KR100481434B1 - Manufacturing method of drug complex - Google Patents

Abstract

The present invention relates to a drug complex in which a polysaccharide derivative having a carboxyl group and a residue of a pharmaceutical compound are combined through a spacer consisting of one amino acid or a spacer consisting of two to eight amino acids bonded to a peptide, or a residue of a polysaccharide derivative having a carboxyl group and a pharmaceutical compound. The present invention relates to a method for producing a drug complex which is bound via an electrical spacer, wherein the method comprises reacting an organic amine salt of a polysaccharide derivative having a carboxyl group with a pharmaceutical compound or a spacer in which a pharmaceutical compound is bound in a non-aqueous system. .

The reaction with a polysaccharide derivative having a carboxyl group and a pharmaceutical compound having a spacer bonded thereto can be efficiently performed, and side reactions can be suppressed when the pharmaceutical compound having a lactone ring is reacted.

Description

Manufacturing method of drug complex

The present invention relates to a method for producing a drug complex in which a pharmaceutical compound such as a polysaccharide derivative and an anti-tumor agent is combined.

Antitumor agents, which are used for the treatment of solid cancers such as lung cancer and gastrointestinal cancer, and blood cancers such as leukemia, are administered systemically through the administration route such as intravenous administration or oral administration, and then transferred to specific tumor sites, It exhibits a therapeutic effect by inhibiting or inhibiting proliferation. However, since the antitumor agent administered systemically is rapidly absorbed from the blood into the hepatic and intraluminal organs, or rapidly excreted in the urinary tract, the blood concentration may be lowered and transition to the tumor site may not be sufficient. In addition, since the conventional antitumor agent itself has low transition selectivity (tumor selectivity) to the tumor site, the antitumor agent is distributed to various cells and tissues throughout the system, and acts as a cytotoxic to normal cells or tissues. Side effects such as vomiting, fever or hair loss occur at a very high rate. Therefore, there is a need for the development of means for effectively and selectively transferring antitumor agents to tumor sites.

As one of such means, a polysaccharide derivative having a carboxyl group is used as a drug carrier, and an anti-tumor agent is bound to the polysaccharide derivative to delay the loss of the anti-tumor agent from the blood and cancer tissue. A method of increasing the directivity to the future has been proposed. For example, in WO 94/19376, a peptide chain (amino acids 1 to 8) is bound to a carboxyl group of a polysaccharide having a carboxyl group, and further, doxorubicin and daunorubicin are linked through the peptide chain. A drug complex in which daunorubicin, mitomycin C, or bleomycin is combined is disclosed. In addition, Japanese Patent Application Laid-Open No. 7-84481 discloses a drug complex in which the antitumor agent is introduced into a carboxymethylated mannoglucan derivative through a Schiff base or an acid amide bond. . These drug complexes are characterized by having more excellent anti-tumor effect and less toxicity and side effects than the anti-tumor agent itself which is not bound to the carrier for drug delivery.

In addition, for the description of drug complexes using polyalcoholized polysaccharide derivatives as carriers for drug delivery, "Research on polysaccharide-peptide-doxorubicin complexes and the relationship between blood stability and anti-tumor effect of polysaccharide derivatives" (10th) Journal of the Japanese DDS Society Abstract, 279, 1994); "Study on Polysaccharide-Peptide-Doxorubicin Complexes, In Vivo Dynamics and Antitumor Effects" (9th Pharmacokinetic Society Banquet Lecture Abstract, 292, 1994); 19th Research & Development Trend Seminar (Organized by Pharmaceutical Organization) Abstract, D-9, 1995; And "Study on Drug Delivery to Tumors with Polysaccharide Carriers" (12th Colloid / Interface Symposium, Japanese Chemistry Society, Lecture Abstract, 51, 1995).

Conventionally, these drug complexes are prepared by preparing a polysaccharide derivative having a carboxyl group such as carboxymethylfuran and carboxymethylmannoglucan with sodium salt, and then a carboxyl group of the polysaccharide derivative and an amino group of an antitumor agent (or a peptide bound to an antitumor agent). N-terminal amino group of the chain) is produced by acid amide bonding. Since the sodium salt of the polysaccharide derivative is not dissolved in the organic solvent at all, the sodium salt of the polysaccharide derivative is prepared from a solution of water or an aqueous organic solvent containing water, and the condensing agent and A method of dissolving an antitumor agent (or an antitumor agent having a peptide chain) in the form of a hydrochloride salt has been adopted.

In this reaction, however, the dehydration and condensation reaction was carried out in a solvent containing water, so that the target product was not obtained in high yield. Antitumor agents and the like used as reaction raw materials are generally expensive, and development of a method for efficiently producing the above-described drug complexes has been required from the viewpoint of production cost. In addition, since the compound described in Japanese Patent Application Laid-Open No. 6-87746 useful as an anti-tumor agent has a lactone ring in its molecule, when a compound having a peptide chain attached thereto is attached by the above reaction, in the presence of a base and water The lactone ring is ring-opened, and the resulting carboxyl group reacts with the N-terminal amide group of the peptide chain bound to the anti-tumor agent, resulting in a significant reduction in the yield of the target product, and a target drug complex cannot be obtained. For this reason, development of the reaction method which can avoid production | generation of the compound which the lactone ring ring-opened was calculated | required.

Disclosure of the Invention

An object of the present invention is to provide a method for efficiently producing a drug complex that can selectively transfer active ingredients such as antitumor agents and anti-inflammatory agents to tumor sites and the like. More specifically, it is possible to efficiently react a polysaccharide derivative having a carboxyl group with a spacer such as a pharmaceutical compound such as an anti-tumor agent or an oligopeptide combined with a pharmaceutical compound, thereby providing a method for producing a drug complex at a low cost in large quantities. Is in.

As a result of intensive efforts to solve the above problems, the present inventors use an organic amine salt in place of the sodium salt of a polysaccharide derivative that has been conventionally used when the polysaccharide derivative having a carboxyl group is reacted with a pharmaceutical compound or a spacer to which the pharmaceutical compound is bound. In other words, the salt of the polysaccharide derivative can be dissolved in high concentration in an organic solvent that is substantially free of water, and the reaction between the polysaccharide derivative and the pharmaceutical compound or the spacer incorporating the pharmaceutical compound can be carried out in an extremely good yield. It was found that by-products and the like can be reduced. The present invention has been completed based on these contents.

That is, the present invention relates to a polysaccharide derivative having a carboxyl group and a drug compound wherein a residue of the pharmaceutical compound is bound via a spacer consisting of 2 to 8 amino acids bound by one amino acid or peptide, or a polysaccharide derivative having a carboxyl group and a pharmaceutical compound. A method for producing a drug complex in which a residue is bonded through a spacer, wherein the organic amine salt of a polysaccharide derivative having a carboxyl group and a pharmaceutical compound or a pharmaceutical compound bonded with a spacer are reacted in a non-aqueous system; (1) preparing a drug complex comprising converting an alkali metal salt of a polysaccharide derivative having a carboxyl group into an organic amine salt and (2) reacting the organic amine salt with a pharmaceutical compound or a pharmaceutical compound bonded with a spacer in a non-aqueous system To provide a way.

According to an exemplary embodiment of the present invention, wherein the method is a polysaccharide derivative having a carboxyl group, carboxy C _1-4 alkyl polyalcohol (carboxyC _1-4 alkyldextranpolyalcohol); The dextran polyalcohol constituting the carboxyC _1-4 alkyldextran polyalcohol (dextranpolyalcohol) is a dextran polyalcohol obtained by treating dextran under conditions that are substantially polyalcoholizable; The above method wherein the carboxyC _1-4 alkyldextran polyalcohol is a carboxymethyldextran polyalcohol; The carboxyC _1-4 alkyldextran polyalcohol is a carboxymethyldextran polyalcohol in the range of molecular weight 5,000 to 500,000, preferably in the range of 50,000 to 450,000, more preferably in the range of 200,000 to 400,000, and the degree of carboxymethylation Wherein the range is from 0.01 to 2.0, preferably from 0.1 to 1.0, more preferably from 0.3 to 0.5 per residue per constituent; And the above method wherein the pharmaceutical compound is an anti-tumor agent or an anti-inflammatory agent.

Moreover, as a preferable aspect of this invention, the said method whose pharmaceutical compound is a compound which can form a lactone ring; In the reaction of an organic amine salt with a spacer combining a pharmaceutical compound or a pharmaceutical compound, the above method using a spacer combining a pharmaceutical compound having a lactone ring or a pharmaceutical compound having a lactone ring; And, the pharmaceutical compound capable of forming a lactone ring is (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro -9-hydroxy-4-ethyl-1H, 12H-benzo [de] pyrano [3 ', 4'; 6,7] indolizino [ 1,2-b] quinoline-10,13 (9H, 15H) -dione is provided.

1 is a diagram showing a GPC chart of a drug complex of Example 8 prepared by the method of the present invention.

2 is a diagram showing an ultraviolet absorption spectrum of the drug complex of Example 8 prepared by the method of the present invention.

3 is a diagram showing a GPC chart of a drug complex of Example 9 prepared by the method of the present invention.

4 is a diagram showing an ultraviolet absorption spectrum of the drug complex of Example 9 prepared by the method of the present invention.

5 is a diagram showing an ultraviolet absorption spectrum of the drug complex of Example 10 prepared by the method of the present invention.

Fig. 6 shows the GPC chart of the drug complex of Example 15 prepared by the method of the present invention.

Fig. 7 shows the ultraviolet absorption spectrum of the drug complex of Example 15 prepared by the method of the present invention.

Fig. 8 shows the GPC chart of the drug complex of Example 28 prepared by the method of the present invention.

Fig. 9 shows the ultraviolet absorption spectrum of the drug complex of Example 28 prepared by the method of the present invention.

Fig. 10 shows the GPC chart of the drug complex of Example 29 prepared by the method of the present invention.

Fig. 11 shows the ultraviolet absorption spectrum of the drug complex of Example 29 prepared by the method of the present invention.

Fig. 12 shows the GPC chart of the drug complex of Example 34 prepared by the method of the present invention.

Fig. 13 shows the ultraviolet absorption spectrum of the drug complex of Example 34 prepared by the method of the present invention.

Fig. 14 shows the GPC chart of the drug complex of Example 39 prepared by the method of the present invention.

Fig. 15 shows the ultraviolet absorption spectrum of the drug complex of Example 39 prepared by the method of the present invention.

Fig. 16 shows the GPC chart of the drug complex of Example 41 prepared by the method of the present invention.

Fig. 17 shows the ultraviolet absorption spectrum of the drug complex of Example 41 prepared by the method of the present invention.

Fig. 18 shows the GPC chart of the drug complex of Example 44 prepared by the method of the present invention.

Fig. 19 shows the ultraviolet absorption spectrum of the drug complex of Example 44 prepared by the method of the present invention.

20 is a diagram showing the body kinetics of the drug complex of Example 15 prepared by the method of the present invention. Each point in a figure shows the average value of three examples.

Best Mode for Carrying Out the Invention

The drug complex produced by the production method of the present invention is a polysaccharide derivative having a carboxyl group and a residue of the pharmaceutical compound is bound through a spacer consisting of 2 to 8 amino acids, one amino acid or peptide-bonded, or a polysaccharide derivative having a carboxyl group And the residues of the pharmaceutical compound are bonded to each other without passing through the spacer.

The residue of the pharmaceutical compound included in the drug complex is derived from a pharmaceutical compound used for the treatment and / or prevention of diseases of mammals including humans, for example, as a medicine such as an anti-tumor agent, an anti-inflammatory agent, an antimicrobial agent, and a partial structure thereof. It is composed by. However, the pharmaceutical compound from which the residue is derived is not limited to the above. In addition, any pharmaceutical compound may be used as long as it has one or two or more reactive functional groups (for example, amino group, carboxyl group, hydroxyl group, thiol group, ester group, etc.) that may be involved in binding to a polysaccharide derivative or spacer. In the present specification, when referred to as a pharmaceutical compound, a prodrug compound that can reproduce the compound in vivo, including the main structure of the compound which itself has a medicinal action as its partial structure, is also included. .

More specifically, in the present specification, the residue of the pharmaceutical compound is a reaction of a reactive group in the pharmaceutical compound with a polysaccharide derivative or spacer and the residue of the drug compound in the pharmaceutical compound (for example, dehydration condensation, etc.). In the case of assuming that it is formed by a), it means a partial structure derived from a pharmaceutical compound present in the compound after bonding. For example, when the pharmaceutical compound is represented by D-NH ₂ , D-COOH, D-COOR, D-OH, D-SH, D-CONH ₂ , D-NH-COOR (where R is a lower alkyl group), Residues of pharmaceutical compounds are D-NH- (D-NH-CO-Q, etc.), D-CO- (D-CO-NH-Q, D-CO-OQ, D-CO-SQ, etc.), D- CO- (D-CO-NH-Q, D-CO-OQ, D-CO-SQ, etc.), DO- (DO-CO-Q, DOQ, etc.), DS- (DS-CO-Q, DSQ, etc.) , D-CONH- (D-CO-NH-CO-Q, etc.), D-NH-CO- (D-NH-CO-OQ, D-NH-CO-NH-Q, etc.) The spacer or polysaccharide derivative is bound to the drug compound moiety, and Q represents the remaining partial structure excluding the reactive functional group and the carboxyl group in the spacer and the polysaccharide derivative, respectively). However, the type of bond between the spacer or the polysaccharide derivative and the residue of the pharmaceutical compound is not limited to the above. The residue of the pharmaceutical compound may be bonded to the carboxyl group of the polysaccharide derivative, the N-terminal amino group or C-terminal carboxyl group of the spacer, or the reactive functional group present in the amino acid constituting the spacer.

As the residue of the pharmaceutical compound, for example, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum Anti-tumor agents (cysplatine or derivatives thereof), taxol or derivatives thereof, camptothecin or derivatives thereof (antitumor agents described in Japanese Patent Application Laid-Open No. 6-87746, preferably Claim 2 (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4- as described in Ethyl-1H, 12H-benzo (de) pyrano [3 ', 4'; 6,7] indolizino [1,2-b] quinoline-10,13 Residues of anti-tumor agents such as (9H, 15H) -dione). In addition, for example, steroid-based anti-inflammatory agents such as succinic acid-hydrocortisone, succinic acid-prednisolone, or mefenamic acid, flufenamic acid, diclofenac (diclofenac) Residues of non-steroidal anti-inflammatory drugs such as ibuprofen and tinoridine are also suitable.

The residue of the pharmaceutical compound may be directly bonded to the polysaccharide derivative, or may be bonded via a spacer. As such a spacer, a spacer consisting of one amino acid or a spacer consisting of two to eight amino acids bonded to peptides can be used. More specifically, when a residue of a pharmaceutical compound and a polysaccharide derivative are bound via a spacer, the spacer is a residue of one amino acid (a residue from which one hydrogen atom and one hydroxyl group are removed from the amino and carboxyl groups of the amino acid, respectively). Or a residue of an oligopeptide of 2 to 8 amino acids bound to a peptide (meaning a residue obtained by removing one hydrogen atom and one hydroxyl group from the N-terminal amino group and the C-terminal carboxyl group, respectively).

Preferred spacers are residues of oligopeptides of 2 to 6 amino acids. The kind of amino acid which comprises a spacer is not specifically limited, For example, L- or D-amino acid, Preferably L-amino acid can be used, In addition to ( alpha )-amino acid, ( beta ) -alanine ( ε -amino) Capronic acid (aminocaproic acid), γ -amino lactic acid may be used. It is preferable that such amino acids other than the α -amino acid are arranged at positions close to the polysaccharide derivative in the spacer.

For example, the binding direction in the case of using the oligopeptide spacer is not particularly limited, but in general, the carboxyl group of the carboxyC _1-4 alkyldextran polyalcohol is bonded to the N terminal of the spacer by an acid amide bond, The C terminal of the spacer can be bonded to the amino group of the compound. Alternatively, for example, when a ridine residue is included as a structural unit of the peptide spacer, and the α -amino group and the ε -amino group of the lidin residue are each combined with a carboxyl group and an acid amide of another amino acid, the terminal end of the peptide spacer is N-terminated. Therefore, it becomes possible to combine the carboxyl group of a pharmaceutical compound. In addition, the spacer includes residues of one or two or more diamine compounds or dicarboxylic acid compounds (for example, residues of diamines such as ethylenediamine and residues of dicarboxylic acids such as succinic acid) as structural units. The sock end may have a spacer at the N end and the sock end may have a spacer at the C end.

The amino acid sequence in the case of using a spacer made of an oligopeptide is not particularly limited. For example, the residue of the dipeptide in which the spacer is represented by -XZ- (X represents a residue of a hydrophobic amino acid, and Z represents a residue of a hydrophilic amino acid. And -XZ- represents one hydrogen atom in the N-terminal amino group and C-terminal carboxyl group of the peptide to which the hydrophobic amino acid (X) and the hydrophilic amino acid (Z) are respectively N-terminal and C-terminal, respectively. Or a residue containing a residue of the dipeptide as a partial peptide sequence can be suitably used. As the hydrophobic amino acid, for example, phenylalanine, tyrosine, leucine, and the like can be used. As the hydrophilic amino acid, for example, glycine, alanine, or the like can be used. The spacer may have a repeating arrangement of such dipeptide residues (eg, -X-Z-X-Z-, -X-Z-X-Z-X-Z-, etc.).

When the spacer including such a dipeptide structure is used, the spacer is hydrolyzed at the tumor site or the inflammatory site where the spacer is considered to be rich in peptidase, and the pharmaceutical compound is liberated at a high concentration at the site. The structure formed by combining the spacer containing the dipeptide and the pharmaceutical compound is a preferred substructure of the drug complex produced by the method of the present invention. Antitumor agents that express concentration-dependent antitumor activity as residues of pharmaceutical compounds (antitumor agents that express stronger antitumor activity at higher concentrations: concentration-dependent antitumor agents such as doxorubicin, etc.) When using the residue of, it is particularly preferable to use a spacer comprising the above-described dipeptide residue represented by -XZ- or a spacer containing the dipeptide residue as a partial peptide sequence.

In addition, even when using a time-dependent anti-tumor agent which requires the duration of action time at a certain concentration or more as a residue of a pharmaceutical compound, a high anti-tumor effect may be achieved by using the spacer described above. For example, the anti-tumor agent described in Unexamined-Japanese-Patent No. 6-87746, Preferably the anti-tumor agent of Claim 2 is mentioned. In general, it is not limited to the above-mentioned spacers, but it is necessary to select a preferred spacer from the viewpoint of the mechanism of action of the anti-tumor agent, the characteristics of the body dynamics and toxic expression, and the viability of the anti-tumor agent in the body. In general, for a carcinoma having a rapid proliferation, it is preferable to select the above-mentioned spacers that can favor a high concentration of the pharmaceutical compound in a short time.

Although the specific example of a spacer is shown in the following table, the spacer used for the manufacturing method of the drug complex of this invention is not limited to the following, It can be said that those skilled in the art can select suitably to give an appropriate glass velocity of a pharmaceutical compound. Nothing. In the table, the peptide sequence has the N terminus on the left side, and the residue of the pharmaceutical compound binds to the C terminus side. D-Phe represents a D-phenylalanine residue and other amino acids represent L-amino acids. In addition, the magnitude of the free rate was determined by the degree of drug expression of the doxorubicin-coupled drug complex in rats with Walker 256 cancer or the concentration of free doxorubicin in the tumor site of rats with Walker 256 cancer. . Among these spacers, for doxorubicin, it is preferable to use a spacer capable of releasing a high concentration of pharmaceutical compound in a short time such as (N-terminal) -Gly-Gly-Phe-Gly-.

TABLE 1

As the polysaccharide derivative having a carboxyl group constituting the polysaccharide derivative portion of the drug complex, for example, any of polysaccharides or derivatives obtained by modifying them chemically or biologically and having a carboxyl group in the molecule may be used. For example, in addition to polysaccharides such as hyaluronic acid, pectic acid, alginic acid, chondroitin, heparin, pullulan, dextran, mannan, chitin A functional group having a carboxyl group is introduced to some or all hydroxyl groups of polysaccharides such as chitin, inulin, levan, xylan, araban, mannoglucan and chitosan. Can be used. For example, the thing which carboxy C _1-4 alkylated the hydroxyl group, the thing which ester-bonded the carboxyl group which is one of polybasic acids, etc. can be used suitably. Moreover, after polyalcoholizing the said polysaccharide, you may use what introduce | transduced the functional group which has a carboxyl group.

Of these polysaccharide derivatives, it is preferable to use carboxyC _1-4 alkyldextran polyalcohols. Carboxy C _1-4 alkyl polyalcohol The degree of polyalcohol include, but are not particularly limited, and under completely polyalcohol Chemistry possible conditions in the dextran polyalcohol constituting the carboxy C _1-4 alkyl polyalcohol substantially Dex It is preferable that it is dextran polyalcohol obtained by processing a tran.

The kind of dextran used for producing the carboxyC _1-4 alkyldextran polyalcohol is not particularly limited, and may include α- D-1,6-bonds at any ratio. For example, it may have α -D-1,6- combination ratio of used, and more than 85%, more than 90%, or 95% dextran. Although the molecular weight of dextran is not specifically limited, For example, about 10,000 to about 2,000,000, Preferably about 50,000 to about 800,000 can be used. Carboxy C _1-4 alkyl C _1-4 alkyl as dextran poly constituting the carboxy C _1-4 alkyl alcohols, linear or branched C _1-4 alkyl, specifically methyl groups, a lacquer, n- propyl (propyl) group, isopropyl group, n-butyl (butyl) group, sec-butyl group and the like can be used, but preferably methyl group.

According to the method of the present invention, in the preparation of the drug complex, a spacer in which the organic amine salt of the polysaccharide derivative having a carboxyl group and the pharmaceutical compound itself or the organic amine salt and the pharmaceutical compound of the polysaccharide derivative having a carboxyl group are combined with water is substantially water. It is characterized by reacting in an organic solvent which does not contain ie, a water-insoluble system. Hereinafter, a preferred embodiment of the method of the present invention, with respect to the case of using a carboxy C _1-4 alkyl polyalcohol as the polysaccharide derivative specifically described, but the scope of the present invention is not limited to this aspect.

When dextran is used as a starting material, dextran polyalcohol in which the dextran is substantially completely polyalcoholized by sequentially acting excess sodium iodide and sodium borohydride on the dextran. However, the method of polyalcoholization of dextran is not limited to the above-mentioned thing, Any method may be employ | adopted as long as it is available to a person skilled in the art. Carboxy-C _1-4 alkylated, for example, index with respect to the hydroxyl groups of the polyalcohol, acid chloride, acid bromide, acid chloride, α-, α-methyl-α-propionic acid chloride, β-propionic acid chloride, α-methyl- Halogenated C _1-4 alkylcarboxylic acids, such as β -propionic acid, α -chlorinated lactic acid, β -chloride lactic acid, and γ -chlorinated lactic acid, preferably by reacting acetic acid chloride to partially or completely carboxyC _1-4 By alkylation.

For example, dextran polyalcohol is dissolved in an inert solvent (eg, water, N, N-dimethylformamide, dimethylsulfoxide) that is not involved in the reaction, and the base (for example, For example, halogenated C _1-4 alkylcarboxylic acid or a salt thereof may be added in the presence of sodium hydroxide, potassium hydroxide, or the like, and reacted for several minutes to several days under ice-cooling to about 100 ° C. Carboxy C _1- the degree of introduction of the ₄ groups are, for example, and can be easily controlled by appropriately selecting the amount of the halogenated C _1-4 alkyl-carboxylic acid and the base used as the reaction temperature and the reagent of carboxy-C _1-4 alkylated, means, such as that is the fact known to those skilled in the art. dextran polycarboxy degree of C _1-4 alkylation for hydroxyl groups of the alcohol is not particularly limited, for example, 0.01 naejineun 2.0 per sugar moiety configuration Range, preferably from 0.1 naejineun 1.0, more preferably 0.3 naejineun in the range of 0.5. The molecular weight of the carboxy C _1-4 alkyl polyalcohol is, when measured by the gel filtration method naejineun 5,000 to 500,000, preferably about 50,000 To 450,000, more preferably 200,000 to 400,000.

The carboxyC _1-4 alkyldextran polyalcohol thus prepared is prepared as an aqueous solution in the form of an alkali metal salt such as sodium salt or potassium salt. In the method of the present invention, when a spacer bonded to a pharmaceutical compound or a residue of a pharmaceutical compound is bonded to a carboxyl group of such a polysaccharide derivative, the polysaccharide derivative in the form of an alkali metal salt is changed to use a polysaccharide derivative in the form of an organic amine salt. It is characterized by. Such a polysaccharide derivative in the form of an organic amine salt can be dissolved in a high concentration in an organic solvent substantially free of water, and the reaction can be carried out in a non-aqueous system, thereby significantly increasing the reaction efficiency.

Examples of the organic amine salts include N-methylpyrrolidine, N-methylpiperidine, and N- in addition to salts of aliphatic amines such as triethylamine, trimethylamine, and triethanolamine. Salts of alicyclic or aromatic amines such as methylmorpholine and dimethylaminopyridine, and quaternary ammonium salts such as tetramethylammonium chloride and tetraethylammonium chloride. The conversion from the sodium salt of the polysaccharide derivative to the organic amine salt can be carried out using an ion exchange resin or the like. For example, the sodium salt of carboxymethyldextran polyalcohol is dissolved in water, injected into a column filled with Bio-Rad AG50W-X2 (200-400 mesh, H ⁺ type) resin and filled with water. After eluting, organic amines, such as triethylamine, can be added and lyophilized. In addition, the sodium salt of carboxymethyldextran polyalcohol is dissolved in water, and the triethylammonium-type resin can be passed through to convert it into one step.

Coupling of the pharmaceutical compound itself and the carboxyl group of the carboxymethyldextran polyalcohol or the spacer of which the pharmaceutical compound is bound to the carboxyl group of the carboxymethyldextran polyalcohol generally refers to the reactive amino group or spacer of the pharmaceutical compound. The N-terminal amino group and the carboxyl group of the carboxymethyldextran polyalcohol may be combined with an acid amide. However, the bond between the pharmaceutical compound or the spacer and the carboxyl group of the carboxymethyldextran polyalcohol is not limited to the above, but may be a bond using another chemical bond or one or more spacers. For example, an acid anhydride may be formed by the C-terminal carboxyl group of the spacer or the carboxyl group of the pharmaceutical compound or the carboxyl group of the carboxymethyldextran polyalcohol, or each carboxyl group may be formed by using a diamine compound such as ethylenediamine as a spacer. You may couple | bond an acid amide to each amino group of.

When the N-terminal amino group of the pharmaceutical compound or the N-terminal amino group of the spacer and the carboxyl group of the carboxymethyldextran polyalcohol are bonded by an acid amide bond, a common dehydrating condensing agent used for synthesis of the peptide chain, for example, N, N, N'-dicycloalkylcarbodiimides such as N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAPC) In addition to carbodiimide derivatives, such as benzotriazole derivatives such as 1-hydroxybenzotriazole (HOBT) and the like, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxy Quinoline (EEDQ) and the like can be used. The reaction may be carried out by an active ester method, an acid halide method or the like.

The reaction is an organic solvent substantially free of water, and any reaction may be used as long as it can dissolve the reactive species (organic amine salt of carboxymethyldextran polyalcohol and a pharmaceutical compound or a spacer incorporating the pharmaceutical compound). For example, it is suitable to use N, N-dimethylformamide, dimethylsulfoxide, acetoamide, N-methylpyrrolidone, sulfolane and the like. The amount of the drug compound residue introduced into the carboxymethyldextran polyalcohol by reacting the drug compound or the spacer bound to the drug compound is not particularly limited, but the physicochemical properties of the drug compound residue and the body kinetics and efficacy of the drug complex Selection should be made in terms of toxicity, toxicity and the like. Generally, a range of about 0.1 to 30% by weight, preferably about 1 to 15% by weight can be selected. The ratio of the pharmaceutical compound residues introduced into the carboxymethyldextran polyalcohol can be easily determined, for example, by absorbance analysis.

As a preferable aspect of the manufacturing method of this invention, the method of introduce | transducing the oligopeptide which combined the pharmaceutical compound which is the anti-tumor agent of Claim 2 of Unexamined-Japanese-Patent No. 6-87746 to carboxymethyldextran polyalcohol is shown in the following schematic diagram. However, the method of the present invention is not limited to that shown in this schematic. The introduction amount of the pharmaceutical compound residue in the schematic diagram below is, for example, about 1 to 15% by weight, preferably about 4 to 8% by weight. Or, in the following schematic diagram, only the structural unit into which one or two carboxymethyl groups are introduced in the structural units of polyalcohols is exemplarily described, but the polysaccharide derivative portion of the drug complex is not constituted by repetition of the structural unit. It must be understood.

The pharmaceutical compound of the above schematic is in equilibrium with a compound (lung ring) which forms a lactone ring in an acidic aqueous medium (for example, pH 3), while in a basic aqueous medium (for example, pH 10). It is known that the equilibrium is inclined to the compound (opening ring) in which the lactone ring is ring-opened, but the drug complex in which such a pulmonary ring and a residue corresponding to the ring opening are introduced has the same anti-tumor effect. However, when the carboxymethyldextran polyalcohol and the spacer (e.g., oligopeptide spacer) in which the pharmaceutical compound is bound are reacted and the ring-opening reactive species is present in the reaction system, the carboxyl group derived from the lactone ring and the spacer-derived species are present. The condensation reaction proceeds between the amino groups, the reaction yield notably decreases, and the desired uniform drug complex may not be obtained. Such side reactions can be avoided by using cyclization as a reactive species in a non-aqueous system where equilibrium is not achieved. Therefore, the method of the present invention is particularly suitable for the preparation of drug complexes containing the above pharmaceutical compounds.

The drug complex prepared by the method of the present invention may have a desired pharmacological activity depending on the kind of residue of the pharmaceutical compound (for example, the residue of the pharmaceutical compound such as an anti-tumor agent or an anti-inflammatory agent) and the like. It can be specifically expressed locally, and can also reduce the toxicity of the pharmaceutical compound itself. For example, the polysaccharide derivative, carboxymethyldextran polyalcohol, has excellent blood retention and integration to tumor and inflammation sites as a carrier for drug delivery, and the drug complex has tumor selectivity and inflammation site selectivity. Have In addition, since it is considered that the protease (peptidase) is expressed in the tumor site or the inflammation site, the drug complex having a spacer made of oligopeptide is easily hydrolyzed into the spacer moiety, and the free pharmaceutical compound exhibits efficacy.

Pharmaceuticals containing the drug complex prepared by the method of the present invention are usually filled in vials or the like in the form of lyophilized products, for parenteral administration such as injectable or injectable preparations that can be dissolved immediately upon use. Although provided clinically as an agent, the form of such a medicament is not limited to this embodiment. For the preparation of the preparation, for example, a pharmaceutical composition prepared using additives for preparations available in the art such as dissolution aids, pH adjusting agents, stabilizers, etc. may be used. The dosage of the drug is not particularly limited, but is usually determined in consideration of the dosage of the pharmaceutical compound constituting the pharmaceutical compound residue, the amount of the residue of the pharmaceutical compound introduced into the drug complex, the condition of the patient or the type of disease, etc. You must do it. For example, when parenterally administering a drug complex in which the antitumor agent residues described in claim 2 of Japanese Patent Application Laid-Open No. 6-87746 are introduced at a rate of about 6% by weight, it is generally a day. It is preferable to administer once in a range of about 1 to 500 mg, preferably about 10 to 100 mg per 1 m 2 of surface area, and repeat every 3 to 4 weeks.

Hereinafter, although an Example demonstrates this invention more concretely, the scope of the present invention is not limited to the following Example. In Examples, "A-NH-" is a pharmaceutical compound having a lactone ring, such as the pharmaceutical compound described in claim 2 of the Japanese Patent Application Laid-open No. Hei 6-87746 (may be referred to as "DX-8951" in Examples). Denotes a drug compound residue when the drug compound closed by the lactone ring is represented by A-NH ₂ , and as one example thereof, a group represented by A-NH- in the above schematic diagram (which forms a lactone ring). In addition, A'-NH- shows that the lactone ring in the chemical compound residue represented by A-NH- is either a closed ring type or a ring-opened type, or a mixed form thereof.

In addition, in the Example, unless otherwise mentioned, the degree of carboxymethylation of the carboxymethyldextran polyalcohol (the degree of substitution of the carboxymethyl group per constituent sugar residue) is a sodium salt of the carboxymethyldextran polyalcohol free acid type. After conversion to, the solution was dissolved in 0.1 N aqueous sodium hydroxide solution and titrated with 0.1 N hydrochloric acid. An aqueous solution of the sodium salt of carboxymethyldextran polyalcohol was injected into a Bio-Rad AG50W-x 2 (H ⁺ type) column, and the passage solution was lyophilized and used as a sample. This sample was dissolved in a predetermined excess amount of 0.1N sodium hydroxide aqueous solution, and phenolphthalein was titrated with 0.1N hydrochloric acid as an indicator. The sample was taken as s (mg), the predetermined excess amount of 0.1N sodium hydroxide aqueous solution was a (ml), and the appropriate amount of 0.1N hydrochloric acid was b (ml), and the degree of carboxymethylation was 13.4 (ab) / [s- 5.8 (ab)]. In addition, the introduction amount (weight%) of a drug was calculated | required by the absorbance analysis (near 362 nm) using the absorption characteristic of a drug. In addition, gel filtration was carried out according to the following conditions (column: TSK gel G4000 PW _XL , eluent: 0.1M NaCl, flow rate: 0.8 ml / min, column temperature: 40 ℃).

Example 1: 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951)

Boc-Gly-Gly-Phe-Gly (600 mg) and N-hydroxysuccinimide (160 mg) were dissolved in N, N-dimethylformamide (20 mL) and 4 ° C. After cooling with N, N'-dicyclohexylcarbodiimide (280 mg) was added. N, N in which methane sulfonate (600 mg: the compound described in Example 50 of the publication) and triethylamine (0.16 ml) of the pharmaceutical compound according to claim 2 of Patent Publication No. Hei 6-87746 are dissolved in this solution. A dimethylformamide (30 mL) solution was added, and the mixture was reacted with stirring at room temperature under light shielding for 16 hours. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane containing 10% acetic acid: methanol = 10: 1 solution) to obtain the compound of Example 1 (1.0 g). )

¹ H-NMR (DMSO-d ₆ ) δ : 8.40 (d, 1H, J = 8.3 Hz), 8.10-8.17 (m, 2H), 7.91-8.01 (m, 1H), 7.78 (d, 1H, J = 10.75 Hz), 7.32 (s, 1H), 6.94-6.96 (m, 1H), 6.50 (s, 1H), 5.57 (t, 1H, J = 4.5 Hz), 5.43 (s, 2H), 5.23 (s, 2H), 3.77 (dd, 2H, J = 5.85 Hz, J = 8.80 Hz), 3.70 (d, 2H, J = 4.40 Hz), 3.65 (d, 2H, J = 5.35 Hz), 3.56 (d, 2H, J = 5.85 Hz), 3.15-3.25 (m, 2H), 2.40 (s, 3H), 2.05-2.25 (m, 1H), 1.86 (m, 2H), 1.35 (s, 9H), 0.88 (t, 3H , J = 7.35).

Mass (FAB); m / e 854 (M + 1)

Example 2: Synthesis of 3'-N- (Boc-Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951)

Boc-Gly-Gly-Gly-Phe (600 mg) and N-hydroxysuccinimide (160 mg) were dissolved in N, N-dimethylformamide (20 mL) and cooled to 4 ° C., followed by N , N'-dicyclohexylcarbodiimide (280 mg) was added. To this solution was added N, N-dimethylformamide (30 mL) solution of DX-8951 methanesulfonate (600 mg) and triethylamine (0.16 mL), which was then reacted under light shielding at room temperature for 16 hours. I was. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluate: dichloromethane: methanol = 10: 1 solution containing 0.5% acetic acid) to obtain the compound of Example 2 (700 mg).

¹ H-NMR (DMSO-d ₆ ) δ : 8.57 (d, 1H, J = 7.8 Hz), 8.19 (d, 1H), 8.05-8.07 (m, 2H), 7.79 (d, 1H, J = 11.2 Hz ), 7.32 (s, 1H), 7.10 (d, 2H, J = 7.8 Hz), 6.93-7.03 (m, 4H), 6.51 (s, 1H), 5.52-5.55 (m, 1H), 5.44 (s, 2H), 5.18 (d, 1H, J = 18.5 Hz), 4.84 (d, 1H, J = 18.5 Hz), 4.57-4.59 (m, 1H), 3.57-3.71 (m, 6H), 3.15-3.25 (m , 2H), 3.00-3.02 (m, 1H), 2.80-2.90 (m, 1H), 2.40 (s, 3H), 2.05-2.25 (m, 1H), 1.86 (m, 2H), 1.35 (s, 9H ), 0.88 (t, 3H, J = 7.35 Hz).

Mass (FAB); m / e 854 (M + 1)

Example 3: Synthesis of 3'-N- (Boc-Gly-Gly-Gly-Gly) -NH-A (A-NH ₂ = DX-8951)

Boc-Gly-Gly-Gly-Gly (120 mg) and N-hydroxysuccinimide (39 mg) were dissolved in N, N-dimethylformamide (20 mL) and cooled to 4 ° C., followed by N, N'-dicyclohexylcarbodiimide (70 mg) was added. N, N-dimethylformamide solution (10 ml) in which DX-8951 methanesulfonic acid salt (150 mg) and triethylamine (0.039 ml) were dissolved was added to the solution, followed by reaction for 16 hours at room temperature under light shielding. I was. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluate: dichloromethane: methanol = 10: 1 solution containing 0.5% acetic acid) to obtain the compound of Example 3 (100 mg).

¹ H-NMR (DMSO-d ₆ ) δ : 8.40 (d, 1H, J = 8.3 Hz), 8.10-8.17 (m, 2H), 7.91-8.01 (m, 1H), 7.78 (d, 1H, J = 10.75 Hz), 7.32 (s, 1H), 6.94-6.96 (m, 1H), 6.50 (s, 1H), 5.57 (t, 1H, J = 4.5 Hz), 5.43 (s, 2H), 5.23 (s, 2H), 3.77 (dd, 2H, J = 5.85 Hz, J = 8.80 hz), 3.70 (d, 2H, J = 4.40 Hz), 3.65 (d, 2H, J = 5.35 Hz), 3.56 (d, 2H, J = 5.85 Hz), 3.15-3.25 (m, 2H), 2.40 (s, 3H), 2.05-2.25 (m, 1H), 1.86 (m, 2H), 1.35 (s, 9H), 0.88 (t, 3H , J = 7.35 Hz).

Mass (FAB); m / e 764 (M + 1)

Example 4: Synthesis of 3'-N- (Gly-Gly-Gly-Gly) -NH-A (A-NH ₂ = DX-8951) trifluoroacetate

3'-N- (Boc-Gly-Gly-Gly-Gly) -NH-A (A-NH ₂ = DX-8951) (79 mg) was dissolved in trifluoroacetic acid (3 mL) and left for 1 hour. . The solvent was removed, and methanol (30 mL) was added twice to boil together, and ethanol (30 mL) was added twice to boil together. The residue was washed with ether to give the compound of Example 4 (80 mg).

¹ H-NMR (DMSO-d ₆ ) δ : 8.59-8.61 (m, 1H), 8.50 (d, 1H, J = 8.3 Hz), 8.21-8.27 (m, 2H), 7.91-8.01 (br, 3H) , 7.81 (d, 1H, J = 11.2 Hz), 7.32 (s, 1H), 6.50-6.52 (br, 1H), 5.57-5.59 (m, 1H), 5.43 (s, 2H), 5.23 (s, 2H ), 3.80-3.82 (m, 3H), 3.70-3.75 (m, 3H), 3.15-3.25 (m, 2H), 2.41 (s, 3H), 2.05-2.25 (m, 1H), 1.86-1.88 (m) , 2H), 0.88 (t, 3H, J = 7.35 Hz).

Example 5 Synthesis of Triethylammonium Salt of Carboxymethyldextran Polyalcohol

Dextran T2000 (10 g, product of Pharmacia, average molecular weight 2,000,000) was dissolved in 0.1 M acetic acid buffer solution (pH 5.5, 1,000 mL), and an aqueous solution of sodium periodate (33.0 g) (1000 mL) was added. After stirring for 10 days at 4 ° C. while blocking light, ethylene glycol (7.0 ml) was added and the mixture was stirred overnight. The pH of the reaction solution was adjusted to 7.5 using 8 M aqueous sodium hydroxide solution under ice-cooling. Sodium borohydride (14 g) was added and dissolved, followed by stirring overnight to room temperature. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid and stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 with 8 M aqueous sodium hydroxide solution under ice-cooling. The low molecular weight fraction was removed by ultrafiltration using the obtained aqueous solution of Biomax-30 membrane (Millipore Co., Ltd.). The polymer fraction was lyophilized to give dextran polyalcohol. After treating this dextran polyalcohol at pH 3.0 for 1 hour, the low molecular weight fraction was removed with a Biomax-50 membrane, the polymer fraction was removed with a Biomax-100 membrane, and lyophilized to purify dextran polyalcohol (2.0 g ) The molecular weight of this substance was 220 K (gel filtration, dextran standard).

This purified dextran polyalcohol (1.8 g) was added to an aqueous solution obtained by dissolving sodium hydroxide (10.5 g) in water (45 mL) and dissolved at room temperature. Monochloroacetic acid (15 g) was added to this solution under ice-cooling to dissolve it, and the mixture was allowed to react at room temperature for 20 hours. The reaction solution was adjusted to pH 8 with acetic acid and the low molecular weight fraction was removed by ultrafiltration using a Biomax-10 membrane. The polymer fraction was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (1.8 g). The molecular weight of this substance was 330K (gel filtration, dextran standard) and the degree of carboxymethylation was 0.8.

The sodium salt of carboxymethyldextran polyalcohol (300 mg) was dissolved in water, injected into a Bio-Rad AG50W-X2 (200-400 mesh, H ⁺ type) column (1.5 X 8.6 cm) and eluted with water. . Triethylamine (0.5 ml) was added to this eluate, and then lyophilized to obtain triethylammonium salt of carboxymethyldextran polyalcohol (380 mg). Sodium salts of carboxymethyldextran polyalcohols (300 mg each) were treated in the same column as above to obtain triethylammonium salts of carboxymethyldextran polyalcohols (380 mg, 400 mg).

Example 6 Synthesis of Sodium Salt of Carboxymethyldextran Polyalcohols

The sodium salt of carboxymethyldextran polyalcohol obtained in Example 5 (0.15 g) was added to an aqueous solution obtained by dissolving sodium hydroxide (1.05 g) in water (4.5 ml), and dissolved at room temperature. Monochloroacetic acid (1.5 g) was added to this solution under ice-cooling, and it was made to melt | dissolve, and it reacted at room temperature for 18 hours. The reaction solution was adjusted to pH 8 with acetic acid, dropped into 90 ml methanol, 3M aqueous sodium chloride solution (0.15 ml) was added, and the precipitate precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was washed with methanol, then dissolved in water (5 ml) and 3M aqueous sodium chloride solution (0.15 ml) was added. This aqueous solution was filtered through a Millipore filter (0.45 µm), the filtrate was added dropwise to 35 ml of ethanol, and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). The precipitate was washed with ethanol, dissolved in water, and dialyzed against purified water using a dialysis membrane (Spectrapore 1, ultra-molecular weight 6,000 to 8,000). The dialysis solution was filtered through a Millipore filter (0.22 μm) and then lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (0.18 g). Carboxymethylation degree (alkali titration method) of the sugar residue sugar of this substance was 1.2.

Example 7: Synthesis of Sodium Salt of Carboxymethyldextran Polyalcohols

The purified dextran polyalcohol (0.2 g) obtained in Example 5 was added to an aqueous solution obtained by dissolving sodium hydroxide (0.84 g) in water (6 mL) and dissolved at room temperature. Monochloroacetic acid (1.2 g) was added to this solution under ice-cooling to dissolve it, and the mixture was allowed to react at room temperature for 18 hours. The reaction solution was adjusted to pH 8 with acetic acid, dropped into 120 ml of methanol, and then added with 3 M aqueous sodium chloride solution (0.2 ml), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). . This precipitate was washed with methanol, then dissolved in water (5 mL) and 3M aqueous sodium chloride solution (0.2 mL) was added. This aqueous solution was filtered through a Millipore filter (0.45 µm), the filtrate was dropped into 35 ml of ethanol, and the precipitate precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was washed with ethanol, dissolved in water, and dialyzed against purified water using a dialysis membrane (Spectrapore 1, ultra-molecular weight 6,000 to 8,000). The dialysis solution was filtered through a Millipore filter (0.22 μm) and then lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (0.20 g). Carboxymethylation degree (alkali titration method) of the sugar residue sugar of this substance was 0.4.

Example 8: Carboxymethyldextran Polyalcohol-Gly-Gly-Gly-Phe-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 5 (380 mg, degree of carboxymethylation 0.8) was dissolved in N, N-dimethylformamide (30 mL). In this solution, N, N-dimethylformamide of trifluoroacetate (49 mg) of 3'-N- (Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) was added. (5 ml) A solution, triethylamine (0.017 ml), and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (380 mg) were added sequentially, while stirring to room temperature overnight. Reacted. The reaction solution was adjusted to pH 10 with 1M aqueous sodium hydroxide solution, and then dropped into 5 ml of 25 ml ethanol. To this mixture, 3M aqueous sodium chloride solution (1 ml) and diethyl ether (5 ml) were added, and the precipitate precipitate was collected by centrifugation (3500 rpm, 8 minutes).

This precipitate was dissolved in water, dialyzed against purified water using a dialysis membrane (Spetrapore 1, ultra molecular weight: 6,000 to 8,000), the dialysis solution was filtered through a Millipore filter (0.22 m), and lyophilized. The obtained crude product was dissolved in water (30 mL), adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution, and treated at 37 ° C. for 1 hour. After the treatment solution was dialyzed in the same manner as above, the dialysis solution was filtered through a Millipore filter (0.22 m) and lyophilized to give 289 mg of the compound of Example 8. The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: TSK Gel PW-4000XL manufactured by Tosoh Co., Ltd., solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and the ultraviolet ray of the compound. Absorption spectra (0.1 M Tris buffer, pH 9.0, 0.25 mg / ml) are shown in FIGS. 1 and 2, respectively. The content of the pharmaceutical compound residue of the present compound was 5.3% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M tris buffer (pH 9.0).

Example 9: Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

DX-8951)

3'-N obtained by de-Boc formation in the same manner as in Example 4 from 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) (50 mg) Trifluoroacetic acid salt of-(Gly-Gly-Phe-Gly) -NH-A was introduced into triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 5 (380 mg) according to the same method as in Example 8. Compound (300 mg) of Example 9 was synthesized. The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: TSK Gel PW-4000XL manufactured by Tosoh Co., Ltd., solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and the ultraviolet ray of the compound. Absorption spectra (0.1 M Tris buffer, pH 9.0, 0.19 mg / ml) are shown in FIGS. 3 and 4, respectively. The content of the pharmaceutical compound residue of the compound was 5.3% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 10 Carboxymethyldextran Polyalcohol-Gly-Gly-Gly-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

3'-N obtained by de-Boc formation in the same manner as in Example 4 from 3'-N- (Boc-Gly-Gly-Gly-Gly) -NH-A (A-NH ₂ = DX-8951) (41 mg) Trifluoroacetic acid salt of-(Gly-Gly-Gly-Gly) -NH-A was introduced into triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 5 (380 mg) according to the same method as in Example 8. The compound of Example 10 (190 mg) was synthesized. The ultraviolet absorption spectrum (0.1 M Tris buffer, pH 9.0, 0.34 mg / ml) of this compound is shown in FIG. The content of the pharmaceutical compound residue of the compound was 5.3% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 11: Antitumor Activity of Drug Complex of the Present Invention

1 x 10 ⁶ mouse fibrosarcoma Meth A cells were transplanted subcutaneously under the right hypodermic volume of BALB / c male mice (7 weeks old) to prepare mice with Meth A tumors (7 per group). The drug complex of Example 9 dissolved in distilled water for injection on day 7 was administered four times every four days in the tail vein of mice with Meth A tumors. At 21 days after transplantation, tumors were removed and weighed, and tumor growth inhibition rate was expressed by the following equation: tumor growth inhibition rate (%) = [1- (average tumor weight of control group / average tumor weight of control group)] X 100 Calculated. As a result, the drug complex of the present invention obtained in Example 9 did not express toxicity (weight loss), and the antitumor effect was greatly enhanced compared with the pharmaceutical compound itself (there was no spacer and the polysaccharide derivative). A pharmaceutical compound residue incorporating only the polysaccharide derivative itself (Example 5) and the spacer (H ₂ N-Gly-Gly-Phe-Gly-NH-A obtained by de-BOCation in the compound of Example 1 according to the method of Example 4) Trifluoroacetic acid of NH ₂ = DX-8951) had no effect.

TABLE 2

Example 12 Antitumor Activity of Drug Complexes of the Present Invention

Mice with Meth A tumors were prepared (6 per group) by the same method as in Example 11, and antitumor effects were compared when the drug complexes of Examples 8 and 9 were administered once on day 7. As a result, the degree of antitumor activity was (polysaccharide derivative) -Gly-Gly-Phe-Gly-NH-A '> (polysaccharide derivative) -Gly-Gly-Gly-Phe-NH-A'> pharmaceutical compound itself. The compound in which the pharmaceutical compound residue was directly bonded to the carboxyl group of the carboxymethyldextran polyalcohol of Example 5 without introducing a spacer (introduction amount of the pharmaceutical compound residue: 6.2% by weight) was ineffective.

TABLE 3

Example 13: Synthesis of Triethylammonium Salt of Carboxymethyldextran Polyalcohol

Dextran T500 (10 g, manufactured by Pharmacia, molecular weight 500 K) was dissolved in 0.1 acetic acid buffer solution (pH 5.5, 1000 mL), and an aqueous solution of sodium periodate (33 g) (1000 mL) was added. After stirring for 10 days at 4 ° C while blocking light, ethylene glycol (7.0 ml) was added, and the mixture was stirred overnight. The reaction solution was adjusted to pH 7.5 using 8 M aqueous sodium hydroxide solution. Sodium borohydride (14 g) was added and dissolved, followed by stirring overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid, stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 with 8 M aqueous sodium hydroxide solution to obtain solution 1. Separately, the above series of operations were performed on dextran T500 (10 g, manufactured by Pharmacia, molecular weight 500 K) to obtain solution 2. In addition, the said series of operation was performed about dextran T250 (each 10g, a Pharmacia company, molecular weight 250K), and the solution 3 and the solution 4 were obtained. These solutions 1-4 were combined and the low molecular weight fraction was removed by the ultrafiltration method using a Biomax-50 membrane. The polymer fraction was lyophilized to give dextran polyalcohol (25 g). The molecular weight (gel filtration, pullulan standard) of this substance was 163K.

This dextran polyalcohol (11 g) was added to the aqueous solution obtained by dissolving sodium hydroxide (46.2 g) in water (330 ml), and it melt | dissolved at room temperature. Monochloroacetic acid (66 g) was added to this solution under ice-cooling to dissolve it, and then reacted overnight at room temperature. The reaction solution was adjusted to pH 9 with acetic acid and then desalted by ultrafiltration using a Biomax-30 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (13 g). The molecular weight of this substance was 228K (gel filtration, pullulan standard), and the degree of carboxymethylation was 0.4.

The sodium salt of carboxymethyldextran polyalcohol (600 mg) was dissolved in water, and injected into a Bod-Rad AG 50W-X2 (200 to 400 mesh, H ⁺ type) column (44 mm in diameter and 210 mm in length). , Eluted with water. Triethylamine (0.93 ml) was added to the eluate, and then lyophilized to obtain the compound of Example 13 (690 mg).

Example 14 Synthesis of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ² = DX-8951) trifluoroacetate

3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ² = DX-8951) (79 mg) obtained in Example 1 was dissolved in trifluoroacetic acid (3 mL), It was left for 1 hour. The solvent was removed, the mixture was boiled twice with methanol (30 ml) and the mixture was boiled twice with ethanol (30 ml), and then the residue was washed with ether to give the compound of Example 14 (80 mg). )

¹ H-NMR (DMSO-d ₆ ) δ : 8.53 (d, 1H, J = 8.3 Hz), 8.40-8.48 (m, 2H), 8.28 (d, 1H, J = 8.3 Hz), 7.95-8.07 (br , 3H), 7.81 (d, 1H, J = 10.2 Hz), 7.30-7.37 (m, 2H), 7.15-7.30 (m, 5H), 6.50-6.55 (br, 1H), 5.50-5.57 (m, 1H ), 5.41 (d, 2H, J = 7.82 Hz), 5.25 (s, 2H), 4.55-4.62 (m, 1H), 3.55-3.92 (m, 6H), 3.15-3.25 (br, 2H), 2.98- 3.03 (m, 1H), 2.73-2.82 (m, 1H), 2.40 (s, 3H), 2.05-2.25 (m, 1H), 1.84-1.92 (m, 2H), 0.88 (t, 3H, J = 7.35 Hz).

Example 15 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-9851)

The sodium salt of carboxymethyldextran polyalcohol obtained in Example 13 (400 mg) was converted into triethylammonium salt (470 mg) and dissolved in N, N-dimethylformamide (30 mL). N, N-di of trifluoroacetic acid salt (62 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) obtained in Example 14 was added to this solution. Methylformamide (5 mL) solution, triethylamine (0.02 mL), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (470 mg) were added sequentially to give light. Blocked and reacted with stirring overnight to room temperature. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3M aqueous sodium chloride solution (2.5 ml) and diethether (20 ml), and the precipitated precipitate was collected by centrifugation. The precipitate was dissolved in 0.5 M aqueous sodium chloride solution, adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling, and dialyzed against purified water using a dialysis membrane (Spectrapore 1, ultra-molecular weight 6000 to 8000). The dialysis solution was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound of Example 15 (600 mg). The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Tosoh TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and ultraviolet absorption of the compound. Spectra (0.1 M Tris buffer, pH 9.0, 0.1 mg / ml) are shown in FIGS. 6 and 7, respectively. The content of the pharmaceutical compound residue of the compound was 5.8% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 16: 3'-N- (Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) trifluoroacetate

Synthesis of

3'-N- (Boc-Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) (79 mg) obtained in Example 2 was dissolved in trifluoroacetic acid (3 mL), It was left for 1 hour. The solvent was removed, two times of boiling with methanol (30 mL) and ethanol (30 mL) was added, and then the residue was washed with ether, and the compound of Example 16 (80 mg) was washed with ether. Got.

¹ H-NMR (DMSO-d ₆ ) δ : 8.62-8.66 (m, 2H), 8.23 (d, 1H, J = 8.3 Hz), 8.18-8.20 (m, 1H), 7.98-8.10 (br, 2H) , 7.79 (d, 1H, J = 10.7 Hz), 7.32 (s, 1H), 7.09 (d, 2H, J = 7.3 Hz), 6.93-7.03 (m, 4H), 6.50-6.60 (br, 1H), 5.52-5.55 (m, 1H), 5.44 (s, 2H), 5.18 (d, 1H, J = 18.5 Hz), 4.80 (d, 1H, J = 18.5 Hz), 4.57-4.59 (m, 1H), 3.57 -3.71 (m, 6H), 3.15-3.25 (m, 2H), 3.00-3.02 (m, 1H), 2.80-2.90 (m, 1H), 2.50 (s, 3H), 2.05-2.25 (m, 1H) , 1.86-2.00 (m, 2H), 0.88 (t, 3H, J = 7.35 Hz).

Example 17 Carboxymethyldextran Polyalcohol-Gly-Gly-Gly-Phe-NH-A '(A-NH ₂

Synthesis of = DX-9851)

The sodium salt of carboxymethyldextran polyalcohol obtained in Example 13 (1.0 g) was converted to triethylammonium salt (1.2 g) and dissolved in N, N-dimethylformamide (90 mL). N, N-di of trifluoroacetic acid salt (158 mg) of 3'-N- (Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) obtained in Example 16 was added to this solution. Methylformamide (15 mL) solution, triethylimine (0.05 mL), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (1.2 g) were added sequentially to give light. Blocked and reacted with stirring overnight to room temperature. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3M aqueous sodium chloride solution (2.5 ml) and diethether (20 ml), and the precipitated precipitate was collected by centrifugation. The precipitate was dissolved in 0.5 M aqueous sodium chloride solution, adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling, and dialyzed against purified water using a dialysis membrane (Spectrapore 1, ultra-molecular weight 6000 to 8000). The dialysate solution was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound of Example 17 (1.4 g). The content of the pharmaceutical compound residue of the present compound was 5.2% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 18 Synthesis of Boc-Gly-Phe-Leu-OH

H-Gly-Phe-Leu-OH (3.0 g) was added to 50% aqueous dioxane solution (48 mL) and ice-cooled. A dioxane (24 mL) solution containing 1N aqueous sodium hydroxide solution (9.45 mL) and (Boc) ₂ O (2.27 g) was added to the solution, followed by stirring overnight. 1N hydrochloric acid (9.45 mL) was added to the reaction solution to remove the solvent. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 5: 1 solution) to obtain the compound (2.5 g) of Example 18.

Example 19 Synthesis of Boc-Gly-Phe-Leu-Gly-OBzl

Boc-Gly-Phe-Leu-OH (2.4 g) and N-hydroxysuccinimide (656 mg) obtained in Example 18 were dissolved in N, N-dimethylformamide (50 mL) and cooled at 4 ° C. After that, N, N-dicyclohexylcarbodiimide (1.17 g) was added and stirred for 2 hours. To this solution was added a solution of N, N-dimethylformamide (40 ml) in which H-Gly-OBzl tosylate (1.9 g) and triethylamine (0.79 ml) were dissolved, followed by stirring at room temperature for 16 hours. . The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 50: 1 solution) to obtain the compound (2.0 g) of Example 19.

¹ H-NMR (DMSO-d ₆ ) δ : 8.20-8.30 (m, 1H), 8.12 (d, 1H, J = 8.3 Hz), 7.83 (d, 1H, J = 8.3 Hz), 7.32-7.37 (m , 5H), 6.89-6.95 (m, 1H), 5.12 (s, 1H), 4.52-4.59 (br, 1H), 4.34 (dd, 1H, J = 7.3 Hz, J = 15.1 Hz), 3.93 (dd, 1H, J = 5.5 Hz, J = 17.2 Hz, 3.84 (dd, 1H, J = 5.5 Hz, J = 17.2 Hz), 3.54 (dd, 1H, J = 5.9 Hz, J = 16.7 Hz), 3.42 (dd , J = 5.9 Hz, J = 16.7 Hz, 3.00 (dd, 1H, J = 4.4 Hz, 13.7 Hz), 2.78 (dd, 1H, J = 8.8 Hz, J = 13.2 Hz), 1.50-1.65 (m, 1H), 1.45 (t, 2H, J = 7.3 Hz), 1.36 (s, 9H), 0.86 (d, 3H, J = 6.4 Hz), 0.82 (d, 3H, J = 6.4 Hz).

Example 20 Synthesis of Boc-Gly-Phe-Leu-Gly-OH

Boc-Gly-Phe-Leu-OBzl (1.7 g) obtained in Example 19 was dissolved in a mixed solution of ethyl acetate (30 mL) and methanol (30 mL), and then 5% Pd-C (1.5 g) was added thereto. , Contact reduction was performed. The reaction solution was filtered, and the filtrate was dried under reduced pressure to obtain the compound (1.15 g) of Example 20.

Example 21 Synthesis of 3′-N- (Boc-Gly-Phe-Leu-Gly) -NH-A (A-NH ₂ = DX-8951)

Boc-Gly-Phe-Leu-Gly-OH (200 mg) and N-hydroxysuccinimide (58 mg) obtained in Example 20 were dissolved in N, N-dimethylformamide (5 mL) and 4 ° C. After cooling with N, N-dicyclohexylcarbodiimide (104 mg) was added and dissolved. To this solution was added N, N-dimethylformamide (5 mL) solution of DX-8951 methanesulfonate (224 mg) and triethylamine (0.059 mL). Reacted. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 10: 1 solution containing 0.5% acetic acid) to obtain the compound of Example 21 (200 mg).

¹ H-NMR (DMSO-d ₆ ) δ : 8.35 (d, 1H, J = 7.8 Hz), 8.08-8.18 (m, 2H), 7.75-7.85 (m, 2H), 7.32 (s, 1H), 7.10 (d, 2H, J = 6.8 Hz), 7.08-7.13 (m, 3H), 6.85-6.95 (br, 1H), 6.40-6.65 (br, 1H), 5.52- 5.55 (m, 1H), 5.46 ( d, 1H, J = 18.5 Hz, 5.37 (d, 1H, J = 18.5 Hz), 5.24 (s, 2H), 4.44-4.52 (m, 1H), 4.15-4.25 (m, 1H), 3.68-3.72 (m, 2H), 3.40-3.52 (m, 2H), 3.15-3.25 (br, 2H), 2.85-2.95 (m, 1H), 2.65-2.75 (m, 1H), 2.40 (s, 3H), 2.05 -2.25 (m, 1H), 1.80-1.91 (m, 2H), 1.50-1.65 (m, 1H), 1.45 (t, 2H, J = 7.3 Hz), 1.35 (s, 9H), 0.88 (t, 3H , J = 7.4 Hz), 0.86 (d, 3H, J = 6.4 Hz), 0.82 (d, 3H, J = 6.4 Hz).

Example 22: 3'-N- (Gly-Phe-Leu-Gly) -NH-A (A-NH ₂ = DX-8951) trifluoroacetate

Synthesis of

3'-N- (Boc-Gly-Phe-Leu-Gly) -NH-A (A-NH ₂ = DX-8951) (97 mg) obtained in Example 21 was dissolved in trifluoroacetic acid (3 mL), It was left for 1 hour. The solvent was removed, two times of boiling together with methanol (30 mL) and two times of boiling together with ethanol (30 mL), and the residue was washed with ether to give the compound of Example 22 (95 mg). )

¹ H-NMR (DMSO-d ₆ ) δ : 8.57 (d, 1H, J = 8.3 Hz), 8.47 (d, 1H, J = 8.3 Hz), 8.32 (d, 1H, J = 7.8 Hz), 8.17 ( t, 1H, J = 5.5 Hz), 7.81-7.91 (br, 3H), 7.79 (d, 1H, J = 10.7 Hz), 7.32 (s, 1H), 7.21-7.23 (m, 5H), 7.12-7.17 (m, 1H), 6.45-6.55 (br, 1H), 5.57 (q, 1H, J = 4.4 Hz), 5.43 (d, 1H, J = 16.1 Hz), 5.34 (d, 1H, J = 16.1 Hz) , 5.23 (s, 2H), 4.67 (dt, 1H, J = 4.0 Hz, J = 9.0 Hz), 4.31 (dd, 1H, J = 8.5 Hz, J = 15.0 Hz), 4.0-4.4 (br, 1H) , 3.74-3.76 (m, 2H), 3.56 (dd, 1H, J = 6.0 Hz J = 16.0 Hz), 3.41 (dd, 1H, J = 6.0 Hz, J = 16.0 Hz), 3.17-3.19 (br, 2H ), 3.02 (dd, 1H, J = 4.0 Hz, J = 14.0 Hz), 2.70 (dd, 1H, J = 10.0 Hz, J = 14.0 Hz), 2.40 (s, 3H), 2.05-2.15 (m, 1H ), 1.85 (dt, 2H, J = 7.0 Hz, J = 14.0 Hz), 1.50-1.55 (m, 1H), 1.45 (t, 2H, J = 6.0 Hz), 1.35 (s, 9H), 0.88 (t , 3H, J = 7.4), 0.85 (d, 3H, J = 6.4 Hz), 0.80 (d, 3H, J = 6.4 Hz).

Example 23 Carboxymethyldextran Polyalcohol-Gly-Phe-Leu-Gly-NH-A '(A-NH ₂

Synthesis of = DX-9851)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 13 (690 mg) was dissolved in N, N-dimethylformamide (50 mL). N, N-di of trifluoroacetate (95 mg) of 3'-N- (Gly-Phe-Leu-Gly) -NH-A (A-NH ₂ = DX-8951) obtained in Example 22 was added to this solution. Methylformamide (10 mL) solution, triethamine (0.03 mL), and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (690 mg) were added sequentially to room temperature. The reaction was stirred overnight. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3M aqueous sodium chloride solution (2.5 ml) and diethether (20 ml), and the precipitated precipitate was collected by centrifugation. This precipitate was dissolved in 0.5 M brine, adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling, and dialyzed against purified water using a dialysis membrane (Spetrapore 1, ultra-molecular weight 6000 to 8000). After the dialysis solution was filtered through a Millipore filter (0.22 m), the filtrate was lyophilized to obtain the compound of Example 23 (600 mg). The content of the pharmaceutical compound residue of the compound was 4.8% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 24: Synthesis of triethylammonium salt of carboxymethyldextran polyalcohol

Dextran T500 (50 g, Pharmacia, molecular weight 500K) was dissolved in 0.1 M acetic acid buffer (pH 5.5, 5000 mL), and an aqueous solution of sodium periodate (165.0 g) (5000 mL) was added. After stirring for 10 days at 4 ° C. while blocking light, ethylene glycol (35.0 ml) was added, and the mixture was stirred overnight. The reaction solution was adjusted to pH 7.5 using 8 M aqueous sodium hydroxide solution. Sodium borohydride (70 g) was added and dissolved, followed by stirring overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid, stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 using an aqueous 8 M sodium hydroxide solution. The obtained aqueous solution was removed by ultrafiltration using a Biomax-50 membrane. The polymer fraction was lyophilized to give dextran polyalcohol (27.1 g). The molecular weight of this substance was 140 K (gel filtration, pullulan standard).

This dextran polyalcohol (5 g) was added to an aqueous solution obtained by dissolving sodium hydroxide (21 g) in water (150 mL) and dissolved at room temperature. Monochloroacetic acid (30 g) was added and dissolved in this solution under ice-cooling, and then reacted overnight at room temperature. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (5.6 g). The molecular weight of this substance was 263 K (gel filtration, pullulan standard) and the degree of carboxymethylation was 0.4.

The sodium salt of carboxymethyldextran polyalcohol (2.0 g) was dissolved in water, and injected into a Bio-Rad AG 50W-X2 (200 to 400 mesh, H ⁺ type) column (44 mm in diameter and 210 mm in length). , Eluted with water. Triethylamine (4 ml) was added to this eluate, and then lyophilized to obtain the compound (2.2 g) in Example 24.

Example 25 Synthesis of Trimethylammonium Salt of Carboxymethyldextran Polyalcohols

The sodium salt of carboxymethyldextran polyalcohol obtained in Example 24 (1.0 g) was dissolved in water, poured into a Bio-Rad AG 50W-X2 (200-400 mesh, Me ₃ NH ⁺ type) column, and eluted with water. . The eluate was lyophilized to obtain the compound of Example 25 (950 mg).

Example 26 Synthesis of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) Hydrochloride

3'-N- (Gly-) obtained from 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) (400 mg) in the same manner as in Example 14. Gly-Phe-Gly) -NH-A trifluoroacetic acid was dissolved in water-MeOH (1: 4) and a Bio-Rad AG 1-X8 (200-400 mesh, Cl ^- type) column (1.5 cm X 8.6) cm) and eluted with the solvent. The eluate was concentrated and then lyophilized to obtain the compound of Example 26 (310 mg).

¹ H-NMR (DMSO-d ₆ ) δ : 8.53 (d, 1H, J = 8.5 Hz), 8.46-8.48 (m, 1H), 8.37-8.39 (m, 1H), 7.95 (d, 1H, J = 8.0 Hz), 7.80 (s, 3H), 7.78 (d, 1H, J = 11.1 Hz), 7.34 (s, 1H), 7.14-7.24 (m, 5H), 6.50 (s, 1H), 5.56-5.60 ( m, 1H), 5.35-5.40 (m, 2H), 5.24 (s, 2H), 4.51-4.56 (m, 1H), 3.86 (dd, J = 4.8, 13.5 Hz, 1H), 3.68-3.79 (m, 3H), 3.54 (s, 2H), 3.15-3.22 (m, 2H), 3.01 (dd, J = 5.6, 13.5 Hz, 1H), 2.78 (dd, J = 9.6, 3.5 Hz, 1H), 2.41 (s , 3H), 2.12-2.23 (m, 2H), 1.81-1.89 (m, 2H), 0.88 (t, 3H, J = 7.2 Hz).

Mass (FAB); m / e 753 (M + 1)

Example 27: Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-9851)

Trimethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 25 (0.1 g) was dissolved in N, N-dimethylformamide (6 mL). To this solution, N, N-dimethylform of hydrochloride (24 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) obtained in Example 26 was obtained. Amide (10 mL) solution, triethylamine (5 μL) and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (0.1 g) were added sequentially and stirred to room temperature overnight. Reacted. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3M aqueous sodium chloride solution (2.5 ml) and diethether (20 ml), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-30 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound of Example 27 (90 mg). The content of the pharmaceutical compound residue of the compound was 11% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 28 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Trimethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 25 (0.1 g) was dissolved in N, N-dimethylformamide (6 mL). In this solution, N, N-dimethylform of hydrochloride (36 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) obtained in Example 26 was obtained. Amide (10 mL) solution, triethylamine (8 μL), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (0.1 g) were added sequentially, and stirred at room temperature overnight. Reacted. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3M aqueous sodium chloride solution (2.5 ml) and diethether (20 ml), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 12 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-30 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 µm) and then lyophilized to obtain the compound of Example 28 (80 mg). The present compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Tosoh TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and ultraviolet absorption spectrum of the compound. (0.1 M Tris buffer, pH 9.0, 36 μg / ml) are shown in FIGS. 8 and 9, respectively. The content of the pharmaceutical compound residue of the present compound was 15% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 29: Carboxymethyldextran Polyalcohol-Gly-Gly-Gly-Phe-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Dextran T250 (20 g, manufactured by EXTRASYNTHESE, average molecular weight 250 K) was dissolved in 0.1 M acetic acid buffer (pH 5.5, 2000 mL) and an aqueous solution of sodium periodate (66.0 g) (2000 mL) was added. After stirring for 10 days at 4 ° C. while blocking light, ethylene glycol (14.0 ml) was added, followed by stirring overnight. The pH of the reaction solution was adjusted to 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. Sodium borohydride (28 g) was added to dissolve and stirred overnight at room temperature. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid and stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 with 8 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was removed by ultrafiltration using a Biomax-30 membrane to obtain Residual Solution 1 that did not pass through the membrane. Separately, dextran T250 (50 g, manufactured by EXTRASYNTHESE, average molecular weight 250 K) was dissolved in 0.1 M acetic acid buffer (pH 5.5, 5000 mL), and an aqueous solution of sodium periodate (165 g) (5000 mL) was added. It was. After stirring for 10 days at 4 ° C. while blocking light, ethylene glycol (35.0 ml) was added, and the mixture was stirred overnight. The pH of the reaction solution was adjusted to 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. Sodium borohydride (70 g) was added and dissolved, followed by stirring at room temperature overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid and stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 with 8 M aqueous sodium hydroxide solution under ice-cooling. The low molecular weight fraction was removed by the ultrafiltration method using the Biomax-30 membrane, and the residual solution 2 which did not pass through the membrane was obtained. The remaining solution 1 and the remaining solution 2 were combined, and the fractions passing through the Biomax-50 membrane by ultrafiltration were removed using the Biomax-30 membrane, and then freeze-dried to obtain dextran polyalcohol (25.7 g). The molecular weight of this substance was 47K (gel filtration, pullulan standard).

This dextran polyalcohol (5 g) was added to an aqueous solution obtained by dissolving sodium hydroxide (35 g) in water (150 mL) and dissolved at room temperature. Monochloroacetic acid (50 g) was added and dissolved in this solution under ice-cooling, followed by reaction at room temperature for 18 hours. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (7.2 g). The molecular weight of this substance was 127 K (gel filtration, pullulan standard) and the degree of carboxymethylation was 0.8. Sodium salt of this carboxymethyldextran polyalcohol (2.2 g) was dissolved in water and injected into a Bio-Rad AG 50W-X2 (200 to 400 mesh, H ⁺ type) column (44 mm in diameter and 210 mm in length). , Eluted with water. Triethylamine (4 ml) was added to this eluate, and then lyophilized to obtain triethylammonium salt of carboxymethyldextran polyalcohol (2.69 g).

Triethylammonium salt (2.67 g) of this carboxymethyldextran polyalcohol was dissolved in N, N-dimethylformamide (200 mL). In this solution, 3'-N- (Boc-Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) (350 mg) synthesized in the same manner as in Example 2 was used. Trifluoroacetate and triethylamine (0.116 mL) of 3'-N- (Gly-Gly-Gly-Phe) -NH-A obtained by debocation in the same manner were added to N, N-dimethylformamide ( 10 ml), a solution obtained by dissolving 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (2.67 g) in N, N-dimethylformamide (10 ml) was obtained. It was added sequentially and reacted with stirring overnight at room temperature. A 3 M aqueous sodium chloride solution (100 ml) was added to the reaction solution, and 8 ml each was dropped into 30 ml of ethanol. To each were added 3 M aqueous sodium chloride solution (1 ml) and diethether (5 ml), and the precipitates were collected by centrifugation (3500 rpm, 8 minutes). The precipitate was washed with acetone, dissolved in water, and added with 3M aqueous sodium chloride solution (10 ml), adjusted to pH 9 with 0.1M aqueous sodium hydroxide solution, and treated at 37 ° C for 1 hour. This treated solution was desalted by ultrafiltration using a Biomax-10 membrane. The remaining solution that did not pass through the membrane was filtered through a Millipore filter (0.22 µm) and then lyophilized to obtain the compound of Example 29 (2.30 g). The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Toso TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and the ultraviolet absorption spectrum of the compound (0.1 M Tris buffer, pH 9.0 and 0.20 mg / ml, are shown in Figs. 10 and 11. The amount of the drug compound residue of the compound was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0). Bar, 5.8% (w / w).

Example 30: Synthesis of triethylammonium salt of carboxymethyldextran polyalcohol

An aqueous solution of sodium periodate (66.0 g) (2000 ml) was added to a solution of 0.1 M acetic acid buffer (pH 5.5) of dextran T10 (20 g, Pharmacia, average molecular weight 10K). After stirring for 10 days at 4 ° C while blocking light, ethylene glycol (14.0 ml) was added, and the mixture was stirred overnight. The pH of the reaction solution was adjusted to 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. Sodium borohydride (28 g) was added and dissolved, followed by stirring overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid, stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 with 8 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was removed by ultrafiltration using a Biomax-5 membrane (Millipoa Corporation), and the remaining solution that had not passed through the membrane was passed through the Biomax-30 membrane. The obtained flow liquid was lyophilized to obtain dextran polyalcohol (8.0 g). The molecular weight of this substance was 13K (gel filtration, pullulan standard).

This dextran polyalcohol (3.7 g) was added to the aqueous solution obtained by dissolving sodium hydroxide (25.9 g) in water (111 mL), and it melt | dissolved at room temperature. Monochloroacetic acid (37 g) was added to this solution under ice cooling, and it was made to melt | dissolve, and it was made to react at room temperature for 20 hours. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-5 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (6.2 g). The molecular weight of this substance was 37K (gel filtration, pullulan standard) and the degree of carboxymethylation was 0.9.

This sodium salt of carboxymethyldextran polyalcohol (6.0 g) was dissolved in water, poured into a Bio-Rad AG 50W-X2 (200-400 mesh, H ⁺ type) column, and eluted with water. Triethylamine (9.3 ml) was added to this eluate, and then lyophilized to obtain the compound (7.2 g) of Example 30.

Example 31 Synthesis of Triethylammonium Salt of Carboxymethyldextran Polyalcohol

Dextran polyalcohol (3.9 g) obtained in Example 30 was added to an aqueous solution obtained by dissolving sodium hydroxide (16.3 g) in water (117 mL) and dissolved at room temperature. Monochloroacetic acid (23.4 g) was added and dissolved in this solution under ice-cooling, followed by reaction at room temperature for 18 hours. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-5 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (5.0 g). The molecular weight of this substance was 28K (gel filtration, pullulan standard) and the degree of carboxymethylation was 0.5. The sodium salt of this carboxymethyldextran polyalcohol (4.8 g) was converted into the triethylammonium salt in the same manner as in Example 30 to obtain the compound (5.6 g) in Example 31.

Example 32: Synthesis of triethylammonium salt of carboxymethyldextran polyalcohol

To an aqueous solution of sodium periodate (66.0 g) (2000 ml) was added to a solution of 0.1 M acetic acid buffer (pH 5.5) of dextran 4 (20 g, manufactured by Funakoshi Co., Ltd., having an average molecular weight of 4K to 6K). It was. After stirring for 10 days at 4 ° C. while blocking light, ethylene glycol (14.0 ml) was added, followed by stirring overnight. The pH of the reaction solution was adjusted to 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. Sodium borohydride (28 g) was added and dissolved, followed by stirring at room temperature overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid and stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. The low molecular weight fraction was removed with the obtained aqueous solution by the ultrafiltration method using the Biomax-3 membrane (made by Millipore). The pass-through liquid obtained was lyophilized to obtain dextran polyalcohol (6.0 g). The molecular weight of this substance was 9K (gel filtration, pullulan standard). This dextran polyalcohol (2.7 g) was added to the aqueous solution obtained by dissolving sodium hydroxide (18.9 g) in water (81 mL), and it melt | dissolved at room temperature. Monochloroacetic acid (27 g) was added and dissolved in this solution under ice-cooling, and then reacted at room temperature for 20 hours. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-5 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (4.2 g). The molecular weight of this substance was 20K (gel filtration, pullulan standard), and the degree of carboxymethylation was 0.9.

The sodium salt of this carboxymethyldextran polyalcohol (4.0 g) was converted into the triethylammonium salt in the same manner as in Example 30 to obtain the compound (4.8 g) in Example 32.

Example 33: Synthesis of triethylammonium salt of carboxymethyldextran polyalcohol

Dextran polyalcohol (2.7 g) obtained in Example 32 was added to an aqueous solution obtained by dissolving sodium hydroxide (11.3 g) in water (81 mL) and dissolved at room temperature. Monochloroacetic acid (16.2g) was added to this solution, and it melt | dissolved under ice cooling, and it was made to react at room temperature for 18 hours. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-5 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (2.7 g). The molecular weight (gel filtration, pullulan standard) of this substance was 16K, and the degree of carboxymethylation was 0.5. The sodium salt of this carboxymethyldextran polyalcohol (2.7 g) was converted into the triethylammonium salt in the same manner as in Example 30 to obtain the compound (3.1 g) of Example 33.

Example 34 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 30 (1.5 g) was dissolved in N, N-dimethylformamide (90 mL). Triethylamine (0.07 ml) and trifluoroacetic acid (210 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) were added to the solution. N, N-dimethylformamide (40 mL) solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (1.5 g) were added sequentially, and the reaction was stirred at room temperature overnight. I was. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To each was added 3 M aqueous sodium chloride solution (2.5 mL) and diethyl ether (20 mL), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-3 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound (1.3 g) of Example 34. The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Toso TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and the ultraviolet absorption spectrum of the compound (0.1 M Tris buffer, pH 9.0, 65 μg / ml) are shown in FIGS. 12 and 13, respectively. The content of the pharmaceutical compound residue of the compound was 6.4% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 35 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt (1.2 g) of carboxymethyldextran polyalcohol obtained in Example 31 was dissolved in N, N-dimethylformamide (90 mL). Triethylamine (0.056 ml) and trifluoroacetic acid (168 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) were added to the solution. N, N-dimethylformamide (30 mL) solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (1.2 g) were added sequentially and reacted with stirring overnight at room temperature. . 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To each was added 3 M aqueous sodium chloride solution (2.5 mL) and diethyl ether (20 mL), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-3 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound (1.0 g) of Example 35. The content of the pharmaceutical compound residue of the compound was 4.8% (w / w) when the amount was determined based on the absorbance at 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 36 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt (1.2 g) of carboxymethyldextran polyalcohol obtained in Example 32 was dissolved in N, N-dimethylformamide (90 mL). Triethylamine (0.056 mL) and trifluoroacetate (168 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) were added to this solution. N, N-dimethylformamide (30 mL) solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (1.2 g) were added sequentially and reacted with stirring overnight at room temperature. . 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To each was added 3 M aqueous sodium chloride solution (2.5 mL) and diethyl ether (20 mL), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-3 membrane. The remaining solution that did not pass through the membrane was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound (1.0 g) of Example 36. The content of the pharmaceutical compound residue of the compound was 5.9% (w / w) when the amount was determined based on the absorbance at 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 37 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 33 (1.5 g) was dissolved in N, N-dimethylformamide (90 mL). Triethylamine (0.07 ml) and trifluoroacetic acid (210 mg) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) were added to the solution. N, N-dimethylformamide (40 mL) solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (1.5 g) were added sequentially and reacted with stirring overnight at room temperature. . 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To each was added 3 M aqueous sodium chloride solution (2.5 mL) and diethyl ether (20 mL), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-3 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound (1.3 g) of Example 37. The content of the pharmaceutical compound residue of the compound was 4.6% (w / w) when the amount was determined based on the absorbance at 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 38: Synthesis of Boc-Gly-Gly-Phe-Gly-NH-A (A-NH ₂ = DW-8286)

Boc-Gly-Gly-Phe-Gly (42 mg) and N-hydroxysuccinimide (12 mg) were dissolved in N, N-dimethylformamide (2 mL) and cooled to 4 ° C., followed by N , N-dichlorohexylcarbodiimide (22 mg) was added. Compound [(1s, 9s) -1-amino-5-chloro-9-ethyl-2,3-dihydro-9-hydroxy-1H, 12H-benzo [de] pyrano, represented by the following formula in this solution: Hydrochloride (50 mg) and triethylamine of [3 ', 4': 6,7] indolizino [1,2-b] quinoline-10,13 (9H, 15H) -dione: DW-8286] 0.01 ml), dissolved N, N-dimethylformamide (6 ml) solution was added and reacted with stirring for 16 hours at room temperature, avoiding light:

The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 10: 1 solution containing 0.5% acetic acid) to obtain the compound of Example 38 (27 mg).

¹ H-NMR (CDC1 ₃ ) δ : 8.10-8.20 (br, 1H), 7.95-8.05 (br, 1H), 7.70-7.80 (br, 2H), 7.50-7.60 (br, 1H), 7.40-7.50 ( br, 1H), 7.10-7.25 (m, 5H), 7.05-7.15 (br, 1H), 5.85-5.95 (br, 1H), 5.50-5.60 (br, 1H), 5.40-5.50 (m, 1H), 5.25-5.35 (m, 1H), 5.05-5.15 (m, 1H), 4.90-5.00 (m, 1H), 4.70-4.80 (br, 1H), 4.10-4.25 (br, 2H), 3.60-3.90 (m , 4H), 3.10-3.40 (m, 3H), 2.95-3.05 (br, 1H), 2.15-2.30 (br, 1H), 1.75-1.90 (br, 2H), 1.39 (s, 9H), 0.80-1.00 (m, 3 H).

Example 39 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of DX-8286)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 24 (175 mg) was dissolved in N, N-dimethylformamide (20 mL). To this solution was removed from the 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DW-8286) (27 mg) obtained in Example 38 in the same manner as in Example 4. N, N-dimethylformamide of 3'-N- (Gly-Gly-Phe-Gly) -NH-A trifluoroacetate (29 mg) and triethylamine (9 µl) obtained by bocation ( 5 ml) solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (175 g) were added sequentially and reacted with stirring overnight at room temperature. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To each was added 3 M aqueous sodium chloride solution (2.5 mL) and diethyl ether (20 mL), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-30 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 m) and then lyophilized to obtain the compound of Example 39 (135 mg). The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Toso TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and the ultraviolet absorption spectrum of the compound (0.1 M Tris buffer, pH 9.0, 99 μg / ml) are shown in FIGS. 14 and 15, respectively. The content of the pharmaceutical compound residue of the present compound was 6.1% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 40: Synthesis of 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DW-8089)

Boc-Gly-Gly-Phe-Gly (163 mg) and N-hydroxysuccinimide (45 mg) were dissolved in N, N-dimethylformamide (10 mL) and cooled to 4 ° C., followed by N , N-dicyclohexylcarbodiimide (79 mg was added. To this solution, the compound represented by the following formula [(1s, 9s) -1-amino-9-ethyl-2,3-dihydro-9-hydride Roxy-1H, 12H-benzo [de] pyrano [3 ', 4': 6,7] indolizino [1,2-b] quinoline-10,13 (9H, 15H) -dione: DW-8089] N, N-dimethylformamide (30 mL) solution containing tosylate (170 mg) and triethylamine (0.054 mL) was added thereto, and reacted with stirring at room temperature overnight, avoiding light.

The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol containing 0.5% acetic acid = 94: 6 solution) to obtain the compound of Example 40 (100 mg).

¹ H-NMR (DMSO-d ₆ ) δ : 8.51 (d, 1H, J = 8.5 Hz), 8.41 (t, 1H, J = 5.6 Hz), 8.29 (s, 1H), 8.17 (d, 1H, J = 8.0 Hz), 8.03 (d, 1H, J = 8.0 Hz), 7.90 (dd, 1H, J = 4.8, 5.6 Hz), 7.79 (t, 1H, J = 5.6 Hz), 7.53 (d, 1H, J) = 7.2 Hz), 7.36 (s, 1H), 7.13-7.25 (m, 5H), 6.94-6.95 (m, 1H), 5.60-5.63 (m, 1H), 5.36-5.47 (m, 2H), 5.21- 5.30 (m, 2H), 4.42-4.47 (m, 1H), 3.63-3.96 (m, 3H), 3.51-3.59 (m, 3H), 3.31-3.40 (m, 1H), 3.09-3.21 (m, 1H ), 3.02 (dd, 1H, J = 4.8, 13.5 Hz), 2.76-2.81 (m, 1H), 2.13-2.17 (m, 2H), 1.85-1.90 (m, 2H), 1.37 (s, 9H), 0.89 (t, 3H, J = 8.0 Hz).

Mass (FAB); m / e 822 (M + 1)

Example 41 Carboxymethyldextran Polyalcohol-Gly-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of DW-8089

Triethylammonium salt (1.6 g) of carboxymethyldextran polyalcohol obtained in Example 24 was dissolved in N, N-dimethylformamide (60 mL). To this solution was removed in the same manner as in Example 4 from 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (A-NH ₂ = DW-8089) (200 mg) obtained in Example 40. Trifluoroacetic acid and triethylamine (0.07 mL) of 3'-N- (Gly-Gly-Phe-Gly) -NH-A obtained by Bocification were added to N, N-dimethylformamide (20 mL). The obtained solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (1.6g) were added sequentially, and it reacted with stirring overnight at room temperature. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To each was added 3M aqueous sodium chloride solution (2.5 mL) and diethether (25 mL), and the precipitates were collected by centrifugation (2500 rpm, 8 minutes). This precipitate was washed with ethanol, dissolved in water, 3M aqueous sodium chloride solution (20 ml) was added, and adjusted to pH 9 with 0.1M aqueous sodium hydroxide solution. This solution was desalted by ultrafiltration using a Biomax-10 membrane. The remaining solution that did not pass through the membrane was filtered through a Millipore filter (0.22 µm) and then lyophilized to obtain the compound of Example 41 (1.20 g). The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Toso TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and ultraviolet absorption spectrum (0.1 M) of the compound. Tris buffer, pH 9.0, 0.26 μg / ml) is shown in FIGS. 16 and 17, respectively. The content of the pharmaceutical compound residue of the compound was 5.0% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 42: Synthesis of triethylammonium salt of carboxymethyldextran polyalcohol

Dextran T150 (20 g, Pharmacia, average molecular weight 150K) was dissolved in 0.1 M acetate buffer (pH 5.5, 2000 mL) and an aqueous solution of sodium periodate (66.0 g) (2000 mL) was added. After stirring for 10 days at 4 ° C. while blocking light, ethylene glycol (14.0 ml) was added, followed by stirring overnight. The pH of the reaction solution was adjusted to 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. Sodium borohydride (28 g) was added and dissolved, followed by stirring at room temperature overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid and stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 using 8 M aqueous sodium hydroxide solution under ice cooling. 500 ml of the obtained aqueous solution was concentrated by the ultrafiltration method using the Biomax-5 membrane (made by Millipore), and the solution 1 was obtained. Separately, the above series of operations were performed on dextran T110 (20 g) to obtain solution 2. Solution 1 and Solution 2 were mixed, the pH of the mixed solution was adjusted to 3.0, left at 40 ° C. for 4 hours, and then the pH was adjusted to 7 to obtain a solution containing low molecular weighted dextran polyalcohol. After passing through the Biomax-30 membrane, desalting was performed using the Biomax-5 membrane, and then lyophilized to obtain dextran polyalcohol (4.6 g). The molecular weight of this substance was 17K (gel filtration, pullulan standard).

This dextran polyalcohol (2.5 g) was added to the aqueous solution obtained by dissolving sodium hydroxide (17.5 g) in water (75 mL), and it melt | dissolved at room temperature. Monochloroacetic acid (25 g) was added and dissolved in this solution under ice-cooling, and it was made to react at room temperature for 20 hours. The reaction solution was adjusted to pH 9 with acetic acid and then desalted by ultrafiltration using a Biomax-5 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (4.0 g). The molecular weight of this substance was 45 K (gel filtration, pullulan standard) and the degree of CMization was 0.9.

The sodium salt of carboxymethyldextran polyalcohol (3.7 g) was dissolved in water, poured into a Bio-Rad AG 50W-X2 (200-400 mesh, H ⁺ type) column, and dissolved in water. Triethylamine (5.8 mL addition) was added to this eluate, and then lyophilized to obtain the compound of Example 42 (4.4 g).

Example 43 Carboxymethyldextran Polyalcohol-Gly-Gly-Gly-Phe-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 42 (4.4 g) was dissolved in N, N-dimethylformamide (300 mL). To this solution trifluoroamine (580 mg) of triethylamine (0.19 mL) and 3'-N- (Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) N, N-dimethylformamide (45 mL) solution containing 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (4.4 g) was added sequentially to avoid room temperature. The reaction was stirred overnight at. The reaction solution was adjusted to pH 10 with 1 M aqueous sodium hydroxide solution, and 5 ml each was dropped into 25 ml ethanol. To this was added 3 M aqueous sodium chloride solution (1 ml) and diethyl ether (5 ml), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in water, dialyzed against purified water using a dialysis membrane (Spectrapore 1, ultra-molecular weight 6000 to 8000), the dialysis solution was filtered through a Millipore filter (0.22 μm), and then lyophilized. The compound was obtained. The content of the pharmaceutical compound residue of the present compound was 4.6% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 44 α -Methylcarboxymethyldextran polyalcohol-Gly-Gly-Gly-Phe-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Methyldextran polyalcohol (2 g) obtained in Example 42 was added to an aqueous solution obtained by dissolving sodium hydroxide (14 g) in water (60 mL) and dissolved at room temperature. ( Alpha ) -bromopropionic acid (19 ml) was added and dissolved in this solution under ice cooling, and it was made to react at room temperature for 18 hours. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that did not pass through the membrane was lyophilized to obtain sodium salt of α -methylcarboxymethyldextran polyalcohol (2.95 g). The molecular weight of this substance was 45K (gel filtration, pullulan standard). The sugar alpha -methylcarboxymethylation degree was calculated | required as follows according to the case of carboxymethyldextran polyalcohol. An aqueous solution of the sodium salt of α -methylcarboxymethyldextran polyalcohol was injected into a Bio-Rad AG 50 W-X2 (H ⁺ type) column, and the passage solution was lyophilized and used as a sample. This sample was dissolved in a predetermined excess amount of 0.1 N aqueous sodium hydroxide solution, and titrated with 0.1 N hydrochloric acid using phenolphthalein as an indicator. The sample was taken as s mg, the predetermined excess amount of 0.1 N sodium hydroxide aqueous solution was a (ml), the appropriate amount of 0.1 N hydrochloric acid was b (ml), and the degree of α -methylcarboxymethylation was 13.4 (ab) / [s. -7.2 (ab)]. As a result, the degree of α -methylcarboxymethylation was 0.8.

This sodium salt of α -methylcarboxymethyldextran polyalcohol (2.2 g) was dissolved in water, and a Bio-Rad AG 50W-X2 (200 to 400 mesh, H ⁺ type) column (44 mm in diameter and 210 mm in length) was dissolved. Injected into, eluted with water. Triethylamine (4 ml) was added to this eluate, followed by lyophilization to obtain triethylammonium salt (2.69 g) of α -methylcarboxymethyldextran polyalcohol.

Triethylammonium salt (2.68 g) of the α -methylcarboxymethyldextran polyalcohol was dissolved in N, N-dimethylformamide (60 mL). To this solution, 3'-N- (Boc-Gly-Gly-Gly-Phe) -NH-A (A-NH ₂ = DX-8951) (350 mg) synthesized in the same manner as in Example 2 was used. Trifluoroacetate and triethylamine (0.116 mL) of 3'-N- (Gly-Gly-Gly-Phe) -NH-A obtained by debocation in the same manner were added to N, N-dimethylformamide ( 10 ml) was obtained by dissolving 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (2.68 g) in N, N-dimethylformamide (10 ml). It was added sequentially and reacted with stirring overnight at room temperature. A 3 M aqueous sodium chloride solution (40 mL) was added to the reaction solution, and 6 mL each was dropped into 30 mL of ethanol. To each were added 3 M aqueous sodium chloride solution (1 ml) and diethether (5 ml), and the precipitates were collected by centrifugation (3500 rpm, 8 minutes). The precipitate was washed with acetone, dissolved in water, and added with 3M aqueous sodium chloride solution (10 ml), adjusted to pH 9 with 0.1M aqueous sodium hydroxide solution, and treated at 37 ° C for 1 hour. This treated solution was desalted by ultrafiltration using a Biomax-10 membrane. The remaining solution that did not pass through the membrane was filtered through a Millipore filter (0.22 µm), and then lyophilized to obtain the compound (2.5 g) of Example 44. The compound was dissolved in 0.1 M aqueous sodium chloride solution and analyzed by GPC (column: Toso TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min), and the ultraviolet absorption spectrum of the compound (0.1 M Tris buffer, pH 9.0, 0.21 mg / ml) are shown in FIGS. 18 and 19, respectively. The content of the pharmaceutical compound residue of the compound was 5.9% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 45: Synthesis of 3'-N- (Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) trifluoroacetate

P-toluenesulfonic acid salt of Phe-Gly-OBzl (3.06 g), Boc-Gly-OH (1.10 g), N-hydroxysuccinimide (941 mg), N-methylmorpholine (0.725 mL), N, After the mixture of N-dimethylformamide (40 mL) was cooled to 4 ° C., N, N′-dicyclohexylcarbodiimide (1.56 g) was added. After reacting with stirring at room temperature overnight, the reaction solution was dried under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 98: 2 solution) to obtain Boc-Gly-Phe-Gly-OBzl (1.93 g).

¹ H-NMR (DMSO-d ₆ ) δ : 8.52 (dd, 1H, J = 5.6, 6.4 Hz), 7.97 (d, 1H, J = 8.8 Hz), 7.30-7.39 (m, 5H), 7.15-7.26 (m, 5H), 6.83 (t, 1H, J = 5.6 Hz), 5.14 (s, 1H), 4.52-4.57 (m, 1H), 3.87-3.96 (m, 2H), 3.57 (dd, 1H, J = 5.6, 16.7 Hz), 3.43 (dd, 1H, J = 5.6, 16.7 Hz), 3.01 (dd, 1H, J = 4.8, 14.3 Hz), 2.77 (dd, 1H, J = 5.6, 14.3 Hz), 1.37 (s, 9H).

The obtained Boc-Gly-Phe-Gly-OBzl (1.78 g) was dissolved in ethyl acetate (60 mL) and subjected to contact reduction for 24 hours in the presence of 5% -Pd-C (1.8 g). The catalyst was filtered off and the filtrate was concentrated under reduced pressure to obtain Boc-Gly-Phe-Gly-OH (1.41 g).

¹ H-NMR (DMSO-d ₆ ) δ : 8.35 (t, 1H, J = 5.6 Hz), 7.94 (d, 1H, J = 8.8 Hz), 7.15-7.26 (m, 5H), 6.85 (dd, 1H) , J = 5.6, 6.4 Hz, 4.52-4.58 (m, 1H), 3.76 (d, 2H, J = 5.6 Hz), 3.56 (dd, 1H, J = 6.4, 16.7 Hz), 3.43 (dd, 1H, J = 5.6, 16.7 Hz), 3.03 (dd, 1H, J = 5.0, 13.5 Hz), 2.79 (dd, 1H, J = 9.5, 13.5 Hz), 1.37 (s, 9H).

Boc-Gly-Phe-Gly-OH (500 mg) and N-hydroxysuccinimide (161 mg) obtained above were dissolved in N, N-dimethylformamide (10 mL). To this solution was added N, N-dimethylformamide (50 mL) solution of DX-8951 methanesulfonate (530 mg) and triethylamine (0.146 mL), and cooled to 4 ° C., followed by N, N-dicyclohexylcarbodiimide (268 mg) was added, and light was interrupted and reacted with stirring overnight at room temperature. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 96: 4 solution) to give 3'-N- (Boc-Gly-Phe-Gly) -NH-. A (A-NH ₂ = DX-8951) (100 mg) was obtained.

¹ H-NMR (DMSO-d ₆ ) δ : 8.39 (d, 1H, J = 8.0 Hz), 8.34 (t, 1H, J = 5.6 Hz), 7.98 (d, 1H, J = 7.2 Hz), 7.78 ( d, 1H, J = 10.3 Hz, 7.33 (s, 1H), 7.13-7.24 (m, 5H), 6.80 (dd, 1H, J = 5.6, 6.4 Hz), 5.55-5.61 (m, 1H), 5.44 (d, 1H, J = 16.0 Hz), 5.41 (d, 1H, J = 16.0 Hz), 5.25 (s, 2H), 4.43-4.46 (m, 1H), 3.69-3.79 (m, 2H), 3.50 ( dd, 1H, J = 5.6, 16.7 Hz), 3.41 (dd, 1H, J = 5.6, 16.7 Hz), 3.16-3.19 (m, 2H), 2.98 (dd, 1H, J = 4.8, 14.3 Hz), 2.79 (dd, 1H, J = 9.5, 14.3 Hz), 2.41 (s, 3H), 2.19-2.25 (m, 1H), 2.10-2.15 (m, 1H), 1.82-1.90 (m, 2H), 1.35 (s , 9H), 0.88 (t, 3H, J = 8.0 Hz).

Mass (FAB); m / e 797 (M + 1)

The obtained 3'-N- (Boc-Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) (100 mg) was dissolved in trifluoroacetic acid (3 mL) and left to stand for 1 hour. The solvent was removed and the mixture was boiled twice with methanol (30 mL) and ethanol (30 mL) was added twice to boil together. The residue was washed with ethanol to give the compound of Example 45 (80 mg). )

¹ H-NMR (DMSO-d ₆ ) δ : 8.52-8.62 (m, 1H), 7.94 (s, 3H), 7.79 (t, 1H, J = 11.1 Hz), 7.34 (s, 1H), 7.15-7.27 (m, 5H), 6.52 (s, 1H), 5.57-5.61 (m, 1H), 5.36-5.46 (m, 2H), 5.24 (s, 2H), 4.66-4.70 (m, 1H), 3.69-3.81 (m, 2H), 3.61-3.68 (m, 1H), 3.40-3.47 (m, 1H), 3.15-3.23 (m, 1H), 3.01 (dd, 1H, J = 4.0, 13.5 Hz), 2.77 (dd , 1H, J = 9.5, 13.5 Hz), 2.12-2.23 (m, 2H), 1.81-1.91 (m, 2H), 0.89 (t, 3H, J = 7.2 Hz).

Mass (FAB); m / e 697 (M + 1)

Example 46: Synthesis of 3'-N- (Phe-Gly) -NH-A (A-NH ₂ = DX-8951) trifluoroacetate

Boc-Phe-Gly (771 mg) and N-hydroxysuccinimide (300 mg) were dissolved in N, N-dimethylformamide (10 mL). N, N-dimethylformamide solution (50 mL) in which DX-8951 methanesulfonate (1058 mg) and triethylamine (0.293 mL) were dissolved was added to this solution, and cooled at 4 占 폚. N-dicyclohexylcarbodiimide (494 mg) was added, and the reaction was stirred overnight at room temperature while blocking light. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 98: 2 solution) to give 3'-N- (Boc-Phe-Gly) -NH-A ( A-NH ₂ = DX-8951) (1.20 g).

¹ H-NMR (DMSO-d ₆ ) δ : 8.29 (d, 1H, J = 8.0 Hz), 8.21 (t, 1H, J = 4.8 Hz), 7.76 (d, 1H, J = 10.3 Hz), 7.32 ( s, 1H), 7.13-7.25 (m, 5H), 6.92 (d, 1H, J = 7.2 Hz), 6.49 (s, 1H), 5.56-5.61 (m, 1H), 5.44 (d, 1H, J = 15.9 Hz), 5.38 (d, 1H, J = 15.9 Hz), 5.25 (s, 2H), 4.08-4.12 (m, 1H), 3.78 (d, 1H, J = 4.8 Hz), 3.16-3.25 (m, 2H), 2.99 (dd, 1H, J = 4.0, 13.5 Hz), 2.72 (dd, 1H, J = 10.3, 13.5 Hz), 2.40 (s, 3H), 2.09-2.35 (m, 2H), 1.80-1.91 (m, 2H), 1.16 (s, 9H), 0.88 (t, 3H, J = 8.0 Hz).

Mass (FAB); m / e 741 (M + 1)

3'-N- (Boc-Phe-Gly) -NH-A (170 mg) obtained above was dissolved in trifluoroacetic acid (4 mL), and left to stand for 1 hour. The solvent was removed, two times of boiling together with methanol (10 mL) and two times of boiling together with ethanol (10 mL), and the residue was washed with ethanol to give the compound of Example 46 (100 mg). )

¹ H-NMR (DMSO-d ₆ ) δ : 8.88 (t, 1H, J = 4.8 Hz), 8.68 (d, 1H, J = 8.7 Hz), 8.05-8.15 (m, 3H), 7.79 (d, 1H , J = 11.1 Hz), 7.26-7.36 (m, 5H), 6.52 (d, 1H, J = 7.2 Hz), 5.57-5.62 (m, 1H), 5.43 (d, 1H, J = 15.9 Hz), 5.38 (d, 1H, J = 15.9 Hz), 5.19-5.28 (m, 1H), 4.10-4.18 (m, 1H), 3.93 (dd, 1H, J = 4.8, 16.7 Hz), 3.82 (dd, 1H, J = 4.8, 16.7 Hz), 3.17-3.24 (m, 2H), 3.14 (dd, 1H, J = 4.8, 13.5 Hz), 2.95 (dd, 1H, J = 8.0, 13.5 Hz), 2.42 (s, 3H) , 2.14-2.25 (m, 2H), 1.83-1.91 (m, 2H), 0.89 (t, 3H, J = 8.0 Hz).

Mass (FAB); m / e 640 (M + 1)

Example 47 Synthesis of 3'-N-Gly-NH-A (A-NH ₂ = DX-8951) Trifluoroacetate

Methanesulfonate (530 mg) and triethylamine (0.28 mL) of DX-8951 were dissolved in N, N-dimethylformamide (10 mL), cooled to 4 ° C., and then N- of Boc-Gly. Hydroxysuccinimide ester (327 mg) was added. The reaction was stirred overnight at room temperature while blocking light. The reaction solution was dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 98: 2 solution) to give 3'-N- (Boc-Gly) -NH-A (A-NH ₂ =). DX-8951) (500 mg) was obtained.

¹ H-NMR (DMSO-d ₆ ) δ : 8.38 (d, 1H, J = 8.3 Hz), 7.77 (d, 1H, J = 10.7 Hz), 7.31 (s, 1H), 6.89-6.91 (m, 1H ), 6.49 (s, 1H), 5.55-5.59 (m, 1H), 5.45 (d, 1H, J = 16.1 Hz), 5.38 (d, 1H, J = 16.1 Hz), 5.27 (d, 1H, J = 19.0 Hz), 5.18 (d, 1H, J = 19.0 Hz), 3.50-3.62 (m, 2H), 3.15-3.19 (m, 2H), 2.41 (s, 3H), 2.18-2.24 (m, 1H), 2.08-2.12 (m, 1H), 1.81-1.91 (m, 2H), 1.31 (s, 9H), 0.87 (t, 3H, J = 8.0 Hz).

Mass (FAB); m / e 593 (M + 1)

The 3'-N- (Boc-Gly) -NH-A (100 mg) obtained above was dissolved in trifluoroacetic acid (2 mL), and left to stand for 1 hour. The solvent was removed and the mixture was boiled twice with methanol (10 ml) and ethanol (10 ml) twice with boiling. The residue was washed with ethanol to give the compound of Example 47 (70 mg). )

¹ H-NMR (DMSO-d ₆ ) δ : 8.88 (d, 1H, J = 8.8 Hz), 8.08 (s, 3H), 7.81 (d, 1H, J = 11.2 Hz), 7.34 (s, 1H), 6.52 (s, 1H), 5.63-5.67 (m, 1H), 5.45 (d, 1H, J = 16.7 Hz), 5.40 (d, 1H, J = 16.7 Hz), 5.36 (d, 1H, J = 19.1 Hz ), 5.25 (d, 1H, J = 19.1 Hz), 3.56 (s, 2H), 3.11-3.19 (m, 2H), 2.43 (s, 3H), 2.23-2.28 (m, 1H), 2.11-2.19 ( m, 1H), 1.81-1.91 (m, 2H), 0.88 (t, 3H, J = 8.0 Hz).

Mass (FAB); m / e 493 (M + 1)

Example 48: Synthesis of trimethylammonium salt of carboxymethyldextran polyalcohol

Dextran T500 (50 g, Pharmacia, molecular weight 500K) was dissolved in 0.1 M acetic acid buffer (pH 5.5, 5000 mL), and an aqueous solution of sodium periodate (165.0 g) (5000 mL) was added. After stirring for 10 days at 4 ° C while blocking light, ethylene glycol (35.0 ml) was added, and the mixture was stirred overnight. The reaction solution was adjusted to pH 7 using 8 M aqueous sodium hydroxide solution. Sodium borohydride (70 g) was added and dissolved, followed by stirring overnight. The reaction solution was ice-cooled, adjusted to pH 5.5 with acetic acid, stirred at 4 ° C. for 1 hour, and then adjusted to pH 7.5 using an aqueous 8 M sodium hydroxide solution. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain dextran polyalcohol (20.2 g). The molecular weight of this substance was 159K (gel filtration, pullulan standard).

The dextran polyalcohol (7.5 g) was added to an aqueous solution obtained by dissolving sodium hydroxide (31.5 g) in water (225 ml), and the mixture was dissolved at room temperature. Monochloroacetic acid (45 g) was added and dissolved in this solution under ice-cooling, and then reacted overnight at room temperature. The reaction solution was adjusted to pH 8 with acetic acid and then desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that had not passed through the membrane was lyophilized to obtain sodium salt of carboxymethyldextran polyalcohol (8.5 g). The molecular weight of this substance was 274 K (gel filtration, pullulan standard) and the degree of carboxymethylation was 0.4. Sodium salt of this carboxymethyldextran polyalcohol (2.0 g) was dissolved in water and poured into a Bio-Rad AG 50W-X2 (200 to 400 mesh, H ⁺ type) column (44 mm in diameter and 210 mm in length). , Eluted with water. Triethylamine (4 ml) was added to this eluate, and then lyophilized to obtain the compound (2.2 g) in Example 48.

Example 49 Carboxymethyldextran Polyalcohol-Gly-Phe-Gly-NH-A '(A-NH ₂

Synthesis of = DX-8951)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 48 (200 mg) was dissolved in N, N-dimethylformamide (7 mL). To this solution, N, N-dimethyls of trifluoroacetate (41 mg) of 3'-N- (Gly-Phe-Gly) -NH-A (A-NH ₂ = DX-8951) obtained in Example 45 was obtained. Formamide (5 mL) solution, triethylamine (0.014 mL) and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (100 mg) were added sequentially and stirred to room temperature overnight. Reacted. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3 M aqueous sodium chloride solution (2.0 mL) and diethether (25 mL), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that did not pass through the membrane was filtered through a Millipore filter (0.22 µm) and then lyophilized to obtain the compound of Example 49 (190 mg). The content of the pharmaceutical compound residue of the compound was 4.5% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 50 Synthesis of Carboxymethyldextran Polyalcohol-Phe-Gly-NH-A '(A-NH ₂ = DX-8951)

The sodium salt of carboxymethyldextran polyalcohol obtained in Example 24 (2.5 g) was dissolved in water, injected into a Bio-Rad AG 50W-X2 (200-400 mesh, Et ₃ NH ⁺ type) column and eluted with water. . The eluate was lyophilized to obtain triethylammonium salt (2.5 g) of carboxymethyldextran polyalcohol.

Triethylammonium salt (200 mg) of this carboxymethyldextran polyalcohol was dissolved in N, N-dimethylformamide (12 mL). To this solution, trifluoroacetate (42 mg) and triethylamine (0.016 mL) of 3'-N- (Phe-Gly) -NH-A (A-NH ₂ = DX-8951) obtained in Example 46 were obtained. N, N-dimethylformamide (5 mL) solution, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (200 mg) was added sequentially to block light at room temperature. The reaction was stirred overnight. Water (300 mL) was added to this reaction solution, and ultrafiltration was performed using the ultrafiltration membrane 10K (made by Filtron). The remaining solution that did not pass through the membrane was adjusted to pH 10 with 0.1 N aqueous sodium hydroxide solution, and passed through a filtration membrane (0.16 µm, manufactured by Pitron). The solution passed through was desalted by ultrafiltration using a Biomax-50 membrane, filtered through a Millipore filter (0.22 μm), and then lyophilized to obtain the compound of Example 50 (180 mg). The content of the pharmaceutical compound residue of the present compound was 6.1% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 51 Synthesis of Carboxymethyldextran Polyalcohol-Gly-NH-A '(A-NH ₂ = DX-8951)

Triethylammonium salt of carboxymethyldextran polyalcohol obtained in Example 48 (370 mg) was dissolved in N, N-dimethylformamide (10 mL). To this solution, N, N-dimethylformamide (3 mL) of 3'-N-Gly-NH-A (A-NH ₂ = DX-8951) trifluoroacetate (57 mg) obtained in Example 47 was obtained. A solution, triethylamine (0.027 mL) and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (185 mg) were added sequentially, and it reacted with stirring overnight at room temperature. 5 ml of this reaction solution was dropped into each 10 ml of ethanol. To this was added 3M aqueous sodium chloride solution (2.0 ml) and diethether (25 mlml), and the precipitated precipitate was collected by centrifugation (3500 rpm, 8 minutes). This precipitate was dissolved in 0.5 M brine and adjusted to pH 9 with 0.1 M aqueous sodium hydroxide solution under ice-cooling. The obtained aqueous solution was desalted by ultrafiltration using a Biomax-50 membrane. The remaining solution that had not passed through the membrane was filtered through a Millipore filter (0.22 µm) and then lyophilized to obtain the compound of Example 51 (290 mg). The content of the pharmaceutical compound residue of the present compound was 0.5% (w / w) when the amount was determined based on the absorbance of 362 nm in 0.1 M Tris buffer (pH 9.0).

Example 52 Antitumor Activity of Drug Complexes of the Invention

Mice with Meth A tumors were prepared in the same manner as in Example 11 (6 per group), and the antitumor effect of the drug complex of Example 15 when administered once in the same manner as in Example 12 was examined. As a result, the drug complex of Example 15 showed remarkable enhancement of antitumor effect and enlargement of effective dose area, compared with the pharmaceutical compound of Example 12 itself.

Example 53 Antitumor Activity of Drug Complexes of the Invention

Tumor masses of human gastric cancer SC-6 were transplanted subcutaneously to the right neck of nude mice (BALB / c-nu / nu, male) to prepare nude mice with SC-6 tumors (5 per group). The drug complex of Example 15 dissolved in distilled water for injection was administered once intravenously 24 days after transplantation, and antitumor activity was compared with the pharmaceutical compound itself. As a result, the drug complex of Example 15 did not show lethality due to toxic death, but exhibited high antitumor effect, compared with the pharmaceutical compound itself.

Example 54 Antitumor Activity of Drug Complexes of the Invention

Nude mice with human lung cancer QG-90 tumors were prepared by the same method as Example 53 (5 per group). The drug complex of Example 15 dissolved in distilled water for injection was administered once intravenously 16 days after transplantation, and antitumor activity was compared with the pharmaceutical compound itself. As a result, the drug complex of Example 15 showed remarkable enhancement of antitumor effect and enlargement of effective dose area, compared with the pharmaceutical compound itself.

Example 55 Antitumor Activity of Drug Complexes of the Invention

In the same manner as in Example 11, mice with Meth A tumors were prepared (6 per group), and the antitumor effect of the single drug complex of Example 41 in the same manner as in Example 12 was compared with the pharmaceutical compound itself. It was. As a result, the drug complex of Example 41 showed remarkable enhancement of antitumor effect and enlargement of effective dose area, compared with the pharmaceutical compound itself.

Example 56 Antitumor Activity of Drug Complexes of the Invention

Antitumor when a mouse with Meth A tumor was prepared (6 per group) in the same manner as in Example 11, and each drug complex of Examples 29, 43 and 44 was administered once in the same manner as in Example 12. The action was investigated.

As a result, it showed higher antitumor effect and wider effective dose range than any drug complex.

Example 57: In vivo kinetics of the drug complex of the present invention

A mouse having a Meth A tumor was prepared in the same manner as in Example 11, and the drug complex of Example 15 was administered once (10 mg / kg: equivalent of a pharmaceutical compound) in the same manner as in Example 12, and each tissue of the drug complex was The concentration change in the inside was investigated. As a result, the drug complex of Example 15 showed remarkably high blood retention, high migration to tumor tissue and high tumor selectivity for liver and small intestine. The results are shown in FIG. 20.

Since the reaction between a polysaccharide derivative having a carboxyl group and a pharmaceutical compound having a pharmaceutical compound or a spacer bonded thereto can be performed efficiently, side reactions can be suppressed when the pharmaceutical compound having a lactone ring is reacted. It is very useful as a manufacturing method.

Claims (7)

A drug complex having a residue of a polysaccharide derivative having a carboxyl group and a pharmaceutical compound having a lactone ring bound through a spacer consisting of 2 to 8 amino acids bound by one amino acid or a peptide, or a drug having a polysaccharide derivative having a carboxyl group and a lactone ring In the method for producing a drug complex in which residues of a compound are bonded through an electric spacer, triethylamine, trimethylamine, triethanolamine, N-methylpyrrolidine, N, of a polysaccharide derivative having a carboxyl group Organic amine salts selected from the group consisting of methylpiperidine, N-methylmorpholine, dimethylaminopyridine, tetramethylammonium chloride and tetraethylammonium chloride; A spacer combining a pharmaceutical compound having a ton ring or a pharmaceutical compound having a lactone ring Is reacted in an organic solvent selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, acetoamide, N-methylpyrrolidone and sulfolane. . A drug complex having a residue of a polysaccharide derivative having a carboxyl group and a pharmaceutical compound having a lactone ring bound through a spacer consisting of 2 to 8 amino acids linked to one amino acid or a peptide, or a drug having a polysaccharide derivative having a carboxyl group and a lactone ring In the method for producing a drug complex in which the residues of the compound are bound via an electrical spacer, (Iii) Alkali metal salts of polysaccharide derivatives having a carboxyl group include triethylamine, trimethylamine, triethanolamine, N-methylpyrrolidine, N-methylpiperidine, and N-methylmorpholine. (methylmorpholine), dimethylaminopyridine, tetramethylammonium chloride and tetraethylammonium chloride and tetraethylammonium chloride; And (Ii) N, N-dimethylformamide, dimethylsulfoxide, acetoamide, N-methylpyrrolidone, comprising a spacer in which an electroorganic amine salt and a pharmaceutical compound having a lactone ring or a pharmaceutical compound having a lactone ring are combined. reacting in an organic solvent selected from the group consisting of pyrrolidone and sulfolane. The method according to claim 1 or 2, The polysaccharide derivative having a carboxyl group is carboxyC _1-4 alkyldextran polyalcohol Characterized by Way. The method of claim 3, wherein Dextran polyalcohol constituting the carboxy C _1-4 alkyldextran polyalcohol is characterized in that the dextran polyalcohol is prepared by sequentially acting excess sodium iodide and sodium borohydride on the dextran Way. The method of claim 3, wherein CarboxyC _1-4 alkyldextran polyalcohol is characterized in that carboxymethyldextran polyalcohol Way. The method according to claim 1 or 2, The pharmaceutical compound having a lactone ring is characterized in that the anti-tumor agent or anti-inflammatory agent Way. The method according to claim 1 or 2, The pharmaceutical compound having a lactone ring is (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de] Pyrano [3 ', 4;: 6,7] indolinino [1,2-b] quinoline-10,13 (9H, 15H) -dione Way.