosphonium hexafluorophosphate-N-methylpyrrolidone solution (4.0ml) was added to each vessel. ) And 0.4 M diisopropylethylamine-N-methylpyrrolidone solution (4.0
ml) was added in this order and left still for 5 minutes. The crown derivative (c ') (80 pieces) obtained in the previous step was divided into 8 pieces according to the information of the attached tags and added to each reaction vessel. After stirring and shaking at room temperature for 16 hours, the reaction solution was removed and washed with N-methylpyrrolidone (8 ml) three times. All crown derivatives (d ') produced (80
Pieces) and add 3 with N, N-dimethylformamide (80 ml).
3 times with methylene chloride (80 ml), N-methylpyrrolidone
(80 ml) and washed twice.

4) Fmoc group deprotection It was carried out according to 2) of Example 9.

5) Amidation reaction It was carried out according to 5) of Example 9.

6) Deprotection of Fmoc group It was carried out according to 2) of Example 9.

7) Amidation reaction It was carried out according to 7) of Example 9.

8) Cleavage reaction It was carried out according to 8) of Example 9.

9) The MS data of the 80 compounds thus obtained are shown below.

[Table 2]

Example 11 160 kinds of compounds were synthesized according to the library synthesis method shown below.

[Chemical 20] [Where AA ₃ is

[Chemical 21] And Ar is

[Chemical formula 22] Is. ]

1) 50% acetyl chloride-methylene chloride solution (50 ml) in a solid-phase supported reaction vessel
Introduce, and add 90 commercially available synphase crowns (trityl alcohol linker, Mimotopes), and add 2 at room temperature.
The mixture was shaken with stirring for 0 hour. The reaction solution was removed and washed with methylene chloride (50 ml) three times to obtain a chlorinated product (a). Prepare another reaction vessel, 2-[([1-[(9H-fluoren-9-ylmethoxy) carbonyl] -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl) amino] -3-methyl- 2-butenoic acid
To a solution of (1.04g, 2.24mmol) in methylene chloride (22.5ml) was added diisopropylethylamine (1.22ml, 7.0mmol), and the mixture was allowed to stand for 5 minutes, then 90 (a) were added and stirred at room temperature for 20 hours. did. The reaction solution is removed by filtration,
The extract was washed twice with methylene chloride (50 ml), three times with N, N-dimethylformamide (50 ml), once with methanol (50 ml) and three times with methylene chloride (50 ml), and dried under reduced pressure for 30 minutes.

2) Fmoc group deprotection It was carried out according to 2) of Example 9.

3) Amidation reaction The above-mentioned 1 to 10 were used as Fmoc amino acids, and this was carried out according to 3) of Example 9.

4) Deprotection of Fmoc group It was carried out according to 2) of Example 9.

5) Amidation reaction The above A to H were used as the aromatic carboxylic acid, and the amidation reaction was carried out according to 3) of Example 9.

6) Cleavage reaction It was carried out according to 8) of Example 9. The above operations 1) to 6) were performed twice to synthesize a total of 160 compounds. In the second time, the above 11 to 20 were used as Fmoc amino acids in 3).

7) MS data of 160 kinds of obtained compounds are shown below.

[Table 3]

[0109]

[Table 4]

Example 12 80 kinds of compounds were synthesized according to the library synthesis method shown below.

[Chemical formula 23] [In the formula, Ar ′ is

[Chemical formula 24] And R is

[Chemical 25] Is. ]

1) Solid phase support It was carried out according to 1) of Example 9.

2) Fmoc group deprotection It was carried out according to 2) of Example 9.

3) Amidation reaction It was carried out according to 5) of Example 9.

4) Deprotection of Fmoc group It was carried out according to 2) of Example 9.

5) Amidation reaction Using A to H shown above as the aromatic carboxylic acid,
This was carried out according to 3) of Example 9.

6) Deprotection of ivDde group 2% hydrazine hydrate-N, N-dimethylformamide (80 ml) was added with the crown derivative (f ″ ′) (80) obtained in the previous step, and the mixture was allowed to stand at room temperature for 1 hour. Semi-stationary. The reaction solution was removed by filtration and washed with N-methylpyrrolidone (80 ml) three times,
After washing 3 times with methylene chloride (80 ml), it was dried under reduced pressure at 40 ° C. for 30 minutes.

7) Modification of lysine side chain Ten reaction vessels were prepared, and the above-mentioned 1 to 5 isocyanate reagents (0.80 mmol) were introduced into each of the vessels 1 to 5 one by one in each reaction vessel. , Methylene chloride in each
(8.0 ml) was added. After introducing the sulfonyl chloride reagents (0.80 mmol) of 6 and 7 shown above into the reaction vessels one by one in each of the vessels 6 and 7, methylene chloride (8.0 m
l) and diisopropylethylamine (418 μl, 2.4 mmol)
Was added. Containers 8 to 10 include the above 8 to 10
After introducing one kind of carboxylic acid (0.8 mmol) into each reaction vessel, N-methylpyrrolidone (8.0 ml), 1-hydroxybenzotriazole (108 mg, 0.8 mmol) and N, N '
-Diisopropylcarbodiimide (125 μl, 0.8 mmol) was added in this order. The crown derivative (g ''') (80 pieces) obtained in the previous step was divided into 8 pieces according to the information of the attached tags and added to each reaction vessel.
After standing at room temperature for 18 hours, the reaction solution was removed and washed with N-methylpyrrolidone (8 ml) three times. All the obtained crown derivatives (h ''') (80 pieces) were combined, and three times with N, N-dimethylformamide (80 ml) and N, N-dimethylformamide-methylene chloride (1: 1, 80 ml). 3 times, methylene chloride (8
The product was dried under reduced pressure at 40 ° C. for 30 minutes.

8) Cleavage reaction It was carried out according to 8) of Example 9.

9) MS data of 80 kinds of the obtained compounds are shown below.

[0120]

[Table 5]

Example 1-2 1) Succinylated SGNEGDEN Particles SGNEGDEN particles are described in JP-A-10-195099, Me.
thods in Enzymology, 254: p595-604, 1995, Colloids
and Surfaces B, 10: p41-49, 1997 and Nature Biotechn.
ology, 18: p877-881, 2000, etc. The amino group content on the surface of the synthesized SGNEGDEN particles is Ka
Method of waguchi et al. (J. Appl. Poly. Sci., 26, 2015-202
2, 1981) and by conductivity titration. SG
NEGDEN particles (1.79% (w / v) water suspension, amino group content 0.415
mmol / g) 280 μl (5 mg) was placed in an Eppendorf tube, and 220 μl of distilled water was added to make a 1% (w / v) suspension. Use a micro high-speed centrifuge (TOMY Seiko MX-160) to disperse this suspension 15
Fine particles were collected by centrifugation at 000 rpm for 5 minutes and suspended again in 500 μl of water (hereinafter, this operation is called a washing operation) was performed twice. Further, the operation of collecting the fine particles by centrifugation and washing with 500 μl of N, N-dimethylformamide (DMF) was repeated 5 times to completely replace the solvent with DMF. Collect the microparticles again by centrifugation and remove 150 μl
Suspended in DMF and added with 300 μl of succinic anhydride and 50 μl of triethylamine dissolved in DMF at 500 mM (final concentrations of succinic anhydride and triethylamine were 300 m each).
M and 10%). The tube was allowed to react overnight at room temperature with vigorous stirring.

The fine particles after the reaction were washed twice with 500 μl of DMF and suspended in 450 μl of DMF. Triethylamine 50 μl,
Add 20 μl acetic anhydride and stir vigorously at room temperature for 2
After reacting for a time, unreacted amino groups were masked. The fine particles after the masking reaction were washed twice with 500 μl of DMF and further twice with 500 μl of water. 500 μl of fine particles after washing
Was suspended in 0.1 N NaOH and reacted at room temperature for 40 minutes. The fine particles after the reaction were washed 5 times with 500 μl of water, suspended in 500 μl of water, and stored at 4 ° C.

[0123]

[Chemical formula 26] 2) Activation of succinylated SGNEGDEN particles 5 mg of succinylated SGNEGDEN particles was placed in an Eppendorf tube and washed once with 500 μl of water. Further, a washing operation with 500 μl of dehydrated dioxane (water content of 50 ppm or less) was performed 5 times to completely replace the solvent with dehydrated dioxane. 1
The fine particles were suspended in ml of dehydrated dioxane, and 23 mg of N-hydroxysuccinimide and 38.4 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added as a powder. The mixture was reacted at room temperature for 2 hours with vigorous stirring. Fine particles after the reaction are dehydrated methanol (water content 50 pp
m or less), washed 5 times with 500 μl, and suspended in a very small amount (20 μl) of dehydrated methanol to obtain N-hydroxysuccinimide-activated fine particles.

[0124]

[Chemical 27]

3) Immobilization of the compound obtained in Example 1-1 onto fine particles Accurately weigh 0.4 mg of the compound obtained in Example 1-1 (hereinafter referred to as "FR254149", molecular weight 806.4), Dissolved in 124 μl of 0.1 N sodium acetate buffer (pH 7.3) (4 m
M). 100 mg of this solution was added to 1 mg of activated fine particles prepared in 2).
μl was added, and the mixture was reacted at room temperature for 1 hour with vigorous stirring. Collect the microparticles by centrifugation, remove the supernatant, and
0 μl of 1 M ethanolamine hydrochloride solution (pH 8.5) was added, and the mixture was reacted for 1 hour at room temperature with vigorous stirring to mask unreacted active ester groups. After the reaction, wash the microparticles 3 times with 100 μl of methanol and then in 50 μl of water.
Stored at 4 ° C.

4) Quantification of the immobilized amount of FR254149 0.1 mg each of the FR254149-immobilized fine particles and the fine particles without the drug immobilized as a control were placed in a hydrolysis test tube,
It was dried in a centrifugal concentrator. At the same time, 10 μl of a 2.5 mM aqueous solution of Compound A (FR225659) was used to prepare a standard curve.
(25 nmol) It was taken in a test tube for hydrolysis and dried in a centrifugal concentrator.

Compound A

[Chemical 28]

A test tube was placed in a Waters gas phase reaction vial, and 300 μl of constant boiling hydrochloric acid was added to the outside of the test tube. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. Heat in a constant temperature aluminum block at 110 ° C for 24 hours,
A hydrolysis reaction was performed. 400 μl of hydrolyzate after reaction
Was extracted with 50% aqueous acetonitrile solution, and the solid components were removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried under reduced pressure again. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure.

The residue was dissolved in 200 μl of 6% acetonitrile aqueous solution, and 10 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below. Analysis condition column: Waters Symmetry column C18 5 μm 4.6 mm I.
D. × 150mm Mobile phase: Liquid A 10 mM ammonium acetate (pH 5.7) Liquid B Acetonitrile A / B (6/94) → (20/80) linear gradient (30 minutes) Flow rate: 1 ml / min Temperature: 40 ° C Detection: UV (254nm)

Regarding the chromatogram of each sample obtained under the above conditions, a standard curve was prepared based on the area integral value of the peak with a retention time of 9.95 minutes (presumed to be PTC-hydroxymethylproline by LE-MS), FR254149 The amount of immobilized fine particles was calculated. As a result, the immobilized amount of FR254149 was 18.9 nmol / mg fine particles.

Example 2-2 In the same manner as in Example 1-2, 1) SGNEGDEN particles were succinylated and 2) succinylated SGNEGDEN particles were activated. 3) Immobilization of the Compound Obtained in Example 2-1 on Microparticles The compound obtained in Example 2-1 (hereinafter referred to as "FR253761", molecular weight 742.9) 1.8 mg was added to 600 μl of 0.2 M sodium acetate buffer. It was dissolved in the liquid (4 mM). 500 μl of this solution was added to 5 mg of the activated fine particles prepared in 2), and the mixture was reacted for 1 hour at room temperature with vigorous stirring. After collecting the microparticles by centrifugation and removing the supernatant, 250 μl of 1M ethanolamine hydrochloride solution (pH 8.5) was added,
The reaction was carried out at room temperature for 1 hour to mask unreacted active ester groups. The fine particles after the reaction were washed with 500 μl of methanol three times and then stored in 500 μl of methanol.

4) Quantification of the immobilized amount of FR253761 0.2 mg of FR253761 immobilized microparticles was placed in a test tube for hydrolysis and dried with a centrifugal concentrator. At the same time, in order to prepare a standard curve, 10 μl (0, 12.5, 25 nmol) of 0 mM, 1.25 mM and 2.5 mM aqueous solutions of the following compound A were placed in a hydrolysis test tube and dried in a centrifugal concentrator. A test tube was placed in a Waters gas-phase reaction vial, and 300 μl of constant boiling hydrochloric acid was added to the outside of the test tube. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. With a constant temperature aluminum block
A hydrolysis reaction was carried out by heating at 110 ° C. for 24 hours. The hydrolyzate after the reaction was extracted with 400 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried under reduced pressure again. The residue is again mixed with ethanol: water: triethylamine: phenylisothiocyanate (7: 1: 1: 1).
20 μl was added and the mixture was stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. 200 μl of 6% acetonitrile aqueous solution
10 μl was injected into the HPLC device and analyzed. HPLC
The analysis conditions are described below.

Analytical condition column: Symmetry column C18 5 μm 4.6 mm I. manufactured by Waters.
D. × 150mm Mobile phase: Liquid A 10 mM ammonium acetate (pH 5.7) Liquid B Acetonitrile A / B (6/94) → (20/80) linear gradient (30 minutes) Flow rate: 1 ml / min Temperature: 40 ° C Detection: UV (254nm)

For the chromatogram of each sample obtained under the above conditions, the peak with a retention time of 9.7 minutes (by LE-MS
A standard curve was created based on the area integral value of PTC-modified hydroxymethylproline), and the immobilized amount of FR253761 immobilized fine particles was calculated. As a result, the immobilized amount of FR253761 was 38.9 nmol / mg fine particles.

Example 3-2 1) SGNEGDEN Particles of SGNEGDEN Particles SGNEGDEN particles were synthesized according to the same literature as in Example 1-2 and measured by conductivity titration. SGNEGDEN particles (2.13% (w / v) water suspension, amino group content 0.415 mmol / g) 4
70 μl (10 mg) was placed in an Eppendorf tube, and 530 μl of distilled water was added to make a 1% (w / v) suspension. This suspension was centrifuged at 15000 rpm in a micro high-speed centrifuge (MX-160 manufactured by TOMY Seiko),
Collect the microparticles by centrifuging for 5 minutes and re-1000 μl
The operation of suspending in water (hereinafter, this operation is called a washing operation) was performed twice. Further, the operation of collecting the fine particles by centrifugation and washing with 500 μl of N, N-dimethylformamide (DMF) was repeated 5 times to completely replace the solvent with DMF. Collect the microparticles again by centrifugation and add 300 μl DMF.
600 μl of succinic anhydride and 100 μl of triethylamine dissolved in DMF at 500 mM were added (final concentrations of succinic anhydride and triethylamine were 300 mM and 1 mM, respectively).
0%). The tube was allowed to react overnight at room temperature with vigorous stirring.

The microparticles after the reaction were washed once with 500 μl of DMF and suspended in 430 μl of DMF. Triethylamine 50 μl,
Add 20 μl acetic anhydride and stir vigorously at room temperature for 2
After reacting for a time, unreacted amino groups were masked. The fine particles after the masking reaction were washed twice with 500 μl of DMF and further twice with 500 μl of water. 500 μl of fine particles after washing
Was suspended in 0.1 N NaOH and reacted at room temperature for 40 minutes. The fine particles after the reaction were washed 5 times with 500 μl of water, suspended in 500 μl of water, and stored at 4 ° C.

2) Activation of succinylated SGNEGDEN particles 5 mg of succinylated SGNEGDEN particles were placed in an Eppendorf tube and washed once with 500 μl of water. Then, it was washed once with 500 μl of methanol. Further, a washing operation with 500 μl of dehydrated dioxane (water content of 50 ppm or less) was performed 3 times to completely replace the solvent with dehydrated dioxane. The fine particles were suspended in 1 ml of dehydrated dioxane, and 23 mg of N-hydroxysuccinimide and 38.4 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added as powder. The mixture was reacted at room temperature for 2 hours with vigorous stirring. Fine particles after the reaction are dehydrated methanol (water content 50 ppm
The following) was washed 5 times with 500 μl and suspended in 100 μl of dehydrated methanol to obtain N-hydroxysuccinimide activated fine particles.

3) Immobilization of the compound obtained in Example 3-1 on fine particles

Example 3-1 (FR259384, molecular weight 935.1)
2.0 mg was dissolved in 535 μl 0.2 M sodium acetate buffer (4 mM). Add this solution to 5 mg of activated fine particles prepared in 2).
500 μl was added, and the mixture was reacted at room temperature for 70 minutes with vigorous stirring. After collecting the fine particles by centrifugation and removing the supernatant,
500 μl of 1 M ethanolamine hydrochloride solution (pH 8.5) was added, and the mixture was reacted for 1 hour at room temperature with vigorous stirring to mask unreacted active ester groups. After the reaction, wash the microparticles 5 times with 500 μl of 50% methanol water, then 500 μl
It was stored in 50% methanol water.

4) Quantification of the immobilized amount of FR259384 0.1 mg of FR259384-immobilized fine particles was placed in a test tube for hydrolysis and dried by a centrifugal concentrator. At the same time, 10 μl (0, 12.5, 25 nmol) of 0 mM, 1.25 mM, and 2.5 mM aqueous solutions of Compound A were placed in a hydrolysis test tube and dried in a centrifugal concentrator to prepare a standard curve. Place the test tube in a Waters gas-phase reaction vial, and add constant boiling hydrochloric acid to the outside of the test tube.
0 μl was added. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. 110 with constant temperature aluminum block
The mixture was heated at ℃ for 24 hours to carry out hydrolysis reaction. The hydrolyzate after the reaction was extracted with 400 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried under reduced pressure again. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 100 μl of 6% acetonitrile aqueous solution, and 10 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analysis condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.
0 = 60/40 (v / v) 0 → 15 minutes B solution = 0 → 70%, 15 minutes → B solution = 100% Flow rate: 1 ml / min Temperature: 40 ℃ Detection: UV (254nm) Obtained under the above conditions For each chromatogram of each sample, create a standard curve based on the area integration value of the peak with a retention time of 12.0 minutes (identified as PTC-modified hydroxymethylproline by LC-MS), and calculate the immobilized amount of FR259384 immobilized fine particles. did. As a result, the amount of FR259384 immobilized was 27.2 nmol / mg fine particles.

5) Confirmation of immobilization of FR259384 1.0 mg of FR259384-immobilized microparticles was subjected to hydrochloric acid gas phase decomposition and phenylisothiocyanate (PTC) conversion by the same method and analyzed by LC-MS. The identification of the degradation product confirmed that FR259384 was immobilized on the microparticles. The HPLC analysis conditions and the identified degradation products are described below. Analytical condition column: Kanto Kagaku Mightysil RP-18 GP (ODS) 3 μm 4.
6 mm I.D. × 50 mm Mobile phase: Solution A 0.1% formic acid / water Solution B 0.1% formic acid / acetonitrile 5% Solution B (0 minutes) → 90% Solution B (7-10 minutes) Flow rate: 1 ml / minute Temperature: 40 ℃ detection: UV (254nm)

[0146]

[Chemical 29]

Example 4-2 1) Immobilization of the compound of Example 4-1 on fine particles Bo of fine particles on which the compound of Example 3-1 (FR259384) was immobilized
FR259383-immobilized fine particles were prepared by removing the c group (deprotection). That is, 1 mg of FR259384-immobilized fine particles was placed in an Eppendorf tube and washed once with 100 μl of acetonitrile. Further, the fine particles were suspended in 80 μl of acetonitrile, and 20 μl of 50% hydrogen fluoride aqueous solution was added. The mixture was reacted for 24 hours at room temperature with vigorous stirring. The fine particles after the reaction were washed 5 times with 500 μl of 50% methanol water and then stored in 100 μl of 50% methanol water.

2) Quantification of the amount of FR259383 immobilized In each tube, 0.1 mg of FR259383 immobilized microparticles and microparticles without a drug immobilized as a control were placed in a hydrolysis test tube,
It was dried in a centrifugal concentrator. At the same time, to prepare a standard curve, a 2.5 mM aqueous solution of Compound A was placed in a test tube for hydrolysis (10 μl, 25 nmol) and dried in a centrifugal concentrator. Waters
A test tube was placed in a gas phase reaction vial, and 300 μl of constant boiling hydrochloric acid was added to the outside of the test tube. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. A hydrolysis reaction was performed by heating at 110 ° C. for 24 hours in a constant temperature aluminum block. The hydrolyzate after the reaction was extracted with 400 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried again under reduced pressure. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 100 μl of 6% acetonitrile aqueous solution, and 10 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analysis condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.
0 = 60/40 (v / v) 0 → 15 minutes B solution = 0 → 70%, 15 minutes → B solution = 100% Flow rate: 1 ml / min Temperature: 40 ℃ Detection: UV (254nm) Obtained under the above conditions For each chromatogram of each sample, a standard curve was created based on the area integration value of the peak with retention time of 12.0 minutes (identified as PTC-modified hydroxymethylproline by LC-MS), and the immobilized amount of FR259383 immobilized microparticles was calculated. did. As a result, the immobilized amount of FR259383 was 15.0 nmol / mg fine particles.

3) Confirmation of immobilization of FR259383 1.0 mg of FR259383-immobilized fine particles were subjected to hydrochloric acid gas phase decomposition and phenylisothiocyanate (PTC) conversion by the same method, and analyzed by LC-MS. The identification of the degradation products confirmed that FR259383 was immobilized on the microparticles. The HPLC analysis conditions and the identified degradation products are described below.

Analytical condition column: Mightysil RP-18 GP (ODS) 3 μm manufactured by Kanto Kagaku 4.
6 mm I.D. × 50 mm Mobile phase: Solution A 0.1% formic acid / water Solution B 0.1% formic acid / acetonitrile 5% Solution B (0 minutes) → 90% Solution B (7-10 minutes) Flow rate: 1 ml / minute Temperature: 40 ℃ detection: UV (254nm)

[0153]

[Chemical 30] Example 5-2 1) Immobilization of the compound obtained in Example 5-1 on fine particles 2.0 mg of the compound of Example 5-1 (FR264450, molecular weight 907.1)
Was dissolved in 551 μl of 0.2 M sodium acetate buffer (4 m
M). This solution is added to 5 mg of activated fine particles prepared in 2).
μl was added, and the mixture was reacted at room temperature for 70 minutes with vigorous stirring. Collect the microparticles by centrifugation, remove the supernatant, and
0 μl of 1 M ethanolamine hydrochloride solution (pH 8.5) was added, and the mixture was reacted for 1 hour at room temperature with vigorous stirring to mask unreacted active ester groups. After the reaction, the microparticles were washed 5 times with 500 μl of 50% methanol water, and then 500 μl of
Stored in 50% methanol water.

2) Quantification of the immobilized amount of FR264450 0.1 mg of FR264450-immobilized fine particles was placed in a test tube for hydrolysis and dried with a centrifugal concentrator. At the same time, in order to prepare a standard curve, 10 μl (0, 12.5, 25 nmol) of FR264452 0 mM, 1.25 mM, and 2.5 mM aqueous solutions were placed in hydrolysis test tubes and dried in a centrifugal concentrator. Place the test tube in a Waters gas-phase reaction vial, and add constant boiling hydrochloric acid to the outside of the test tube.
0 μl was added. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. 110 with constant temperature aluminum block
The mixture was heated at ℃ for 24 hours to carry out hydrolysis reaction. The hydrolyzate after the reaction was extracted with 400 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried under reduced pressure again. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 100 μl of 6% acetonitrile aqueous solution, and 20 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analysis condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.
0 = 60/40 (v / v) 0 → 15 minutes B solution = 0 → 70%, 15 minutes → B solution = 100% Flow rate: 1 ml / min Temperature: 40 ℃ Detection: UV (254nm) Obtained under the above conditions For the chromatogram of each sample, a standard curve was created based on the area integral value of the peak with a retention time of 17.4 minutes (identified as PTC-modified lysine by LC-MS).
50 The amount of immobilized fine particles was calculated. As a result, FR26
The immobilized amount of 4450 was 9.4 nmol / mg fine particles.

3) Confirmation of FR264450 immobilization 1.0 mg of FR264450-immobilized fine particles was subjected to hydrochloric acid gas phase decomposition and phenylisothiocyanate (PTC) conversion by the same method, and analyzed by LC-MS. The identification of the degradation product confirmed that FR264450 was immobilized on the microparticles. The HPLC analysis conditions and the identified degradation products are described below.

Analytical condition column: Mightysil RP-18 GP (ODS) 3 μm manufactured by Kanto Kagaku 4.
6 mm I.D. × 50 mm Mobile phase: Solution A 0.1% formic acid / water Solution B 0.1% formic acid / acetonitrile 5% Solution B (0 minutes) → 90% Solution B (7-10 minutes) Flow rate: 1 ml / minute Temperature: 40 ℃ detection: UV (254nm)

[0159]

[Chemical 31]

Example 6- 21 1) Preparation of immobilized fine particles of the compound (FR264451) obtained in Example 6-1 FR264450-immobilized by removing the Boc group of FR264450-immobilized fine particles (deprotection). Fine particles were prepared. That is, 5 mg of FR264450-immobilized fine particles were placed in an Eppendorf tube and washed once with 500 μl of acetonitrile.
Further, suspend the microparticles in 400 μl of acetonitrile, and
0 μl of 50% aqueous hydrogen fluoride was added. The mixture was reacted at room temperature for 9 hours with vigorous stirring. 500 μl of fine particles after reaction
After washing 5 times with 0% methanol water, it was stored in 500 μl of 50% methanol water.

2) Quantification of the immobilized amount of FR264451 Fixed amount of FR264451-immobilized microparticles and 0.1 mg of microparticles on which no drug was immobilized as a control were placed in a hydrolysis test tube,
It was dried in a centrifugal concentrator. At the same time, 10 μl of 0, 1.25 and 2.5 mM FR264452 in water was used to create a standard curve.
(0, 12.5, 25 nmol) It was taken in a test tube for hydrolysis and dried in a centrifugal concentrator. A test tube was placed in a Waters gas-phase reaction vial, and 300 μl of constant boiling hydrochloric acid was added to the outside of the test tube. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. A hydrolysis reaction was performed by heating at 110 ° C. for 24 hours in a constant temperature aluminum block. The hydrolyzate after the reaction is 40
The mixture was extracted with 0 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and the mixture was dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried under reduced pressure again. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 100 μl of 6% acetonitrile aqueous solution, and 20 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analysis condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.
0 = 60/40 (v / v) 0 → 15 minutes B solution = 0 → 70%, 15 minutes → B solution = 100% Flow rate: 1 ml / min Temperature: 40 ℃ Detection: UV (254nm) Obtained under the above conditions For the chromatogram of each sample, a standard curve was created based on the area integral value of the peak with a retention time of 17.4 minutes (identified as PTC-modified lysine by LC-MS).
51 The amount of immobilized fine particles was calculated. As a result, FR26
The immobilized amount of 4451 was 9.7 nmol / mg fine particles.

3) Confirmation of immobilization of FR264451 1.0 mg of FR264451-immobilized fine particles was subjected to hydrochloric acid gas phase decomposition and phenylisothiocyanate (PTC) conversion by the same method and analyzed by LC-MS. The identification of the degradation products confirmed that FR264451 was immobilized on the microparticles. The HPLC analysis conditions and the identified degradation products are described below.

Analytical condition column: Mightysil RP-18 GP (ODS) 3 μm manufactured by Kanto Kagaku 4.
6 mm I.D. × 50 mm Mobile phase: Solution A 0.1% formic acid / water Solution B 0.1% formic acid / acetonitrile 5% Solution B (0 minutes) → 90% Solution B (7-10 minutes) Flow rate: 1 ml / minute Temperature: 40 ℃ detection: UV (254nm)

[0166]

[Chemical 32]

Example 7-21 1) Immobilization of the compound (FR264452) obtained in Example 7-1 on fine particles 2.0 mg of FR264452 (921.0 molecular weight) was dissolved in 543 μl of 0.2 M sodium acetate buffer (4 mM). ). 500 μl of this solution was added to 5 mg of the activated fine particles prepared in 2), and the mixture was reacted for 70 minutes at room temperature with vigorous stirring. After collecting the microparticles by centrifugation and removing the supernatant, add 500 μl of 1M ethanolamine hydrochloride solution (pH 8.5) and react with vigorous stirring for 2 hours at room temperature to mask unreacted active ester groups. did. The fine particles after the reaction were washed 5 times with 500 μl of 50% methanol water, and then stored in 500 μl of 50% methanol water.

2) Quantification of the immobilized amount of FR264452 0.1 mg of FR264452-immobilized fine particles was placed in a test tube for hydrolysis and dried with a centrifugal concentrator. At the same time, in order to prepare a standard curve, 10 μl (0, 12.5, 25 nmol) of FR264452 0 mM, 1.25 mM, and 2.5 mM aqueous solutions were placed in hydrolysis test tubes and dried in a centrifugal concentrator. Place the test tube in a Waters gas-phase reaction vial, and add constant boiling hydrochloric acid to the outside of the test tube.
0 μl was added. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. 110 with constant temperature aluminum block
The mixture was heated at ℃ for 24 hours to carry out hydrolysis reaction. The hydrolyzate after the reaction was extracted with 400 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried again under reduced pressure. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 100 μl of 6% acetonitrile aqueous solution, and 20 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analysis condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.
0 = 60/40 (v / v) 0 → 15 minutes B solution = 0 → 70%, 15 minutes → B solution = 100% Flow rate: 1 ml / min Temperature: 40 ℃ Detection: UV (254nm) Obtained under the above conditions For the chromatogram of each sample, a standard curve was created based on the area integral value of the peak with a retention time of 17.4 minutes (identified as PTC-modified lysine by LC-MS).
52 The amount of immobilized fine particles was calculated. As a result, FR26
The immobilized amount of 4452 was 17.5 nmol / mg fine particles.

2) Confirmation of immobilization of FR264452 1.0 mg of FR264452-immobilized fine particles were subjected to hydrochloric acid gas phase decomposition and phenylisothiocyanate (PTC) conversion by the same method and analyzed by LC-MS. The identification of the degradation product confirmed that FR264452 was immobilized on the microparticles. The HPLC analysis conditions and the identified degradation products are described below. Analytical condition column: Kanto Kagaku Mightysil RP-18 GP (ODS) 3 μm 4.
6 mm I.D. × 50 mm Mobile phase: Solution A 0.1% formic acid / water Solution B 0.1% formic acid / acetonitrile 5% Solution B (0 minutes) → 90% Solution B (7-10 minutes) Flow rate: 1 ml / minute Temperature: 40 ℃ detection: UV (254nm)

[0172]

[Chemical 33]

Example 8-2 1) Preparation of immobilized particles of the compound (FR264453) obtained in Example 8-1 FR264453 was immobilized by removing the Boc group of FR264452 immobilized particles (deprotection). Fine particles were prepared. That is, 5 mg of FR264452-immobilized fine particles were placed in an Eppendorf tube and washed once with 500 μl of acetonitrile.
Further, suspend the microparticles in 500 μl of acetonitrile, and
0 μl of 50% aqueous hydrogen fluoride was added. The mixture was reacted for 12 hours at room temperature with vigorous stirring. 500 μl of fine particles after reaction
After washing 5 times with 50% methanol water, it was stored in 500 μl of 50% methanol water.

2) Quantification of the immobilized amount of FR264453 Fixed amount of FR264453-immobilized microparticles and 0.1 mg of microparticles without a drug immobilized as a control were taken in a hydrolysis test tube,
It was dried in a centrifugal concentrator. At the same time, 10 μl of 0, 1.25 and 2.5 mM FR264452 in water was used to create a standard curve.
(0, 12.5, 25 nmol) It was taken in a test tube for hydrolysis and dried in a centrifugal concentrator. A test tube was placed in a Waters gas-phase reaction vial, and 300 μl of constant boiling hydrochloric acid was added to the outside of the test tube. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. A hydrolysis reaction was performed by heating at 110 ° C. for 24 hours in a constant temperature aluminum block. The hydrolyzate after the reaction is 40
The mixture was extracted with 0 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and the mixture was dried under reduced pressure.

20 μl of a mixture of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried again under reduced pressure. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 100 μl of 6% acetonitrile aqueous solution, and 20 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analysis condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.
0 = 60/40 (v / v) 0 → 15 minutes B solution = 0 → 70%, 15 minutes → B solution = 100% Flow rate: 1 ml / min Temperature: 40 ℃ Detection: UV (254nm) Obtained under the above conditions For the chromatogram of each sample, a standard curve was created based on the area integral value of the peak with a retention time of 17.4 minutes (identified as PTC-modified lysine by LC-MS).
53 The amount of immobilized fine particles was calculated. As a result, FR26
The immobilized amount of 4453 was 11.3 nmol / mg fine particles.

3) Confirmation of immobilization of FR264453 1.0 mg of FR264453-immobilized fine particles were subjected to hydrochloric acid gas phase decomposition and phenylisothiocyanate (PTC) conversion by the same method, and analyzed by LC-MS. The identification of the degradation products confirmed that FR264453 was immobilized on the microparticles. The HPLC analysis conditions and the identified degradation products are described below. Analytical condition column: Kanto Kagaku Mightysil RP-18 GP (ODS) 3 μm 4.
6 mm I.D. × 50 mm Mobile phase: Solution A 0.1% formic acid / water Solution B 0.1% formic acid / acetonitrile 5% Solution B (0 minutes) → 90% Solution B (7-10 minutes) Flow rate: 1 ml / minute Temperature: 40 ℃ detection: UV (254nm)

[0178]

[Chemical 34]

Example 13 1) Iodoacetylation of SGN particles SGN particles (244 mg) were treated with N, N-dimethylformamide (3.0 m).
l) suspended. In the suspension, 1,3-diisopropylcarbodiimide (69 ml) and α-iodoacetic acid (83 ml) were added.
mg) was added. After reacting for 15 hours at room temperature, the iodoamidated latex particles were obtained by washing thoroughly with N, N-dimethylformamide. It was confirmed by Kaiser test that all amino groups were iodoacetylated.

2) Preparation of immobilized fine particles Iodoacetylated latex particles (10 mg) were added to DMF.
(0.2 ml) suspended in 2-([[1- (5-[[amino (imino) methyl] amino] -2-[[4-hydroxy-3- (3-isopropenyl-4-
(Quinolinyl) benzoyl] amino] -3-chloro-4-hydroxypentanoyl) -3-hydroxy-3-methyl-2-pyrrolidinyl] carbonyl] amino) -3-methyl-2-butenoic acid (10 mg, hereinafter “FR225659” ) And diisopropylethylamine (0.05 m
l) was added and reacted at room temperature for 40 hours. The obtained particles are N,
By thoroughly washing with N-dimethylformamide, FR225659-immobilized latex particles were obtained.

3) Quantification of the immobilized amount of FR225659 0.2 mg of FR225659-immobilized fine particles was taken in a test tube for hydrolysis and dried by a centrifugal concentrator. At the same time, 10 μl (0, 12.5, 25 nmol) of 0 mM, 1.25 mM, and 2.5 mM aqueous solutions of Compound A were placed in a hydrolysis test tube and dried in a centrifugal concentrator to prepare a standard curve. Place the test tube in a Waters gas-phase reaction vial, and add constant boiling hydrochloric acid to the outside of the test tube.
0 μl was added. A vacuum pump was used to reduce the pressure in the gas phase reaction vial, and the valve was closed. 110 with constant temperature aluminum block
The mixture was heated at ℃ for 24 hours to carry out hydrolysis reaction. The hydrolyzate after the reaction was extracted with 400 μl of 50% acetonitrile aqueous solution, the solid component was removed through a 0.1 μm filter, and then dried under reduced pressure.

20 μl of a mixed solution of ethanol: water: triethylamine (2: 2: 1) was added to the residue, and the mixture was stirred and dried under reduced pressure again. Ethanol: water: triethylamine:
Phenyl isothiocyanate (7: 1: 1: 1) mixed solution 20μ
l was added and stirred, reacted at room temperature for 20 minutes, and then dried under reduced pressure. The residue was dissolved in 200 μl of 6% acetonitrile aqueous solution, and 10 μl was injected into the HPLC device and analyzed. The HPLC analysis conditions are described below.

Analytical condition column: Wako Wakopac WS-PTC column 4.0 mm I.D. × 200
mm Mobile phase: Solution A acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 6/94 (v / v) Solution B acetonitrile / 60 mM sodium acetate buffer, pH 6.0 = 60/40 (v / v) 0 → 15 minutes Solution B = 0 → 70%, 15 minutes → Solution B = 100% Flow rate: 1 ml / min Temperature: 40 ° C Detection: UV (254 nm) Chromatogram of each sample obtained under the above conditions, retention time 12.0 minutes A standard curve was prepared based on the area integrated value of the peak (identified as PTC-modified hydroxymethylproline by LC-MS), and the immobilized amount of FR225659-immobilized fine particles was calculated. As a result, the immobilized amount of FR253761 was 2.5 nmol / mg fine particles.

Test Example 1: Effect on rat hepatocyte gluconeogenesis Male Wistar rats (150 to 2) fasted for 24 hours
From 00g), hepatocytes were prepared by the collagenase perfusion method. 5% (v / v) fetal bovine serum and 0.1 mg / m
Cells were cultured for 6 hours in 96 well tissue culture plates in William E medium containing 1 kanamycin. Cells were washed with phosphate buffered saline, glucose-free, 20
mM pyruvic acid, 1 × 10 ⁻⁷ M glucagon, 0.1 mg /
Incubated in Dulbecco's modified Eagle's medium supplemented with ml kanamycin and 1% (v / v) fetal bovine serum. After 15 hours, glucose produced in the medium was enzymatically measured. The glucose value derived from pyruvic acid was defined as the gluconeogenesis rate. Table 6 shows the 50% inhibitory concentrations of the compounds obtained in Examples for rat hepatocyte gluconeogenesis.

[0185]

[Table 6]

Further, in the evaluation system of glucagon-induced gluconeogenesis inhibition for the compounds of Examples 9 to 12 according to the above, the compounds H01, H02, H06, H07, H of Example 11 were used.
In H.10, H12, H14 to H18, and H10 of Example 12, a suppression effect of 50% or more was observed at 1 * 10 < ^-5 > M.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/10 A61K 37/02 (72) Inventor Hiroshi Tanaka 2-6-2 Mugame, Kanazawa City, Yokohama City, Kanagawa Prefecture − 406 (72) Inventor Hiroshi Handa 1-17-16 Sakurajosui, Setagaya-ku, Tokyo (72) Inventor Hiroyuki Setoi 3-4-7 Doshomachi, Chuo-ku, Osaka City, Osaka Prefecture (72) Invention Person Hidetaka Hatori 3-4-7 Doshomachi, Chuo-ku, Osaka-shi, Osaka Prefecture Fujisawa Pharmaceutical Co., Ltd. (72) Inventor Tatsuya Yoshimitsu 3-4-7, Dosho-machi, Chuo-ku, Osaka City, Osaka Prefecture Fujisawa Pharmaceutical Co., Ltd. (72) Inventor Mikio Kobayashi 3-4-7 Doshocho, Chuo-ku, Osaka-shi, Osaka Prefecture F-term inside Fujisawa Pharmaceutical Co., Ltd. (reference) 2G045 AA40 4C084 AA07 AA17 BA10 BA14 BA15 BA23 BA 32 CA59 DB34 ZA70 ZC35 4H045 AA10 AA30 BA12 EA50 FA30 GA45

Claims (5)

[Claims] 1. Formula (I): [Wherein R ¹ is an aryl group or a heterocyclic group, and the aryl group and the heterocyclic group are selected from the group consisting of a heterocyclic group, an aryl group, a hydroxy group and a lower alkyl group. It may be substituted with one or more substituents, and among these substituents, the heterocyclic group and the aryl group may be further substituted with a lower alkenyl group or a halogen atom, and the hydroxy group may be further substituted. X is a bond, a lower alkylene group or a lower alkenylene group, R ² represents a lower alkyl group, or may form a heterocycle with an adjacent nitrogen atom, a lower alkyl group. Is one or more selected from the group consisting of an amino group, a hydroxy group, a halogen atom, a carboxy group, a carbamoyl group, a heterocyclic group, an aryl group and a lower alkylthio group. May be substituted by a substituent, amino group and hydroxy group of these substituents may be further substituted, R ³ is a hydrogen atom or a halogen atom, R ⁴ is a hydrogen atom or a hydroxyl group Represents a lower alkyl group which may be substituted with, or R ³ and R ⁴ may be bonded to each other to form a heterocycle, which is a hydroxy group and / or a lower alkyl group. R ⁵ is a hydrogen atom, a lower alkyl group or a lower alkenyl group which may be substituted, and R ⁶ is a hydrogen atom, a hydroxy group, a lower alkyl group, a hydroxy (lower) alkyl group or an amino (lower) alkylamino group. And a hydroxy group, a hydroxy (lower) alkyl group and an amino (lower) alkylamino group may be further substituted, and n is an integer of 0 to 2 Indicates a number. However, R ¹ is a quinolin-4-yl group substituted with a lower alkenyl group and a phenyl group substituted with a hydroxy group, X is a bond, and R ² is an amino group substituted with an amidino group. A lower alkyl group substituted with a hydroxy group and a halogen atom, R ³
And R ⁴ are bonded to each other to form a pyrrolidine ring substituted with a hydroxy group and a lower alkyl group, R ⁵ is a lower alkenyl group, and R ⁶ is a hydroxy group,
n is not 0. ] Compounds and salts thereof. 2. R ¹ is a phenyl group, which may be substituted with one or more substituents selected from the group consisting of a heterocyclic group and a hydroxy group,
Among these substituents, the heterocyclic group may be further substituted with a lower alkenyl group, the hydroxy group may be further substituted, X is a bond, and R ² is optionally substituted. A lower alkyl group optionally substituted by one or more substituents selected from the group consisting of an amino group, a hydroxy group, a halogen atom, a carboxy group, a carbamoyl group, an aryl group and a lower alkylthio group, and R ³ and R ⁴ is bonded to each other to form a heterocycle which may be substituted with a hydroxy group and / or a lower alkyl group, R ⁵ is a lower alkyl group or a lower alkenyl group, and R ⁶ is a hydroxy group or an amino group. The compound or a salt thereof according to claim 1, which is a lower alkylamino group and n is 0. 3. Formula (I) is of formula (Ic) [In the formula, R ⁶ is a hydroxy group or an amino lower alkylamino group, R ⁷ is a hydrogen atom or an amino lower alkyl group, R ⁸ is a hydrogen atom or a lower alkyl group, and R ⁹ is a hydrogen atom or a hydroxy group. And R ² and R ⁵ have the same meanings as defined in claim 2. ] The compound and its salt of Claim 2 which are these. 4. Formula (Ia): embedded image [Wherein R ^1a represents an aryl group or a heterocyclic group, and these aryl group and heterocyclic group are selected from the group consisting of a heterocyclic group, an aryl group, a hydroxy group and a lower alkyl group. It may be substituted with one or more substituents, and among these substituents, the heterocyclic group and the aryl group may be further substituted with a lower alkenyl group or a halogen atom, and the hydroxy group may be further substituted. Xa is a bond, a lower alkylene group or a lower alkenylene group, and R ^2a represents a lower alkyl group, or may form a heterocycle with an adjacent nitrogen atom, a lower alkyl group. Is selected from the group consisting of an amino group, a hydroxy group, a halogen atom, a carboxy group, a carbamoyl group, a heterocyclic group, an aryl group and a lower alkylthio group. May be substituted by the above substituents, amino group and hydroxy group of these substituents may be further substituted, R ^3a is hydrogen atom or halogen atom, R ^4a is a hydrogen atom or a hydroxy Represents a lower alkyl group which may be substituted with a group, or R ^3a and R ^4a may combine with each other to form a heterocycle, which is a hydroxy group and / or a lower alkyl group. R ^5a is a hydrogen atom, a lower alkyl group or a lower alkenyl group, and R ^6a is a hydrogen atom, a hydroxy group, a lower alkyl group, a hydroxy (lower) alkyl group or an amino (lower) alkylamino which may be substituted with And a hydroxy group, a hydroxy (lower) alkyl group and an amino (lower) alkylamino group may be further substituted, and n is 0. Shows a two-integer. ] The immobilization product in which the compound of [4] is bonded to the fine particles made of a styrene-glycidyl methacrylate polymer through a spacer. 5. A method for screening a protein, which comprises screening a protein capable of binding to a compound of formula (Ia) using the immobilized product according to claim 4.