HK40034412A - Use of bis-iminobiotin compound for drug delivery purposes - Google Patents

Description

Use of bis-imino-biotin compounds for drug delivery

Technical Field

The present invention relates to the use of bis-imino-biotin compounds as compounds useful in the medical field for drug delivery.

Background

Biotin (represented by formula 1 below) is known to be a molecule present in a living body, and is strongly bonded to streptavidin (Kd ═ 10)^-15M), has been widely used in biological research.

Modified bisbiotin and streptavidin modified by their strong interaction and bonding force modulation are known to be used in medicine.

Iminobiotin (represented by the following formula 2) is a modified biotin, which is a compound in which the cyclic urea structure of biotin is a cyclic guanidine. Iminobiotin is known as a structure capable of regulating the binding force with streptavidin, and a bis-iminobiotin compound represented by general formula 3 is known as a structure applicable to a pre-targeting (pre-targeting) drug (patent document 1).

In the prior art, amines and carboxylic acids having polyethylene glycol and amide bonds in the side chain W bonded to the spacer group V of the bis-imino-biotin compound represented by general formula 3 and having a compound for bonding a fluorescent compound or an active ingredient of a drug at the end thereof have been reported. However, when a fluorescent compound or the like is added to the terminal, it is necessary to protect a functional group having high reactivity and then deprotect the functional group.

[ chemical formula 1]

For example, the bis-imino-biotin compounds represented by the following formulae 4 and 5 described in patent document 1 have a fluorophore and a drug in the molecule, and are reported to be useful in drug delivery (drug delivery) by a pretargeting method and in diagnostic techniques. However, synthesis of these molecules requires a plurality of steps, and selective modification of functional groups and removal of protecting groups at the final stage of synthesis are sometimes problematic.

[ chemical formula 2]

Therefore, it is required to develop a compound in which the bisiminobiotin moiety is common and only the terminal is easily converted. Therefore, the synthesis of the bisiminobiotin compound has become a plurality of steps, and there is room for improvement from the viewpoint of industrial production, and a method for more easily bonding a bisiminobiotin moiety to a drug or a fluorescent compound has been desired.

Documents of the prior art

Patent document

Patent document 1: international publication No. 2015/125820

Disclosure of Invention

Problems to be solved by the invention

However, the prior art does not disclose a method for easily bonding a bisiminobiotin moiety to a drug or a fluorescent compound.

The technical subject of the present invention is to provide: a bis-iminobiotin compound having a structure for simply bonding a bis-iminobiotin moiety to a drug or a fluorescent compound, which is useful for use in drug delivery to a substance labeled with streptavidin.

Means for solving the problems

In order to solve the problems of the present invention, the inventors of the present application studied molecular design using the click reaction defined by k.b. sharpless.

In the click reaction, Huisgen cyclization, in which an alkyne is reacted with an azide group to form a triazole, is one of the most commonly used reactions. It is considered that the end of the side chain can be easily modified without complicated introduction and removal of a protecting group by synthesizing a compound having an alkyne or azide group at the end of bisiminobiotin.

Based on the above, the inventors of the present invention have obtained the molecular structure of the bis-imino-biotin compound of the present invention, and have completed the present invention.

The bis-imino-biotin compound of the present invention is represented by the following general formula 9.

[ chemical formula 3]

(wherein A, D, E represents a spacer group in which 2 bicyclo rings are bonded, E represents a structure capable of forming a branched chain, each of which may have a substituent and may form a ring structure; J represents a functional group for click reaction, G represents a spacer group in which E and J are bonded, and R represents hydrogen, acetyl, benzyl, trifluoroacetyl, or tert-butoxycarbonyl [ hereinafter, referred to as Boc group ]

Among them, the bis-imino-biotin compounds represented by the above general formula 9 do not include the following compounds 10 to 24 and compounds 10 to 25.

[ chemical formula 4]

The bis-imino-biotin compound represented by the above general formula 9 is useful as a substrate for delivering a drug to a streptavidin-labeled substance.

The bis-imino-biotin compound bonded with a drug, which is obtained by bonding a drug to the bis-imino-biotin compound represented by the above general formula 9, can be used as a drug delivery agent for delivering a drug to a substance labeled with streptavidin.

The method for delivering a drug to a streptavidin-labeled substance using the bis-imino-biotin compound represented by the above general formula 9 comprises the steps of: bonding a drug to a drug delivery base material, bonding the drug delivery base material to which the drug is bonded to the streptavidin-labeled substance, bonding the drug to the streptavidin-labeled substance via the drug delivery base material,

the base material for drug delivery is characterized by being a bis-imino-biotin compound represented by the above general formula 9.

The bis-imino-biotin compound represented by the above general formula 9 can be used as a base material for drug delivery in a method for producing a drug delivery agent for delivering a drug to a streptavidin-labeled substance.

The method for producing a drug delivery agent for delivering a drug to a streptavidin-labeled substance, using the bis-imino-biotin compound represented by the above general formula 9, is characterized by comprising the steps of: the drug is bonded to the bis-imino-biotin compound represented by the general formula 9 using the functional group J.

As the drug, a biologically active substance or a fluorescent compound can be used.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides a bis-iminobiotin compound having a structure for simply bonding a bis-iminobiotin moiety to a drug, which is useful for use in delivering a drug to a substance labeled with streptavidin.

Detailed Description

According to one embodiment of the bisiminobiotin compound of the present invention, the following reaction can be carried out.

[ chemical formula 5]

In the above reaction, the bis-imino-biotin compound represented by formula 8 having a terminal having a triazole ring can be obtained by reacting the terminal having an alkyne structure of the bis-imino-biotin compound represented by formula 6 with the terminal having an azide group of the compound represented by formula 7 under copper catalysis.

As Y in formula 7 or 8, a drug such as a bioactive substance or a fluorescent compound can be used. The compound of formula 8 can be applied to pretargeting and target searching as in the above formulae 4 and 5 due to its molecular structure.

Since the Huisgen cyclization reaction frequently used in the click reaction selectively occurs for a functional group, it is not necessary to modify the portion other than formula 6 and formula 7 in the above reaction by a protecting group or the like. Thus, two molecules can be bonded at the end of the synthesis. Further, even a compound having a bisiminobiotin moiety as a common structure in place of an alkyne structure and an azide structure can be reacted in the same manner, and thus flexible synthesis suitable for a compound can be performed.

Therefore, in order to synthesize a bis-imino-biotin compound useful as an active ingredient of the drug delivery agent of formula 8, a compound of formula 6 as a base material for drug delivery is required.

The present invention will be described in detail below.

The bis-imino-biotin compound useful as a substrate for drug delivery is represented by the following general formula 9.

[ chemical formula 6]

(wherein A, D, E represents a spacer group in which 2 bicyclo rings are bonded, E represents a structure capable of forming a branched chain, each of which may have a substituent and may form a ring structure; J represents a functional group for click reaction, G represents a spacer group in which E and J are bonded, and R represents hydrogen, an acetyl group, a benzyl group, a trifluoroacetyl group, or a Boc group.)

Among them, the bis-imino-biotin compounds represented by the above general formula 9 do not include the following compounds 10 to 24 and compounds 10 to 25.

[ chemical formula 7]

The drug delivery substrate is characterized by containing the bis-imino-biotin compound shown above and being applicable to the delivery of drugs to streptavidin-labeled substances.

As the moiety bonded to A, D and G in E, a nitrogen atom, a carbon atom, an amide group, a benzene ring, or a heterocyclic ring (e.g., furan ring, pyrimidine ring, pyrrole ring, pyridine ring, etc.) can be preferably used.

A. The ring structure and the substituent of D and E can be selected within a range in which the intended effect of the present invention can be obtained.

As the portion formed by A, D and E, the following portions are preferable: a is formed by a1-a2-a3-a4, D is formed by D1-D2-D3-D4, A-E-D is represented by a1-a2-a3-a4-E-D1-D2-D3-D4, and a1, a2, a3, a4, E, D1, D2, D3 and D4 are independently selected from the columns in Table 1 below.

[ Table 1]

TABLE 1

In the present invention, "bonding" described in tables 1 to 4 means direct bonding in which adjacent groups are directly bonded.

A-E-D is preferably a structure selected from combinations 1 to 113 in tables 2 to 4 below.

[ Table 2]

TABLE 2

[ Table 3]

TABLE 3

[ Table 4]

TABLE 4

More preferred bis-imino-biotin compounds according to the present invention include compounds represented by the following general formulae (10-1) to (10-38).

[ chemical formula 8]

[ chemical formula 9]

[ chemical formula 10]

[ chemical formula 11]

[ chemical formula 12]

[ chemical formula 13]

[ chemical formula 14]

[ chemical formula 15]

G is a linking group for linking E to J, and is represented by G1-G2-G3-G4-G5-G6-G7, and G1, G2, G3, G4, G5, G6 and G7 are each independently a linking group selected from the columns of Table 4 below.

[ Table 5]

TABLE 5

g1

g2

g3

g4

g5

g6

g7

NHCO

(CH2)2

Bonding of

Bonding of

Bonding of

Bonding of

Bonding of

CO

CO

(OCH2CH2)3

CO

NH

(CH2)1

NH

O

Ph

O

NHCO

O

(CH2)2

O

NH

(CH2)1

Ph

(CH2)1

CO

(CH2)3

CO

(CH2)1

(CH2)3

Heterocyclic rings

(CH2)2

Ph

(CH2)4

(CH2)2

(CH2)4

(CH2)1

(CH2)3

Heterocyclic rings

(CH2)5

(CH2)3

(CH2)5

(CH2)2

(CH2)4

(OCH2CH2)1

(CH2)6

(CH2)4

(CH2)6

(CH2)3

(CH2)5

(OCH2CH2)2

(CH2)5

(OCH2CH2)1

(CH2)4

(CH2)6

(OCH2CH2)3

(CH2)6

(OCH2CH2)2

(CH2)5

(OCH2CH2)1

(OCH2CH2)4

Ph

(OCH2CH2)3

(CH2)6

(OCH2CH2)2

(OCH2CH2)5

Heterocyclic rings

(OCH2CH2)4

(OCH2CH2)1

(OCH2CH2)3

(OCH2CH2)6

Bonding of

(OCH2CH2)5

(OCH2CH2)2

(OCH2CH2)4

(OCH2CH2)7

(OCH2CH2)6

(OCH2CH2)4

(OCH2CH2)5

(OCH2CH2)8

(OCH2CH2)7

(OCH2CH2)5

(OCH2CH2)6

(OCH2CH2)9

(OCH2CH2)8

(OCH2CH2)6

(OCH2CH2)7

(OCH2CH2)9

(OCH2CH2)7

(OCH2CH2)8

Bonding of

(OCH2CH2)8

(OCH2CH2)9

(OCH2CH2)9

Examples of the heterocyclic ring in table 5 include furan ring, pyrimidine ring, pyrrole ring, and pyridine ring.

Preferred examples of the linking group as G include linking groups represented by the following general formulae (3-1) to (3-22).

In the following general formulae, J and E are shown to indicate bonding positions to J and E, and G is a portion other than J and E.

[ chemical formula 16]

In the above general formulae 3-1 to 3-22, - (CH)₂) m in m-represents an integer of 1 to 6, independently of each other, - (OCH)₂CH₂) n in n-represents an integer of 1 to 9 independently.

J is a functional group for performing a click reaction. Examples of the functional group include an azide group, a group having an alkyne structure, a tetrazine group, a trans-cyclooctyne group, and the like. Of these, preferred are azide groups and groups having an alkyne structure, and the group can be selected and used depending on the structure of the site of G bonded to J.

Examples of the group having an alkyne structure include an alkynyl group, an alkynyloxy group, an alkynylamino group, and the like, and examples of the alkynyl group included in these groups include an alkynyl group having 2 to 3 carbon atoms such as an ethynyl group and a propargyl group.

Preferred examples of the azide group and the group having an alkyne structure include groups represented by the following formulae (4-1) to (4-10). In each of the following formulae, the position indicated by G represents the bonding position with G.

[ chemical formula 17]

In the compound of chemical formula 6, the structure and the substituent of R, A, D, E, G, J can be selected within a range that can achieve the intended effects of the present invention.

As R, a hydrogen atom can be preferably used.

A, D, it is more preferable that the compound has one structure selected from 6 structures shown in Table 6 below, each of which has an alkyl chain or an alkyl chain having a substituent.

[ Table 6]

TABLE 6

	A，D
			(Iminobiotin side)	-(OH2)4-	(E side)
	-(CH2)5-
				-(CH2)6-
	-(CH2)7-
				-CH(COOH)CH2CH2CH2CH2-
	-CH(COOH)CH2CH2CH2-

More preferably, E is a structure selected from the following 3 structures having diaminobenzoic acid, monoaminodibenzoic acid, or benzylamine as a central skeleton (wherein (A) represents a bond to A, (D) represents a bond to D, and (G) represents a bond to G).

[ chemical formula 18]

As G, one structure selected from 6 structures shown in table 7 below having ethylene glycol is more preferable.

[ Table 7]

TABLE 7

	G
			(E side)	-NHCH2CH2OCH2-	(J side)
	-NH(CH2CH2O)3CH2-
				-NH(CH2CH2O)3CH2CH2CONHCH2-
	-NH(CH2CH2O)3CH2CH2COOCH2-
				-NHCH2CH2CO-
	-CH2O(CH2CH2O)2CH2CH2-

The J is more preferably one structure selected from the following 3 structures having an alkynyl group or an azido group (wherein (G) represents a bond to G).

[ chemical formula 19]

Further preferable compounds as the bisiminobiotin compound of the present invention are shown in the following compounds 13-1 to 13-23.

[ chemical formula 20]

[ chemical formula 21]

[ chemical formula 22]

[ chemical formula 23]

[ chemical formula 24]

[ chemical formula 25]

The spacer group structure E is particularly preferably a structure (wherein (a) represents a bond to a, (D) represents a bond to D, and (G) represents a bond to G).

[ chemical formula 26]

As more preferable compounds having E as a spacer structure formed of the above structure, the above compounds 13-1, 13-2, 13-4 and 13-6 can be mentioned.

The drug delivery agent of the present invention comprises a drug-bonded bisiminobiotin represented by the following general formula 14 as an active ingredient.

[ chemical formula 27]

(wherein A, D, E, G, R is as defined in general formula 9, and K represents a drug.)

The drug used for derivatization is not particularly limited as long as it has a structure that can be bonded to the compound of formula 9 using the functional group J of formula 9 and can be applied to a drug to be pre-targeted. Examples of the drug include a bioactive substance and a fluorescent compound.

Specific examples of the bioactive substances include anticancer drugs, drugs for central nervous system, drugs for immune diseases, drugs for circulatory system, and the like.

Specific examples of the fluorescent compound include coumarin (coumarin) analogues, cyanine (cyanine) analogues, rhodamine (rhodamine) analogues, and fluorescein analogues.

The drug delivery agent may contain a diluent such as a carrier, an excipient, a solvent, etc. for formulation, in addition to the compound represented by formula 9. The drug delivery agent can be used to deliver a drug to a streptavidin-labeled substance or site in or outside an organism such as an animal including a human.

Hereinafter, a method for producing the compound represented by the general formula 9 will be described.

The ester bond and amide bond in the structure of formula 9 can be formed by the following reaction.

The amide of formula 17 can be formed by condensation of an amine of formula 15 with a carboxylic acid of formula 16.

In addition, the amide bond or ester bond of E and G in the structure of formula 9 can be formed by condensation of formulae 17 and 18.

[ chemical formula 28]

The following describes the conditions applicable to the condensation reaction in the production of the general formula 9.

As the solvent used in the condensation reaction, any one of N, N-dimethylformamide, acetonitrile, tetrahydrofuran, tetrahydropyran, dichloromethane, 1, 4-dioxane, chloroform, toluene, and benzene, or 2 or more kinds of them may be used in combination.

As the solvent, it is preferable to use any one of N, N-dimethylformamide, acetonitrile, tetrahydrofuran, dichloromethane, and 1, 4-dioxane, or 2 or more kinds of them are used in combination.

As the solvent, N-dimethylformamide, acetonitrile, tetrahydrofuran, dichloromethane, or 1, 4-dioxane is more preferably used as a single solvent.

As the solvent, N-dimethylformamide, acetonitrile or tetrahydrofuran is more preferably used as a single solvent.

As the condensing agent for the condensation reaction, a carbodiimide-based condensing agent (e.g., N, N '-dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, etc.), a benzotriazole-based condensing agent (e.g., O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, etc.), a triazole-based condensing agent (e.g., 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium chloride, etc.), a carbodiimide-based condensing agent, a compound, and a compound, One kind of uronium type condensing agent (for example, (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino-carbonium hexafluorophosphate or the like) or 2 or more kinds of these are used in combination.

As the condensing agent, any of a carbodiimide-based condensing agent, a benzotriazole-based condensing agent, a triazole-based condensing agent, and a uronium-based condensing agent is preferably used.

As the condensing agent, any of a carbodiimide-based condensing agent, a benzotriazole-based condensing agent, and a uronium-based condensing agent is more preferably used.

Further, as the condensing agent, it is preferable to use any of O- (7-azabenzotriazol-1-yl) -N, N '-tetramethyluronium hexafluorophosphate, O- (benzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino-carbonium hexafluorophosphate.

In the condensation reaction of carboxylic acid activation, can use acyl halide (for example, acyl chloride, acyl fluoride, acyl bromide etc.), active ester (for example, N-hydroxy succinimide ester, N-hydroxy sulfo succinimide ester, 1-acyloxy-7-nitrogen heterocyclic, 1-acyloxy benzotriazole, etc.) in at least 1.

In the activation of carboxylic acid, preferably using acyl halide, succinate, active ester in at least 1.

In the activation of carboxylic acid, more preferably using acyl chloride, N-hydroxy succinimide ester, N-hydroxy sulfosuccinimide ester in at least 1.

In the activation of carboxylic acid, more preferably using acyl chloride, N-hydroxy succinimide ester in at least 1.

As the additive for the condensation reaction, any of tertiary amine (for example, triethylamine, diisopropylethylamine, trimethylamine, etc.), 4-dimethylaminopyridine, and imidazole, or 2 or more of them may be used in combination.

As the additive, it is preferable to use one of triethylamine, diisopropylethylamine, trimethylamine, and 4-dimethylaminopyridine, or 2 or more of them in combination.

More preferably, any of triethylamine, diisopropylethylamine, trimethylamine, and 4-dimethylaminopyridine is used as the additive.

As the additive, triethylamine or diisopropylethylamine is more preferably used.

The reaction temperature of the condensation reaction can be selected from the range of-78 ℃ to 150 ℃, preferably 0 ℃ to 150 ℃, and more preferably 0 ℃ to 50 ℃.

The concentration of the substrate for the condensation reaction may be selected from the range of 0.001mol/L to no solvent, preferably 0.01mol/L to 10 mol/L.

The reaction time of the condensation reaction may be selected from the range of 1 minute to 100 hours, and more preferably from 30 minutes to 24 hours.

For purification of the condensation reaction, crystallization, filtration and washing, silica gel column chromatography, gel filtration chromatography, and the like can be used.

As a preferred purification method, at least 1 of crystallization, filtration washing and silica gel column chromatography is used.

Hereinafter, a method for producing the compound for drug delivery represented by general formula 14 will be described.

The linkage between G and K of formula 14 may be formed by the following reaction.

For example, when J in the structure of formula 9' is an alkyne, the azide represented by formula 7 is reacted to form the structure represented by formula 18. Alternatively, when J in the structure of formula 9 "is an azide, the azide represented by formula 19 is reacted to form the structure represented by formula 20. (wherein Y, Z represents a structure of a biologically active substance or a fluorescent compound bonded to an alkyne or azide)

[ chemical formula 29]

The conditions applicable to the cyclization reaction in the production of general formula 14 will be described below.

As the solvent used in the cyclization reaction, any one of N, N-dimethylformamide, acetonitrile, tetrahydrofuran, tetrahydropyran, dichloromethane, 1, 4-dioxane, dimethyl sulfoxide, chloroform, toluene, benzene, water, acetic acid, methanol, ethanol, isopropanol, N-butanol, and t-butanol, or 2 or more kinds of them may be used in combination.

As the solvent, it is preferable to use any one of N, N-dimethylformamide, acetonitrile, tetrahydrofuran, 1, 4-dioxane, chloroform, toluene, benzene, water, acetic acid, methanol, ethanol, isopropanol, N-butanol, and t-butanol, or 2 or more kinds of them in combination.

As the solvent, it is more preferable to use any one of N, N-dimethylformamide, acetonitrile, 1, 4-dioxane, water, acetic acid, methanol, ethanol, and tert-butanol, or to use 2 or more of them in combination.

As the solvent, any of N, N-dimethylformamide, acetonitrile, 1, 4-dioxane, water, acetic acid, ethanol, and tert-butanol is more preferably used.

As the catalyst used in the cyclization reaction, any of monovalent copper salts (for example, copper (I) chloride, copper (I) bromide, copper (I) iodide, copper (I) acetate, and the like), divalent copper salts (for example, copper (II) chloride, copper (II) bromide, copper (II) iodide, copper (II) acetate, copper (II) sulfate, and the like), silver salts (silver chloride, silver bromide, silver iodide, silver acetate, and the like), ruthenium salts, or 2 or more kinds of them may be used in combination.

Alternatively, the cyclization reaction may be carried out without adding a catalyst.

As the catalyst, it is preferable to use any one of monovalent copper salt, divalent copper salt, and silver salt, or to use 2 or more of them in combination.

As the catalyst, any of copper (I) chloride, copper (I) bromide, copper (I) iodide, copper (I) acetate, copper (II) chloride, copper (II) bromide, copper (II) iodide, copper (II) acetate, and copper (II) sulfate is more preferably used.

As the additive for the cyclization reaction, any of ascorbic acid, sodium ascorbate, potassium ascorbate, and calcium ascorbate, or 2 or more of them may be used in combination. Alternatively, the cyclization reaction may be carried out without adding an additive.

As the additive, any of ascorbic acid, sodium ascorbate, and potassium ascorbate is preferably used.

As the additive, sodium ascorbate is more preferably used.

The reaction temperature of the cyclization reaction can be selected from the range of-78 ℃ or higher and 150 ℃, preferably 0 ℃ or higher and 100 ℃ or lower, and more preferably 0 ℃ or higher and 60 ℃ or lower.

The concentration of the substrate for the cyclization reaction can be selected from the range of 0.001mol/L to no solvent, preferably 0.01mol/L to 10 mol/L.

The reaction time of the cyclization reaction can be selected from the range of 1 minute to 168 hours, preferably 30 minutes to 24 hours.

For purification of the cyclization reaction, crystallization, filtration and washing, silica gel column chromatography, and gel filtration chromatography can be used.

As a preferred purification method, at least 1 kind of crystallization, filtration washing, silica gel column chromatography is used.

Examples

Hereinafter, examples of the present invention will be described, but the present invention is not limited thereto. NMR analysis values were measured using EX-270(270MHz) manufactured by Kokai electronic division.

For HPLC analysis, the analysis was performed under the following 2 conditions.

(analysis Condition A)

Column: YMC-Pack ODS-AM 150 x 6mm

Flow rate: 1mL/min.

Column temperature: 40 deg.C

Detection wavelength: 254nm

The mobile phase: 0.1% aqueous trifluoroacetic acid/acetonitrile

(analysis Condition B)

Column: YMC Triart C1875 mm 2mm

Flow rate: 0.3mL/min.

Column temperature: 35 deg.C

Detection wavelength: 254nm

The mobile phase: 0.1% aqueous trifluoroacetic acid/acetonitrile

For the gradient conditions, for example, those described as 0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(12 min) 35/65, represent the following conditions: the 0.1% aqueous trifluoroacetic acid solution was reduced from 85% to 35% over 12 minutes and then returned to 85%.

Hereinafter, examples are described as synthetic schemes (reaction formulae).

[ example 1-1]

(Synthesis of bis (Boc-iminobiotin) Compound 24)

[ chemical formula 30]

To 135mg (0.13mmol) of bis (Boc-iminobiotin) 22 was added 1mL of dehydrated dimethylformamide, and further 22mg (1.05 equiv) of carbonyldiimidazole. Stirring was carried out at 40 ℃ for 1 hour, and 36mg (1 equivalent) of amine 23(Shigma-Aldrich, CAS No; 1255942-06-3) dissolved in 0.7mL of dehydrated chloroform was added and stirring was carried out at room temperature for 5 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, 10mL of chloroform was added, and the pH was adjusted to 5 with 0.1N hydrochloric acid. A precipitate formed and water and chloroform were removed. The precipitate was dissolved in methanol, dried over magnesium sulfate, and concentrated to give a residue. Purification was performed by silica gel column (chloroform/methanol: 20/1-5/1) to obtain 128mg of amide 24. (yield 75%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：8.2(1H,br.t),8.0(1H,s),7.95(1H,s),7.75(2H,t),7.3-7.7(12H,m),5.05(1H,d),4.55-4.65(2H,m),4.3-4.4(3H,m),4.1(1H,m),3.64(1H,d),3.4-3.5(4H,m),3.2-3.3(2H,m),3.16(4H,d),2.95-3.1(4H,br.t),2.8-2.9(4H,m),2.5-2.6(1H,m),2.2-2.35(4H,t),2.0-2.1(4H,t),1.8-2.0(1H,m),1.2-1.7(24H,m),1.4(18H,s)

HPLC retention time (analytical conditions a): 14.7 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(18 min) 5/95)

[ examples 1-2]

(Synthesis of bis-Iminobiotin Compound 26)

[ chemical formula 31]

To 15mg (11.6. mu. mol) of the amide 25 synthesized in example 1-1, 0.5mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure at 60 ℃ to obtain 13.3mg of a trifluoroacetate salt of diiminoibiotin 26. (yield 95%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：10.0(2H,s),8.2(1H,br.t),8.0(1H,s),7.95(1H,s),7.75(2H,t),7.72-7.3(15H,m),5.05(1H,d),4.61-4.55(2H,m),4.47-4.40(3H,m),4.19(1H,m),3.60(1H,d),3.53-3.43(4H,m),3.34-3.22(2H,m),3.16(4H,d),3.11-2.89(4H,br.t),2.82-2.75(4H,m),2.61-2.57(1H,m),2.30(4H,t),2.05(4H,t),1.98-1.90(1H,m),1.72-1.27(24H,m)

HPLC retention time (analytical conditions a): 10.3 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(18 min) 5/95)

[ examples 1 to 3]

(Synthesis of bis-Iminobiotin-fluorescein Compound 28)

[ chemical formula 32]

13mg (12. mu. mol) of the trifluoroacetate salt of bisiminobiotin 26 synthesized in example 1-2 was dissolved in 1mL of dehydrated dimethylformamide, and 0.72mL of a chloroform solution was added. Further, 11mg (2 equivalents) of a chloroform solution of 6-carboxyfluorescein azide 27 was added. After stirring overnight at room temperature, the reaction mixture was concentrated under reduced pressure at 60 ℃. The residue was washed twice with 2mL of 1N aqueous hydrochloric acid solution and then dried under reduced pressure to give amorphous 25mg of bisiminobiotin-fluorescein 28.

¹H-NMR(DMSO-d6)：10.4(1H,br.s),10.0(2H,s),8.3(3H,m),8.1(1H,d),8.0(1H,s),7.95(1H,s),7.75(2H,t),7.72-7.3(15H,m),7.53(1H,d),7.00(1H,d),6.46(1H,d),6.22(3H,m),6.11(1H,d),5.05(2H,m),4.61-4.55(2H,m),4.47-4.40(3H,m),4.19(1H,m),3.60(1H,d),3.53-3.43(6H,m),3.34-3.22(2H,m),3.16(4H,d),3.11-2.89(4H,br.t),2.82-2.75(4H,m),2.62-2.57(3H,m),2.30(4H,t),2.05(4H,t),1.98-1.90(1H,m),1.72-1.27(24H,m)

HPLC retention time (analytical conditions a): 12.6 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(18 min) 5/95)

[ example 2-1]

(Synthesis of bis (Boc-iminobiotin) Compound 30)

[ chemical formula 33]

Using 500mg (0.49mmol) of bis (Boc-iminobiotin) 22 and 36mg (1.27 equivalent) of amine 29 in place of 135mg of bis (Boc-iminobiotin) 22 and 36mg of amine 23 in example 1-1, a reaction was carried out according to the synthesis method described in example 1-1 to obtain 128mg of bis (Boc-iminobiotin) 30. (yield 49%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：9.97(2H,s),8.4(1H,br.t),8.0(1H,s),7.95(1H,s),7.74(2H,t),7.65(3H,d),4.6-4.5(2H,m),4.35-4.25(2H,m),4.16(2H,d),4.1(1H,m),3.56(2H,t),3.45-3.35(3H,m),3.25-3.15(1H,m),2.95-3.1(4H,br.q),2.9-2.75(4H,m),2.45-2.2(4H,t),2.1-2.0(4H,t),1.2-1.7(24H,m),1.4(18H,s)

HPLC retention time (analytical conditions a): 11.3 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(12 min) 5/95)

[ examples 2-2]

(Synthesis of bis-Iminobiotin Compound 31)

[ chemical formula 34]

Using 15mg (13.5. mu. mol) of bis (Boc-iminobiotin) -acetylene 30 synthesized in example 2-1 in place of 15mg (11.6. mu. mol) of the amide 25 used in example 1-2, the synthesis method described in example 1-2 was followed to obtain 13.8mg of a trifluoroacetate salt of bisiminobiotin 31. (yield 100%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：9.99(2H,s),8.40(1H,t),8.35(2H,s),8.09(1H,s),7.79(2H,t),7.70(4H,br.s),7.66(2H,s),4.65-4.61(2H,m),4.47-4.42(2H,m),4.16(2H,d),3.56(2H,t),3.45-3.38(3H,m),3.27-3.20(2H,m),3.06(4H,q),2.94-2.73(4H,m),2.31(4H,t),2.06(4H,t),1.71-1.24(24H,m)

HPLC retention time (analytical conditions a): 9.68 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(12 min) 5/95)

[ example 3-1]

(Synthesis of bis (Boc-iminobiotin) Compound 33)

[ chemical formula 35]

Instead of using 135mg of bis (Boc-iminobiotin) 22 and 36mg of amine 23 in example 1-1, 355mg (355. mu. mol) of bis (Boc-iminobiotin) 32 and 55.2. mu.L (1.5 equivalents) of amine 29 were used to carry out the reaction according to the synthesis method described in example 1-1, thereby obtaining 55.6mg of bis (Boc-iminobiotin) 33 as a target reaction product. (yield 15%)

(analysis value of target reaction product)

1H-NMR(DMSO-d6)：9.99(2H,s),8.39(1H,br.t),8.07(1H,s),7.93(2H,s),7.77(2H,t),7.65(4H,br.s),4.53-4.84(2H,m),4.27-4.23(2H,m),4.17(2H,d),3.56(2H,t),3.44-3.38(3H,m),3.21-3.14(2H,m),3.09-3.01(4H,br.q),2.83-2.80(4H,m),2.31(4H,t),2.05(4H,t),1.62-1.42(20H,m),1.36(18H,s)

HPLC retention time (analytical conditions B): 4.99 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(7 min) 5/95)

[ examples 3-2]

(Synthesis of bis-Iminobiotin Compound 34)

[ chemical formula 36]

Using 5.1mg (4.7. mu. mol) of bis (Boc-iminobiotin) 33 synthesized in example 3-1 in place of 15mg (11.6. mu. mol) of amide 25 used in example 1-2, the reaction was carried out by the synthesis method described in example 1-2 to obtain 5.2mg of a trifluoroacetate salt of bisiminobiotin 34. (yield 100%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：10.00(2H,s),8.40(1H,t),8.35(2H,s),8.07(1H,s),7.79(2H,t),7.70(4H,br.s),7.66(2H,s),4.66-4.61(2H,m),4.47-4.42(2H,m),4.16(2H,d),3.56(2H,t),3.45-3.39(3H,m),3.27-3.20(2H,m),3.05(4H,q),2.94-2.73(4H,m),2.31(4H,t),2.06(4H,t),1.71-1.24(20H,m)

HPLC retention time (analytical conditions B): 3.76 min (0.1% trifluoroacetic acid in water/acetonitrile 85/15(7 min) 5/95)

[ example 4-1]

(Synthesis of bis (Boc-iminobiotin) -azide Compound 36)

[ chemical formula 37]

The synthesis method described in example 1-1 was followed using 50mg (50. mu. mol) of bis (Boc-iminobiotin) 32 and 15.0. mu.L (1.5 equivalents) of amine 35 instead of 135mg of bis (Boc-iminobiotin) 22 and 36mg of amine 23 used in example 1-1 to obtain 16.0mg of bis (Boc-iminobiotin) -azide 36. (yield 27%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：10.0(2H,s),8.35(1H,t),8.05(1H,s),7.93(2H,s),7.77(2H,t),7.66(4H,m),4.55(2H,m),4.27(2H,m),3.54-3.51(12H,m),3.39-3.37(2H,m),3.18-3.16(2H,m),3.06-3.04(4H,m),2.83-2.75(4H,m),2.30(4H,t),2.05(4H,t),1.70-1.41(22H,m),1.35(18H,s)

HPLC retention time (analytical conditions B): 5.64 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(7 min) 20/80)

[ example 4-2]

(Synthesis of bis-Iminobiotin-azide Compound 37)

[ chemical formula 38]

Using 16mg (13. mu. mol) of bis (Boc-iminobiotin) -azide 36 synthesized in example 4-1 in place of 15mg (11.6. mu. mol) of amide 25 used in example 1-2, the reaction was carried out by the synthesis method described in example 1-2 to obtain 14.5mg of a trifluoroacetate salt of bisiminobiotin-azide 37. (yield 100%)

(analysis value of target reaction product)

¹H-NMR(DMSO-d6)：10.00(2H,s),8.47-8.44(1H,m),8.35(1H,m),8.21(2H,m),8.05(1H,m),7.81-7.76(5H,m),7.67(2H,d),4.66-4.61(2H,m),4.47-4.42(2H,m),3.60-3.50(12H,m),3.57(2H,m),3.25(2H,m),3.05(4H,q),2.94-2.75(4H,m),2.31(4H,t),2.06(4H,t),1.71-1.24(22H,m)

HPLC retention time (analytical conditions B): 4.34 min (0.1% aqueous trifluoroacetic acid/acetonitrile 85/15(7 min) 20/80).

Claims (13)

1. A drug delivery substrate for delivering a drug to a streptavidin-labeled substance, comprising a bis-imino-biotin compound represented by the following general formula 9:

[ chemical formula 1]

Wherein A, D, E represents a spacer group in which 2 bicyclic rings are bonded, and E represents a structure capable of forming a branched chain, each of which may have a substituent, and may form a ring structure; j represents a functional group for performing a click reaction, G represents a spacer group bonding E to J, R represents hydrogen, an acetyl, benzyl, trifluoroacetyl or Boc group,

wherein the following compounds 10-24 and compounds 10-25 are excluded:

[ chemical formula 2]

2. The drug delivery substrate according to claim 1, wherein,

in the case of the compound of the general formula 9,

r is the hydrogen atom in the formula (I),

a and D are one of 4 structures in the following table,

[ Table 1]

TABLE 1

A，D (Iminobiotin side) -(CH2)4- (E side) -(CH2)5- -(CH2)6- -(CH2)7- -CH(COOH)CH2CH2CH2CH2- -CH(COOH)CH2CH2CH2-

E is any of the following 3 structures, wherein (A) represents a bond with A, (D) represents a bond with D, (G) represents a bond with G,

[ chemical formula 3]

G is any one of 6 structures in the following table,

[ Table 2]

TABLE 2

G (E side) --NHCH2CH2OCH2-- (J side) -NH(CH2CH2O)3CH2- -NH(CH2CH2O)3CH2CH2CONHCH2- -NH(CH2CH2O)3CH2CH2COOCH2- -NHCH2CH2CO- -CH2O(CH2CH2O)2CH2CH2-

J is any of the following 3 structures, (G) represents a bond with G,

[ chemical formula 4]

3. The drug delivery substrate according to claim 2, wherein E is a structure in which (A) represents a bond to A, (D) represents a bond to D, (G) represents a bond to G,

[ chemical formula 5]

4. The drug delivery substrate according to claim 2, wherein the bis-imino-biotin compound is 1 compound selected from the group consisting of 13-1 to 13-23,

[ chemical formula 6]

[ chemical formula 7]

[ chemical formula 8]

[ chemical formula 9]

[ chemical formula 10]

[ chemical formula 11]

5. The drug delivery substrate according to claim 4, wherein the bis-imino-biotin compound is any one of the following compounds,

[ chemical formula 12]

6. A drug delivery agent for delivering a drug to a streptavidin-labeled substance, characterized by comprising a drug-bonded bis-imino-biotin compound represented by general formula 14,

[ chemical formula 13]

In the formula (I), the compound is shown in the specification,

A. d, E is a spacer linking 2 bicyclic rings, G represents a spacer linking E to K,

a and D are one of 4 structures in the following table,

[ Table 3]

TABLE 3

A，D (Iminobiotin side) -(CH2)4- (E side) -(CH2)5- -(CH2)6- -(CH2)7- -CH(COOH)CH2CH2CH2CH2- -CH(COOH)CH2CH2CH2-

E is any of the following 3 structures, wherein (A) represents a bond with A, (D) represents a bond with D, (G) represents a bond with G,

[ chemical formula 14]

G represents any one of 6 structures in the following table,

[ Table 4]

TABLE 4

G (E side) -NHCH2CH2OCH2- (J side) -NH(CH2CH2O)3CH2- -NH(CH2CH2O)3CH2CH2CONHCH2- -NH(CH2CH2O)3CH2CH2COOCH2- -NHCH2CH2CO- -CH2O(CH2CH2O)2CH2CH2-

R is the hydrogen atom in the formula (I),

k represents a drug.

7. Use of the bis-imino-biotin compound of any one of claims 1 to 5 as a substrate for delivering a drug to a streptavidin-labeled substance.

8. Use of the drug-bonded bis-imino-biotin compound of claim 6 as a drug delivery agent for delivering a drug to a streptavidin-labeled substance.

9. A drug delivery method for delivering a drug to a streptavidin-labeled substance, comprising the steps of:

the drug is bonded to the drug delivery substrate,

bonding the drug delivery base material to which the drug is bonded to the streptavidin-labeled substance, bonding the drug to the streptavidin-labeled substance via the drug delivery base material,

wherein the drug delivery substrate is the bis-imino-biotin compound according to any one of claims 1 to 5.

10. A method for producing a drug delivery agent for delivering a drug to a streptavidin-labeled substance, comprising the steps of: a drug is bonded to the bis-imino-biotin compound of any one of claims 1 to 5 using functional group J.

11. The method for producing a compound for drug delivery according to claim 10, wherein the drug is a bioactive substance or a fluorescent compound.

12. Use of the bis-imino-biotin compound of any one of claims 1 to 5 in a method for manufacturing a drug delivery agent for delivering a drug to a streptavidin-labeled substance.

13. The use according to claim 12, wherein the drug is a biologically active substance or a fluorescent compound.