WO2016093352A1 - Multi-branched compound having nucleic acid antimetabolite bonded thereto - Google Patents

Abstract

[Problem] The present invention addresses the problem of providing a polymerized prodrug of a nucleic acid antimetabolite, said prodrug having an improved antitumor effect and having few side effects, particularly myelosuppression. In particular, the invention addresses the problem of providing a nucleic acid antimetabolite that exhibits a tumor growth inhibition effect over a long period of time and does not prolong myelosuppression. [Solution] Provided is a multi-branched compound to which a nucleic acid antimetabolite is bonded, said compound being characterized by being appropriately provided, in a terminal functional group of a multi-branched polymer carrier, with a substituent group that includes a plurality of polyethylene glycol segments and succinic acid monoamide units to which a nucleic acid antimetabolite is bonded. The multi-branched compound, when administered inside the body, is retained in the blood and is distributed in the body, the nucleic acid antimetabolite bonded to said compound can be made to act with an appropriate release profile, drug efficacy can be improved, and side effects can be prevented. In particular, the present invention makes it possible to provide a drug that prevents the prolongation of myelosuppression, which is the main side effect of nucleic acid antimetabolites.

Description

Hyperbranched compounds bound with nucleic acid antimetabolite

The present invention relates to a hyperbranched compound to which a nucleic acid antimetabolite is bound and its use.

Various nucleic acid metabolism antagonists have been developed for the purpose of treating malignant tumors or viral diseases. For example, as an antitumor agent (anticancer agent), there are cytarabine, gemcitabine, doxyfluridine, azacitidine, decitabine, nelarabine and the like. Moreover, as an antiviral agent, zalcitabine, lamivudine, etc. are used clinically.

These nucleic acid antimetabolites have extremely strong pharmacological activity in the in vitro evaluation. However, these drugs are susceptible to metabolism and excretion in the living body, and there is a problem that in vivo evaluation, the drug efficacy of the original drug cannot be fully exhibited. Many of these drugs require high doses in clinical therapeutic regimes. For example, gemcitabine has a strong activity comparable to that of powerful antitumor agents such as paclitaxel and doxorubicin, in vitro cell growth inhibitory activity evaluation (IC ₅₀ value). On the other hand, the clinical use of gemcitabine requires high dose administration of 1,000 mg / m ² as the use per body surface area. This is thought to be due to the fact that the in vivo utilization rate is lowered by cytidine deaminase, a metabolic enzyme of 2′-deoxycytidine, by metabolizing and inactivating the 4-position amino group of the nucleobase portion of gemcitabine. (Non-Patent Document 1).

For the purpose of improving the bioavailability by suppressing the metabolism / inactivation of the drug, a polymerized drug in which the drug is bound to a polymer carrier has been studied. The high molecular weight drug is expected to improve the therapeutic effect by changing the pharmacokinetics based on the high molecular weight. Non-Patent Document 2 describes a polymerized derivative in which cytarabine, which is a nucleic acid antimetabolite, is bound to polyglutamic acids having an average molecular weight of about 30 kilodaltons. However, there is a concern that a high molecular weight derivative of a drug is likely to be recognized by a foreign body in a living body and a large amount of the drug is trapped in a phagocytic tissue such as a liver. Moreover, there is a case where hypersensitivity reaction is caused by inducing immunity. In such a case, there is a concern that repeated administration as a drug cannot be performed.

As a method for avoiding foreign matter recognition of the polymerized derivative, a method using polyethylene glycol is known. For example, Patent Document 1 describes a polymerized derivative in which a cytidine derivative is bound to polyethylene glycols. Non-Patent Document 3 describes a polymerized derivative in which aspartic acid is branched in both ends of polyethylene glycols and cytarabine is bound thereto. Furthermore, Patent Document 2 describes a polymerized derivative having a structure in which an amino acid is branched at the end of a polyethylene glycol chain and the drug is released after each branch undergoes a benzyl elimination reaction.

Patent Document 3 and Patent Document 4 describe a polymerized derivative in which a nucleic acid antimetabolite and a hydrophobic substituent are bonded to a terminal functional group of a block copolymer of polyethylene glycols and a polyacidic amino acid. Yes. Patent Document 5 describes a polymerized derivative in which a nucleic acid metabolism antagonist is bonded to a terminal functional group of a block copolymer of polyethylene glycols and a polyacidic amino acid via a linker having a hydrophobic substituent. Yes.
The polymer conjugates of these nucleic acid antimetabolites are bipolar polymers having both a hydrophobic segment having a hydrophobic substituent introduced into the terminal functional group and polyethylene glycol, which is a hydrophilic segment. For this reason, the polymer conjugate of the nucleic acid antimetabolite is considered to form a self-aggregate having the hydrophobic segment as the inner core and the hydrophilic segment as the outer side due to intermolecular aggregation of the hydrophobic segment in an aqueous solution.

Patent Document 6 describes a polymer derivative in which a polylysine dendrimer based on lysine, which is a branched diamine compound, is used as a carrier and a polyethylene glycol segment and a nucleic acid antimetabolite are bound to the terminal functional group of the outermost shell.
This polymer derivative using dendrimer uses gemcitabine as a nucleic acid metabolism antagonist. The gemcitabine is introduced into the dendrimer carrier through glutaric acid as a linker and an ester bond with the 5′-hydroxy group of gemcitabine.

The polymer conjugate of the above-mentioned nucleic acid antimetabolite having various polyethylene glycols has the property of undergoing hydrolysis in a phosphate buffered saline (PBS) solution and slowly releasing the bound nucleic acid antimetabolite. Have. Therefore, these polymerized nucleic acid antimetabolites have a characteristic that they continue to exert a tumor growth inhibitory effect for a long period of time at a low dose as compared with conventional nucleic acid antimetabolites. However, there is a concern that side effects caused by the same mechanism of action as the medicinal effects may be expressed over a long period of time. Nucleic acid antimetabolites have a problem as a side effect of bone marrow suppression observed as manifestation of leukopenia and the like. The polymerized nucleic acid antimetabolite has been a major issue in establishing a useful therapeutic method that achieves both improvement in therapeutic effect and reduction in side effects.

Special table 2003-524028 gazette JP-T-2004-532289 International Publication No. 2006/120914 International Publication No. 2008/056596 International Publication No. 2008/056654 International Publication 2012/167309

Cancer Science, Japan Cancer Association, Vol. 95, p. 105-111 (2004) "Cancer Science", published by the Japanese Cancer Association, 2004, volume 95, pages 105-111. Cancer Research, American Association for Cancer Research, Vol. 44, p. 25-30 (1984) “Cancer Research” (USA), American Cancer Society, 1984, Vol. 44, pages 25-30 Journal of Controlled Release, Elsevier, Vol. 79, p. 55-70 (2002) "Journal of Controlled Release" (UK), published by Elsevier, 2002, 79, 55-70

An object of the present invention is to provide a nucleic acid antimetabolite with improved antitumor effects and reduced side effects, particularly reduced myelosuppression. Specifically, it provides a nucleic acid metabolism antagonist that does not prolong myelosuppression while exerting a tumor growth inhibitory effect for a long time.

As a result of intensive studies to solve the above problems, the present inventors have found that a nucleic acid in which a succinic acid monoamide unit having a nucleic acid antimetabolite bonded to a terminal functional group of a multi-branched polymer carrier and a polyethylene glycol segment are combined. It has been found that antimetabolite-bound hyperbranched compounds can improve the antitumor effect and avoid the prolongation of myelosuppression, which is a side effect.

［式中、［Ｑ］は（ｍ＋ｎ＋ｏ＋ｐ）個の末端官能基化された多分岐高分子担体であり、（ｍ＋ｎ＋ｏ＋ｐ）は４～２００の整数であり、［Ｆ］は前記末端官能基であってアミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基、及び／又は置換基を有していても良い炭素数（Ｃ１～Ｃ６）のアルキル基を有する保護基で修飾された、アミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基であり、Ｆは前記末端官能基から水素原子又は水酸基が除かれた結合基であり、Ｒ_１は核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを含む置換基であり、Ｒ_２はポリエチレングリコールセグメントを含む置換基であり、Ｒ_３はコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基を含む置換基であり、ｍは０～１９９の整数であり、ｎは１～２００の整数であり、ｏは０～１９９の整数であり、ｐは０～１９９の整数である。］で示される核酸代謝拮抗剤結合多分岐化合物。
　本発明は、一般式（１）におけるＲ_１に係る核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを必須の置換基として具備し、Ｒ_２、Ｒ_３及び［Ｆ］に係る置換基を任意に具備する核酸代謝拮抗剤結合多分岐化合物である。
　なお、本発明の核酸代謝拮抗剤結合多分岐化合物は、ポリエチレングリコールセグメントを含む置換基を具備することが好ましい。該ポリエチレングリコールセグメントを含む置換基は、一般式（１）においてＲ_２として具備していても良く、Ｒ_１に係る核酸代謝拮抗剤が結合したコハク酸モノアミドユニットに該ポリエチレングリコールセグメントを含む置換基を具備させてもいても良い。
　また、前記［Ｆ］は、多分岐高分子担体［Ｑ］の末端官能基であっても良く、また該末端官能基が置換基を有していても良い炭素数（Ｃ１～Ｃ６）のアルキル基を有する保護基の修飾体であっても良く、末端官能基とその保護基修飾体の混合体で存在している態様を含む。 That is, the present invention relates to the following [1] to [14].
[1] A nucleic acid antimetabolite-bound hyperbranched compound is represented by the general formula (1)

[Wherein [Q] is (m + n + o + p) terminal functionalized multi-branched polymer carrier, (m + n + o + p) is an integer of 4 to 200, and [F] is the terminal functional group. A protecting group having one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and / or an optionally substituted alkyl group having a carbon number (C1 to C6). Is one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and F is a linking group obtained by removing a hydrogen atom or a hydroxyl group from the terminal functional group. , R ₁ is a substituent containing a succinic acid monoamide units nucleic acid antimetabolites are bonded, R ₂ is a substituent containing a polyethylene glycol segment, R ₃ is Zanmoto及acid monoamide derivative And / or a substituent containing a succinimide residue, m is an integer from 0 to 199, n is an integer from 1 to 200, o is an integer from 0 to 199, and p is from 0 to 199. It is an integer. ] The nucleic acid antimetabolite binding hyperbranched compound shown by this.
The present invention comprises, as an essential substituent, a succinic acid monoamide unit to which a nucleic acid antimetabolite according to R ₁ in the general formula (1) is bonded, and optionally substituents according to R ₂ , R ₃ and [F]. It is a nucleic acid antimetabolite-binding hyperbranched compound.
In addition, it is preferable that the nucleic acid antimetabolite binding hyperbranched compound of the present invention has a substituent containing a polyethylene glycol segment. The substituent containing the polyethylene glycol segment may be included as R ₂ in the general formula (1), and the substituent containing the polyethylene glycol segment in the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ is bonded. May be provided.
[F] may be a terminal functional group of the multi-branched polymer carrier [Q], and the terminal functional group may have a substituent (C1-C6) alkyl. It may be a modified form of a protecting group having a group, and includes a mode in which the terminal functional group and a modified form of the protecting group are present as a mixture.

[2] The nucleic acid antimetabolite is a nucleic acid antimetabolite having an amino group at a nucleobase, and the nucleic acid antimetabolite is bonded to a carboxy group of succinic acid monoamide through an amide bond by the amino group. The nucleic acid antimetabolite-binding hyperbranched compound according to [1].
By aligning the binding mode of the nucleic acid antimetabolite-binding multibranched compound with the amide bond, the desired drug release profile can be achieved, and the present invention relating to sustained efficacy and reduced side effects can be achieved. The effect can be obtained stably.

[3] The nucleic acid antimetabolite-binding hyperbranched compound according to [1] or [2], wherein the mass content of the nucleic acid antimetabolite is 2% by mass or more and 60% by mass or less.

[4] The mass content of the polyethylene glycol segment in the nucleic acid antimetabolite-binding hyperbranched compound is 20% by mass or more and 90% by mass or less, and the polyethylene glycol segment is bound by 2 to 100 units. [3] The nucleic acid antimetabolite-binding hyperbranched compound according to [3].
The nucleic acid antimetabolite-binding hyperbranched compound can achieve appropriate pharmacokinetics by retaining an appropriate amount of the polyethylene glycol segment, and can achieve the onset of drug efficacy and reduction of side effects.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｘ_４は末端官能基Ｆとの結合基であり、Ｒ_４、Ｒ_５及びＲ_６はそれぞれ独立して水素原子又は炭素数（Ｃ１～Ｃ８）のアルキル基であり、Ｒ_７は水素原子、置換基を有していても良い炭素数（Ｃ１～Ｃ２０）の直鎖状、分岐鎖状又は環状のアルキル基、置換基を有していても良い炭素数（Ｃ７～Ｃ２０）の直鎖状、分岐鎖状又は環状のアラルキル基、置換基を有していても良い芳香族基及びカルボキシ基が保護されたアミノ酸残基からなる群から選択される１種以上の基である。］である前記［１］～［４］の何れか一項に記載の核酸代謝拮抗剤結合多分岐化合物。
　すなわち、該［５］の発明態様は、前記一般式（１）において、前記Ｒ_１に係る前記コハク酸モノアミドユニットが、アスパラギン酸モノアミドユニットを用いることが好ましい。この場合、核酸代謝拮抗剤結合多分岐化合物にポリエチレングリコールセグメントを具備させる場合は、Ｒ_２へポリエチレングリコールセグメントを含む置換基を導入する態様である。 [5] A succinic acid monoamide unit to which a nucleic acid antimetabolite of R ₁ is bound is represented by the general formula (2) and / or (3)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, X ₄ is a binding group to the terminal functional group F, and R ₄ , R ₅ and R ₆ are each independently a hydrogen atom or carbon. An alkyl group having a number (C1 to C8), and R ₇ is a hydrogen atom, a linear, branched or cyclic alkyl group having a carbon number (C1 to C20) which may have a substituent, a substituent A linear, branched or cyclic aralkyl group which may have a carbon number (C7 to C20), an aromatic group which may have a substituent, and an amino acid residue in which a carboxy group is protected One or more groups selected from the group consisting of The nucleic acid antimetabolite-binding hyperbranched compound according to any one of [1] to [4] above.
That is, in the invention aspect of [5], in the general formula (1), the succinic acid monoamide unit according to R ₁ is preferably an aspartic acid monoamide unit. In this case, case of including a polyethylene glycol segment in nucleic acid metabolism antagonist binding hyperbranched compound, a mode of introducing a substituent containing a polyethylene glycol segment into R _2.

[6] Any one of [1] to [5], wherein the succinic acid monoamide unit to which the nucleic acid antimetabolite of R ₁ is bound is a polyaspartic acid derivative in which a nucleic acid antimetabolite is bound to a side chain carboxy group. A nucleic acid antimetabolite-binding hyperbranched compound according to 1.
That is, polyaspartic acid may be used as the succinic acid monoamide unit. The use of the polyaspartic acid is preferable because one R ₁ substituent can be provided with a plurality of nucleic acid metabolism antagonists.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｒ_１２は水酸基及び／又は－Ｎ（Ｒ_１４）ＣＯＮＨ（Ｒ_１５）であり、該Ｒ_１４及び該Ｒ_１５は同一でも異なってもいてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、Ｒ_１３は水素原子、炭素数（Ｃ１～Ｃ８）のアシル基及び炭素数（Ｃ１～Ｃ８）のアルコキシカルボニル基からなる群から選択される基であり、Ｘ_２は末端官能基Ｆとの結合基であり、ａ、ｂ、ｃ、ｄ及びｅはそれぞれ独立して０～３０の整数を示し、（ａ＋ｂ）は１～３０の整数を示し、ポリアスパラギン酸誘導体の総重合数である（ａ＋ｂ＋ｃ＋ｄ＋ｅ）は１～３０であり、前記［Ｄ］が結合したアスパラギン酸単位、前記Ｒ_１２が結合したアスパラギン酸単位及び側鎖カルボキシ基が分子内環化型のアスパラギン酸単位が、それぞれ独立してランダムな配列である。］である前記［６］に記載の核酸代謝拮抗剤結合多分岐化合物。 [7] The polyaspartic acid derivative of R ₁ is represented by the general formula (4) or (5)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, R ₁₂ is a hydroxyl group and / or —N (R ₁₄ ) CONH (R ₁₅ ), and R ₁₄ and R ₁₅ are the same A linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be different or may be substituted with a tertiary amino group, R ₁₃ is a hydrogen atom, a carbon number ( A group selected from the group consisting of an acyl group of C1 to C8) and an alkoxycarbonyl group of carbon number (C1 to C8), X ₂ is a bonding group to the terminal functional group F, and a, b, c, d and e each independently represent an integer of 0 to 30, (a + b) represents an integer of 1 to 30, and the total polymerization number of the polyaspartic acid derivative (a + b + c + d + e) is 1 to 30, D] is bound to an aspartic acid unit, and R ₁₂ is bound to The aspartic acid unit and the side chain carboxy group in the intramolecular cyclization type aspartic acid unit are each independently a random sequence. The nucleic acid antimetabolite-binding hyperbranched compound according to [6] above.

[8] The succinic acid monoamide unit to which the nucleic acid antimetabolite of R ₁ is bonded is a binding substituent of the succinic acid monoamide unit in which the nucleic acid antimetabolite is bonded to a polyethylene glycol segment and a side chain carboxy group [1] The nucleic acid antimetabolite-binding hyperbranched compound according to any one of [4] to [4].
That is, the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ of the general formula (1) is bonded may be a substituent having a polyethylene glycol segment. Use of such a substituent in which two functional groups are integrated is preferable because the ratio of the succinic acid monoamide unit to which the polyethylene glycol segment and the nucleic acid antimetabolite are bound can be made constant. Moreover, it is preferable because the terminal functional group of the multi-branched polymer carrier can be used efficiently.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｒ_１６はポリエチレングリコールセグメントであり、Ｒ_１７は水酸基及び／又は－Ｎ（Ｒ_１８）ＣＯＮＨ（Ｒ_１９）であり、該Ｒ_１８及び該Ｒ_１９は同一でも異なっていてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、Ｘ_３は末端官能基Ｆとの結合基であり、ｆ、ｇ、ｈ、ｉ及びｊはそれぞれ独立して０～３０の整数を示し、（ｆ＋ｇ）は１～３０の整数を示し、ポリアミノ酸誘導体の総重合数である（ｆ＋ｇ＋ｈ＋ｉ＋ｊ）は１～３０であり、前記［Ｄ］が結合したアスパラギン酸単位、前記Ｒ_１７が結合したアスパラギン酸単位及び側鎖カルボキシ基が分子内環化型のアスパラギン酸単位が、それぞれ独立してランダムな配列である。］である前記［８］に記載の核酸代謝拮抗剤結合多分岐化合物。
　ポリエチレングリコールセグメントと核酸代謝拮抗剤が結合したコハク酸モノアミドユニットが結合して一体となった置換基において、コハク酸モノアミドユニットとしてポリアスパラギン酸を用いることにより、１つの置換基に複数の核酸代謝拮抗剤を具備させることができることから好ましい。 [9] The bond-type substituent of R ₁ is a block copolymer-type substituent in which a polyaspartic acid segment in which a nucleic acid antimetabolite is bonded to a polyethylene glycol segment and a side chain carboxy group is bonded. Or (7)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, R ₁₆ is a polyethylene glycol segment, R ₁₇ is a hydroxyl group and / or —N (R ₁₈ ) CONH (R ₁₉ ), R ₁₈ and R ₁₉ may be the same or different and each is a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group; ₃ is a linking group to the terminal functional group F, f, g, h, i and j each independently represents an integer of 0 to 30, (f + g) represents an integer of 1 to 30, and a polyamino acid derivative (F + g + h + i + j) is 1 to 30, and the aspartic acid unit to which the [D] is bonded, the aspartic acid unit to which the R ₁₇ is bonded and the side chain carboxy group is an intramolecularly cyclized aspartic acid. Each unit is German It is a random sequence. The nucleic acid antimetabolite-binding hyperbranched compound according to [8] above.
By using polyaspartic acid as a succinic acid monoamide unit in a succinic acid monoamide unit in which a polyethylene glycol segment and a nucleic acid antimetabolite are bonded together, a plurality of nucleic acid metabolism antagonists are used for one substituent. It is preferable because an agent can be provided.

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_１は末端官能基Ｆとの結合基を示す。］である前記［１］～［９］の何れか一項に記載の核酸代謝拮抗剤結合多分岐化合物。 [10] The polyethylene glycol segment of R ₂ has the general formula (8)

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₁ represents a bonding group with the terminal functional group F. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of [1] to [9] above.

［式中、Ｘ_４は末端官能基Ｆとの結合基であり、Ｒ_９は水酸基及び／又は－Ｎ（Ｒ_１０）ＣＯＮＨ（Ｒ_１１）を示し、Ｒ_１０、Ｒ_１１は同一でも異なっていてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基を示し、Ｒ_４、Ｒ_５、Ｒ_６及びＲ_７は前記と同義である。］からなる置換基群から選ばれる１種以上の基である前記［１］～［１０］の何れか一項に記載の核酸代謝拮抗剤結合多分岐化合物。
　Ｒ_３のコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基を含む置換基が、アスパラギン酸モノアミド由来の残基又はイミド残基であることが好ましい。該Ｒ_３は、前記一般式（２）及び／又は（３）で示される核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを含む置換基から、該核酸代謝拮抗剤が解離した残基の態様を示す。
　すなわち、本発明は一般式（１）において、核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを含む置換基（Ｒ_１）と、ポリエチレングリコールセグメントを含む置換基（Ｒ_２）並びにコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基を含む置換基（Ｒ_３）の少なくとも３種類の置換基が、それぞれ別の置換基として具備する態様であって良い。 [11] The succinic monoamide derivative residue and / or succinimide residue of R ₃ is represented by the general formulas (9), (10) and (11)

[Wherein, X ₄ is a bonding group to the terminal functional group F, R ₉ represents a hydroxyl group and / or —N (R ₁₀ ) CONH (R ₁₁ ), and R ₁₀ and R ₁₁ may be the same or different. Or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group, wherein R ₄ , R ₅ , R ₆ and R ₇ are Is synonymous with. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of the above [1] to [10], which is one or more groups selected from the group consisting of
The substituent containing a succinic acid monoamide derivative residue and / or a succinimide residue of R ₃ is preferably a residue or imide residue derived from aspartic acid monoamide. The R ₃ represents an aspect of a residue in which the nucleic acid antimetabolite is dissociated from a substituent containing a succinic acid monoamide unit to which the nucleic acid antimetabolite represented by the general formula (2) and / or (3) is bound. Show.
That is, the present invention relates to a substituent (R ₁ ) containing a succinic acid monoamide unit to which a nucleic acid antimetabolite is bound, a substituent (R ₂ ) containing a polyethylene glycol segment and a succinic acid monoamide derivative residue in the general formula (1). At least three kinds of substituents (R ₃ ) containing a group and / or a succinimide residue may be provided as separate substituents.

［式中、－Ｒｆは、式（１３）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基を示す。］で表されるいずれか１種以上の核酸代謝拮抗剤である、前記［１］～［１１］の何れか一項に記載の核酸代謝拮抗剤結合多分岐化合物。 [12] The nucleic acid antimetabolite is represented by the formula (12):

[Wherein, -Rf represents the formula (13):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of the above [1] to [11], which is any one or more nucleic acid antimetabolite represented by the formula:

 
［式中、－Ｒｆは、式（１５）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基を示す。］で表されるいずれか１種以上の核酸代謝拮抗剤である、前記［１］～［１２］の何れか一項に記載の核酸代謝拮抗剤結合多分岐化合物。 [13] The nucleic acid metabolism antagonist is represented by the formula (14):

[Wherein, -Rf represents the formula (15):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of the above [1] to [12], which is any one or more nucleic acid antimetabolite represented by the formula:

[14] A medicament comprising the nucleic acid antimetabolite-binding hyperbranched compound according to the above [1] to [13].

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention comprises a substituent containing a polyethylene glycol segment and a substituent containing a succinic acid monoamide unit bound to a nucleic acid antimetabolite on the terminal functional group of the multibranched polymer carrier. It is characterized by doing. The hyperbranched compound can exhibit a pharmacological activity by releasing the bound nucleic acid antimetabolite with an appropriate release profile while remaining in the blood and being distributed in the body when administered in vivo. As a result, side effects can be avoided while improving drug efficacy. In particular, it is possible to provide an anti-nucleic acid antimetabolite that does not prolong myelosuppression, which is a major side effect of anti-nucleic acid anti-metabolite, and can exert its medicinal effects continuously.

The present invention relates to a nucleic acid antimetabolite-binding multibranched compound comprising a substituent containing a succinic acid monoamide unit in which a multi-branched polymer carrier is used and a plurality of terminal groups are bound to a nucleic acid antimetabolite. Details of the present invention will be described below.

［式中、［Ｑ］は（ｍ＋ｎ＋ｏ＋ｐ）個の末端官能基化された多分岐高分子担体であり、（ｍ＋ｎ＋ｏ＋ｐ）は４～２００の整数であり、［Ｆ］は前記末端官能基であってアミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基、及び／又は置換基を有していても良い炭素数（Ｃ１～Ｃ６）のアルキル基を有する保護基で修飾された、アミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基であり、Ｆは前記末端官能基から水素原子又は水酸基が除かれた結合基であり、Ｒ_１は核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを含む置換基であり、Ｒ_２はポリエチレングリコールセグメントを含む置換基であり、Ｒ_３はコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基を含む置換基であり、ｍは０～１９９の整数であり、ｎは１～２００の整数であり、ｏは０～１９９の整数であり、ｐは０～１９９の整数である。］で示される核酸代謝拮抗剤結合多分岐化合物。である。 The nucleic acid antimetabolite-binding hyperbranched compound of the present invention has the general formula (1)

[Wherein [Q] is (m + n + o + p) terminal functionalized multi-branched polymer carrier, (m + n + o + p) is an integer of 4 to 200, and [F] is the terminal functional group. A protecting group having one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and / or an optionally substituted alkyl group having a carbon number (C1 to C6). Is one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and F is a linking group obtained by removing a hydrogen atom or a hydroxyl group from the terminal functional group. , R ₁ is a substituent containing a succinic acid monoamide units nucleic acid antimetabolites are bonded, R ₂ is a substituent containing a polyethylene glycol segment, R ₃ is Zanmoto及acid monoamide derivative And / or a substituent containing a succinimide residue, m is an integer from 0 to 199, n is an integer from 1 to 200, o is an integer from 0 to 199, and p is from 0 to 199. It is an integer. ] The nucleic acid antimetabolite binding hyperbranched compound shown by this. It is.

［式中、［Ｆ］は末端官能基であってアミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基であり、［Ｑ］、ｍ、ｎ、ｏ及びｐは前述と同義である。］で表される多分岐高分子担体に、Ｒ_１で表される核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを含む置換基を具備し、更に、任意にＲ_２で表されるポリエチレングリコールセグメントを含む置換基、並びにＲ_３で表されるコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基を含む置換基を結合させた核酸代謝拮抗剤結合多分岐化合物である。 That is, the nucleic acid antimetabolite-binding hyperbranched compound of the present invention has the general formula (16)

[Wherein [F] is a terminal functional group and one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and [Q], m, n, o, and p is as defined above. A polyethylene glycol segment having a substituent containing a succinic acid monoamide unit to which a nucleic acid antimetabolite represented by R ₁ is bound, and optionally represented by R _2. And a nucleic acid antimetabolite-binding hyperbranched compound to which a substituent containing a succinic acid monoamide derivative residue and / or a succinimide residue represented by R ₃ is bonded.

The multi-branched polymer carrier represented by the general formula (16) is a polymer compound having a multi-branched carrier containing a plurality of terminal functional groups as a polymer core. The terminal functional group includes a substituent containing a succinic acid monoamide unit to which a nucleic acid antimetabolite is bound, a substituent containing a polyethylene glycol segment, and a substituent containing a succinic acid monoamide derivative residue and / or a succinimide residue. Used as a binding functional group for binding.

The multi-branched polymer carrier includes a plurality of branched chains having a branched structure of an arbitrary shape from a core molecular species, and ends of the branched chains include a plurality of reactive functional groups represented by [F]. It is a molecular species having a molecular weight of 1 kilodalton or more.
The branch point of the branched chain is not particularly limited, and may be a so-called star polymer structure in which a plurality of molecular chains are extended from the core molecular species, and a branched chain having one or more branch points is the core. It may be a molecular species having a structure extended from the molecular species.
The branched chain has a structure extending at least 2 from the core molecular species. The plurality of molecular chains extending from the core molecular species may be the same or different from each other. Since the terminal reactive functional groups are preferably the same, it is preferable to use the same molecular chain.

When the branched chain is a branched chain having a plurality of branch points, the multi-branched polymer carrier is preferably a molecular species called a dendrimer or a hyperbranched polymer. Dendrimers or hyperbranched polymers, also called dendritic polymers, are polymer structures in which a plurality of regularly branched branches extend from a core molecular species. These are molecular species having a substantially spherical structure or a radial structure centered on the core molecular species, and having a reactive functional group at the end of the branched chain in the outer shell. In addition, the repeating unit of the hyperbranched structural unit which a dendrimer has is called a dendron. Dendrimers refer to regular repeating branched structures. On the other hand, the hyperbranched polymer refers to a polymer having an irregular repeating branch structure in which the regularity of the repeating branch structure is not as precise as that of the dendrimer and has a different molecular weight and degree of branching.

As a branched skeleton of the multi-branched polymer carrier, a polyamide structure branched by an amide bond, a polyamine structure branched by a tertiary amine, a polyester structure branched by an ester bond, a polyether structure branched by an ether bond, or Examples thereof include a structure having a mixed structure.
In dendrimers and hyperbranched polymers, the number of repeats of the branched structure is called generation (G), and the number of branched functional groups extending from the core molecular species (G0) is taken as the number of generations. Represent as In the present invention, when a dendrimer or hyperbranched polymer is used as the multi-branched polymer carrier, the number of generations is not particularly limited, but the number of generations is preferably G2 to G6, more preferably G3 to G5.

The terminal reactive functional group of the multi-branched polymer carrier is one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group. These functional groups are distributed in the outermost shell of the multi-branched polymer carrier and are used as a linking group for chemical modification of the outer shell structure by a succinic acid monoamide unit or a polyethylene glycol segment to which a nucleic acid antimetabolite is bound.
F in the general formula (1) is a linking group in which a hydrogen atom is removed from an amino group, a hydroxyl group or a mercapto group, or a linking group in which a hydroxyl group is removed from a carboxy group. In the multi-branched polymer carrier, the number of terminal reactive functional groups (m + n + o + p) is 4 or more and 200 or less. Preferably, the terminal reactive functional group has 4 or more and 150 or less, particularly preferably 8 or more and 100 or less. Dendrimers and hyperbranched polymers, which are multi-branched polymer carriers used in the present invention, have a multiple of terminal functional groups depending on the number of generations. Dendrimers and hyperbranched polymers are known and may be appropriately selected and used.
The terminal reactive functional groups may be the same functional group or different functional groups. However, since the terminal reactive functional group is a linking group that undergoes chemical modification with a nucleic acid antimetabolite-binding succinic acid monoamide or a polyethylene glycol segment, it is preferably the same functional group. The terminal reactive functional group is preferably a hydroxyl group or a carboxy group.

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention is intended to control pharmacokinetics by using a polymer carrier, and thus an appropriate molecular weight range is set for the polymer carrier. The multi-branched polymer carrier is preferably a molecule having a molecular weight of 1 kilodalton or more and 100 kilodalton or less. More preferably, it is a multi-branched polymer carrier of 3 kilodalton or more and 70 kilodalton or less.

The multi-branched polymer carrier can be prepared by a known method. That is, as a method of synthesizing a polymer carrier having a plurality of branches based on the core, a divergent method in which molecules are sequentially bonded to the core molecules for each generation, or branched in advance. There are two known synthesis methods, a convergent method in which a branch is reacted with a core molecule. The multi-branched polymer carrier used in the present invention can be prepared by any synthesis method.

Commercially available products may be used as the dendrimer, dendron and hyperbranched polymer used as the multi-branched polymer carrier. As the dendrimer, for example, a polyamidoamine (PAMAM) dendrimer (Generation 0 to 7, several kinds of terminal functional groups such as a hydroxyl group, an amino group, a carboxy group, and a trimethoxysilyl group) (Sigma-Aldrich) may be used. Examples of the dendron include polyester-poly-hydroxy-1-acetylene bis-MPA dendron (hydroxy group: 8-32, Generation 3-5), polyester-poly-hydroxy-1-carboxy bis-MPA dendron (hydroxyl group: 8-32, Generation 3-5) (Sigma-Aldrich) may be used. As the hyperbranched polymer, hyperbranched bis-MPA polyester-poly-hydroxy (hydroxyl groups 16 to 64, Generation 2 to 4) (Sigma-Aldrich) may be used.
A commercially available multi-branched polymer carrier may be used in which these terminal reactive functional groups are converted into arbitrary functional groups by chemical modification by a normal organic reaction. For example, when converting a multi-branched polymer carrier having a hydroxyl group or amino group as a terminal reactive functional group to a multi-branched polymer carrier having a terminal carboxy group, for example, by reacting with any acid anhydride compound, ester The terminal reactive functional group can be converted to a carboxy group via an oxidization reaction or an amidation reaction.

In the nucleic acid antimetabolite-binding multibranched compound of the present invention, the nucleic acid antimetabolite is bonded to the carboxy group of the succinic acid monoamide unit via an amide bond and / or an ester bond. Preferably, the nucleic acid antimetabolite having an amino group and / or a hydroxyl group is used, and the structural unit is bonded by an amide bond with the carboxy group by the amino group or an ester bond with the carboxy group by the hydroxyl group. The bonding mode may be any of an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite to be used, the binding mode of the succinic acid monoamide to the carboxy group may be appropriately selected.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｘは前記末端官能基Ｆとの結合基であり、Ｙは酸素原子又はＮ－Ｒ_４であり、前記Ｒ_４並びにＲ_５及びＲ_６はそれぞれ独立して水素原子又は炭素数（Ｃ１～Ｃ８）のアルキル基であり、Ｒ_７は水素原子、置換基を有していても良い炭素数（Ｃ１～Ｃ２０）の直鎖状、分岐鎖状又は環状のアルキル基、置換基を有していても良い炭素数（Ｃ７～Ｃ２０）の直鎖状、分岐鎖状又は環状のアラルキル基、置換基を有していても良い芳香族基及びカルボキシ基が保護されたアミノ酸残基からなる群から選択される１種以上の基である。］で示される核酸代謝拮抗剤が結合したコハク酸モノアミドユニットが好ましい。
　前記一般式（１７）及び（１８）で示されるコハク酸モノアミドユニットは、光学活性体であっても良く、光学活性体の任意の混合物であっても良い。 The succinic acid monoamide unit to which the nucleic acid antimetabolite represented by R ₁ in the general formula (1) is bound is represented by the following general formula (17) and / or (18)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, X is a binding group to the terminal functional group F, Y is an oxygen atom or N—R ₄ , and R _{4 and} R ₅ and R ₆ are each independently a hydrogen atom or an alkyl group having a carbon number (C1 to C8), and R ₇ is a hydrogen atom or a straight chain having a carbon number (C1 to C20) optionally having a substituent. A linear, branched or cyclic aralkyl group having a carbon number (C7 to C20), which may have a linear, branched or cyclic alkyl group, or a substituent, or a substituent. One or more groups selected from the group consisting of amino acid residues in which an aromatic group and a carboxy group are protected. ] The succinic acid monoamide unit which the nucleic acid antimetabolite shown by these couple | bonded is preferable.
The succinic acid monoamide unit represented by the general formulas (17) and (18) may be an optically active substance or an arbitrary mixture of optically active substances.

In the succinic acid monoamide unit represented by the general formula (17) or (18), in the formula, the alkyl group having carbon atoms (C1 to C8) in R ₄ , R ₅ and R ₆ is linear or branched. Alternatively, it is a cyclic alkyl group (C1 to C8).
Examples of the linear alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-hexyl group.
Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like.
Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and the like.

R _{7 according} to the general formula (17) or (18) has a linear, branched or cyclic alkyl group having a carbon number (C1 to C20) which may have a substituent, and a substituent. A linear, branched or cyclic aralkyl group having a carbon number (C7 to C20) which may be present, and an aromatic group which may have a substituent.
Examples of the substituent which may have here include mercapto group, hydroxyl group, halogen atom, nitro group, cyano group, carbocyclic or heterocyclic aryl group, alkylthio group, arylthio group, alkylsulfinyl group, arylsulfinyl group, alkylsulfonyl Group, arylsulfonyl group, sulfamoyl group, alkoxy group, aryloxy group, acyloxy group, alkoxycarbonyloxy group, carbamoyloxy group, substituted or unsubstituted amino group, acylamino group, alkoxycarbonylamino group, ureido group, sulfonylamino group, Examples thereof include a sulfamoylamino group, a formyl group, an acyl group, a carboxy group, an alkoxycarbonyl group, a carbamoyl group, and a silyl group. The substitution position on the aromatic ring may be the ortho position, the meta position, or the para position.
Moreover, you may have comprised the polyethyleneglycol segment as a substituent. R ₇ has a polyethylene glycol segment as a substituent, and has a carbon number (C1 to C20) linear, branched or cyclic alkyl group, carbon number (C7 to C20) linear, branched or When it is a group selected from the group consisting of a cyclic aralkyl group and an aromatic group, R _{1 according} to the general formula (1) contains a succinic acid monoamide unit to which a nucleic acid antimetabolite is bound, and a polyethylene glycol segment. It becomes the substituent which it has together.
Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C20) which may have a substituent include a methyl group, an ethyl group, an isopropyl group, a t-butyl group, a cyclohexyl group, Examples include n-octyl group, dodecyl group, and octadecyl group.
Examples of the linear, branched or cyclic aralkyl group having a carbon number (C7 to C20) which may have a substituent include, for example, benzyl group, 2-phenylethyl group, 4-phenylbutyl group, 8 -Phenyloctyl group and the like.
Examples of the aromatic group having a carbon number (C5 to C20) which may have a substituent include a phenyl group, a 4-methoxyphenyl group, a 4-dimethylaminophenyl group, and a 4-hydroxyphenyl group. .

R ₇ may be an amino acid residue in which a carboxy group is protected. Examples of amino acid residues in which the carboxyl group is protected include glycinyl-methyl ester group, alanyl-methyl ester group, leucinyl-methyl ester group, valinyl-methyl ester group, phenylalanyl-methyl ester group, alanyl-ethyl ester Group, leucinyl-ethyl ester group, alanyl-butyl ester group, leucinyl-butyl ester group and the like.
Alternatively, it may be an amino acid residue in which a polyethylene glycol segment is bonded to a carboxy group by an amide bond or an ester bond.

The succinic acid monoamide unit to which the nucleic acid antimetabolite is bound may be a polymer of succinic acid monoamide unit to which the nucleic acid antimetabolite is bound. That is, when the succinic acid monoamide unit is an aspartic acid monoamide unit, a polyaspartic acid derivative may be used. When polyaspartic acid is used, one of the carboxy groups naturally becomes a monoamide, and therefore can be mentioned as a preferred substituent.
In the case of a polymer of the succinic acid monoamide unit, a polymer having a polymerization number of 1 to 50 is preferable. More preferably, the polymerization number is 2-30. The polymer of the succinic acid monoamide unit polymer may be a polymer based on α-amide bonds or a polymer based on β-amide bonds, and may be a polymer based on a mixture of α and β-amide bonds. Or either.

When a polymer of a succinic acid monoamide unit is used as the succinic acid monoamide unit to which the nucleic acid antimetabolite binds, the nucleic acid antimetabolite needs only have one or more molecules bonded to the polymer substituent. The above plural molecules may be bonded. This is an advantageous embodiment because the drug content can be increased by binding a plurality of nucleic acid antimetabolites. In this case, the binding mode of the nucleic acid antimetabolite is the same as that described above, and is bonded via an amide bond and / or an ester bond.
The C-terminal carboxy group of the succinic acid monoamide unit polymer is preferably modified with an appropriate protecting group. Examples thereof include ester protecting groups such as methyl ester group, ethyl ester group, butyl ester group, and benzyl ester group, or amide protecting groups such as methylamide group, ethylamide group, butylamide group, and benzylamide group.
Alternatively, it may be an amino acid residue in which a polyethylene glycol segment is bonded to a carboxy group by an amide bond or an ester bond. In this case, R _{1 according} to the general formula (1) is a substituent that includes a succinic acid monoamide unit to which a plurality of nucleic acid antimetabolites are bonded, and also has a polyethylene glycol segment.

When Y is an oxygen atom, the succinic monoamide unit is a unit having malic acid as a basic skeleton. On the other hand, when Y is N—R ₄ , the succinic monoamide unit is a unit having aspartic acid as a basic skeleton. That is, when R ₅ and R ₆ are hydrogen atoms, malic acid monoamide derivatives or aspartic acid monoamide derivatives can be used as the succinic acid monoamide unit. The succinic acid monoamide unit is preferably an aspartic acid monoamide derivative.

X in the general formula (17) and / or (18) represents a substituent containing a succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ is bonded and a terminal reactive functional group of the multi-branched polymer carrier [F ]. As the linking group, an oxygen atom or N—R ₄ of the terminal functional group of the substituent containing the succinic acid monoamide unit and a functional group capable of binding to the terminal reactive functional group [F], respectively, are bonded to both terminals. As long as it is a linking group possessed by, there is no particular limitation.

When the terminal functional group of the substituent containing the succinic acid monoamide unit is an oxygen atom, the bonding group according to X is a bonding functional group in which one terminal group is an ether bond, ester bond, urethane bond or carbonate bond Have On the other hand, when the terminal functional group of the substituent containing the succinic acid monoamide unit is N—R ₄ , one terminal group has a binding functional group capable of amine bonding, amide bonding, urea bonding, or urethane bonding. The other terminal group has a binding functional group that can form an ester bond, an amide bond, a thioester bond, a urea bond, or a urethane bond with the terminal reactive functional group [F]. That is, the linking group according to X is an alkylene group having the above-mentioned terminal functional group and optionally having a substituent (C1-C8).

The linking group according to X is an ether bond when the terminal functional group of the substituent containing the succinic acid monoamide unit is an oxygen atom, or the terminal functional group is N—R ₄ to form an amino bond, As a linking group that bonds to the terminal reactive functional group [F] with an amide bond, an ester bond or a thioester bond, for example, — (CH ₂ ) _x —NH— (x represents an integer of 1 to 8), — (CH ₂ ) _X —O— (x represents an integer of 1 to 8), — (CH ₂ ) _x —S— (x represents an integer of 1 to 8), — (CH ₂ ) _x —CO— (x represents And an integer of 1 to 8).
When the terminal functional group of the substituent containing the succinic acid monoamide unit is an oxygen atom, an ester bond is formed, or the terminal functional group is NR ₄ to form an amide bond, and the other is a terminal reactive functional group [F]. As a linking group that bonds to an amide bond, an ester bond or a thioester bond, for example, —CO— (CH ₂ ) _x —NH— (x represents an integer of 1 to 8), —CO— (CH ₂ ) _x —O— (X represents an integer of 1 to 8), —CO— (CH ₂ ) _x —S— (x represents an integer of 1 to 8), —CO— (CH ₂ ) _x —CO— (x is 1) Represents an integer of ˜8).
When the terminal functional group of the substituent containing the succinic acid monoamide unit is an oxygen atom, a urethane bond is formed, or the terminal functional group is NR ₄ to form a urea bond, and the other is a terminal reactive functional group [F]. As a linking group that bonds to an amide bond, an ester bond or a thioester bond, for example, —CONH— (CH ₂ ) _x —NH— (x represents an integer of 1 to 8), —CONH— (CH ₂ ) _x —O— (X represents an integer of 1 to 8), —CONH— (CH ₂ ) _x —S— (x represents an integer of 1 to 8), —CONH— (CH ₂ ) _x —CO— (x is 1) Represents an integer of ˜8).
Further, when the terminal functional group of the substituent containing the succinic acid monoamide unit is an oxygen atom, a carbonate bond is formed, or the terminal functional group is N—R ₄ to form a urethane bond, and the other is a terminal reactive functional group [ F] can be linked to an amide bond, an ester bond or a thioester bond, for example, —COO— (CH ₂ ) _x —NH— (x represents an integer of 1 to 8), —COO— (CH ₂ ) _x — O— (x represents an integer of 1 to 8), —COO— (CH ₂ ) _x —S— (x represents an integer of 1 to 8), —COO— (CH ₂ ) _x —CO— (x Represents an integer of 1 to 8).
X is preferably an ether bond when the terminal functional group of the substituent containing the succinic acid monoamide unit is an oxygen atom, or the terminal functional group is N—R ₄ to form an amino bond, and the terminal reactive functional group A linking group that bonds with [F] by amide bonding, and is — (CH ₂ ) _x —NH— (x represents an integer of 1 to 8).

In addition, an amino acid derivative may be used as the linking group related to X. When the amino acid derivative is used as a linking group, the linking group is used in such a manner that the N-terminal amino group of the amino acid derivative is amide-bonded to the side chain carboxy group, and the C-terminal carboxy group is substituted with the succinic acid monoamide unit. In this embodiment, the terminal functional group of the group is an oxygen atom and an ester bond, or the terminal functional group is an amide bond of N—R ₄ .
When an amino acid derivative is used as the linking group related to X, the amino acid used may be a natural amino acid or a non-natural amino acid, and any of L-form and D-form can be used without particular limitation. For example, hydrocarbon amino acids such as glycine, β-alanine, alanine, leucine and phenylalanine, acidic amino acids such as aspartic acid and glutamic acid, basic amino acids such as lysine, arginine and histidine can be used.

Further, the X may be a “bond”. The term “bond” means that the terminal reactive functional group of the multi-branched polymer carrier and the terminal functional group of the substituent containing the succinic acid monoamide unit directly form an ester bond or an amide bond without using a bonding group. Refers to the embodiment.

[D] according to the general formula (17) or (18) is a binding residue of a nucleic acid antimetabolite. The nucleic acid antimetabolite has an amino group and / or a hydroxyl group in the molecule, and the nucleic acid antimetabolite binds to the carboxy group of the succinic acid monoamide unit via an amide bond and / or an ester bond. That is, [D] is an amide bond residue and / or an ester bond residue of the nucleic acid antimetabolite. The binding mode of the nucleic acid antimetabolite may be either an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite used, the mode of binding to the carboxy group may be appropriately selected.

The nucleic acid antimetabolite used in the present invention is a nucleoside derivative having pharmacological activity such as antitumor activity or antiviral activity. As the nucleic acid metabolism antagonist, it is preferable to use pyrimidine base nucleoside derivatives, purine base nucleoside derivatives, triazine base nucleoside derivatives, and the like. The nucleic acid metabolism antagonist is preferably a compound having an amino group and / or a hydroxyl group in the molecule. Such a nucleic acid antimetabolite is preferable because it can be introduced into the carboxy group of the succinic acid monoamide unit through an amide bond and / or an ester bond with the amino group and / or hydroxyl group.

Particularly, it is preferable to use a nucleic acid antimetabolite having an amino group at the nucleoside of the nucleoside, and a pyrimidine base nucleoside derivative having an amino group, a purine base nucleoside derivative having an amino group, and a triazine base nucleoside derivative having an amino group are preferable. A nucleic acid antimetabolite having an amino group is preferable because it can be introduced into the carboxy group of the succinic acid monoamide unit by an amide bond by the amino group.

As a nucleic acid metabolism antagonist, a plurality of compounds having antitumor activity and antiviral activity are known, and these may be appropriately selected and used. As the nucleic acid antimetabolite that can be used, a nucleic acid antimetabolite having an amino group and / or a hydroxyl group is preferable. Examples of the nucleic acid antimetabolite include pyrimidine antimetabolite, purine antimetabolite, triazine Antimetabolites etc. are mentioned.

［式中、－Ｒｆは、式（１３）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基残基を示す。］。 In the nucleic acid metabolism antagonist, the nucleobase moiety is any one or more selected from the following formula (12), and the group (Rf) bonded thereto is selected from the following formula (13) A nucleic acid antimetabolite that is a combination of more than one species is particularly preferred.

[Wherein, -Rf represents the formula (13):

R ₂₀ represents a hydrogen atom or an acyl group residue of a fatty acid ester. ].

The acyl group of the fatty acid ester in R ₂₀ is an acyl residue in which a monocarboxylic acid having a carbon number (C4 to C30) is ester-bonded. The hydrocarbon having a carbon number (C4 to C30) may be a saturated fatty acid that is a saturated hydrocarbon, or an unsaturated fatty acid that is an unsaturated hydrocarbon containing one or more double bonds. These fatty acid esters are known as fat-soluble derivatives of the nucleic acid antimetabolite, and can be used as an active ingredient of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention.
Wherein the fatty acyl group residue of a fatty acid ester as R _20, examples of the saturated fatty acid, butanoic acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic An acid, docosanoic acid, etc. are mentioned.
Examples of the unsaturated fatty acid include 9-hexadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, cis, cis-9,12-octadecadienoic acid, and 9,12,15-octadecatrienoic acid. 6,9,12-octadecatrienoic acid, 5,8,11,14-eicosatetraenoic acid, and the like.

A known compound may be used as the nucleic acid metabolism antagonist. For example, cytarabine, gemcitabine, azacitidine, decitabine, nelarabine, 2′-methylidene-2′-deoxycytidine (DC), having the structure shown in the following formula (19): Troxacitabine, 3′-ethynylcytidine, 2′-cyano-2′-deoxy-1-β-D-arabinofuranosylcytosine (CNDAC), 2′-deoxy-5,6-dihydro- 5-azacytidine (DHAC), 5′-fluoro-2′-deoxycytidine (NSC-48006), 4′-thio-β-D-arabinofuranosylcytosine (OSI-7836), cladri Cladribine, Clofarabine or Fludarabine, cytarabine-5′-elaidic acid ester (CP-4055), gemcitabine-5′-elaidic acid ester (CP-4126), azacitidine-5′-elaidic acid And esters (CP-4200). The nucleic acid antimetabolite used in the hyperbranched compound of the present invention is not limited to these compounds, but can be mentioned as a preferred compound to be applied.

As the nucleic acid antimetabolite in the present invention, a cytidine antimetabolite is preferably used. The nucleobase moiety is a cytidine base represented by the following formula (14), and the group (Rf) bonded thereto is represented by the following formula ( The nucleic acid antimetabolite is particularly preferably a combination of any one or more selected from the group of substituents 15). Here, R ₂₀ is a compound represented by a hydroxyl group or an acyl group of a fatty acid ester.

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基を示す。］。一般式（１５）のＲ_２０における脂肪酸エステルのアシル基は、前述と同義である。 [Wherein, -Rf represents the formula (15):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. ]. The acyl group of the fatty acid ester in R ₂₀ of the general formula (15) has the same meaning as described above.

These cytidine antimetabolites are gemcitabine and its fatty acid ester derivative, cytarabine and its fatty acid ester derivative, and 3'-ethynylcytidine and its fatty acid ester derivative. Examples of fatty acid ester derivatives include cytarabine-5'-elaidic acid ester (CP-4055) and gemcitabine-5'-elaidic acid ester (CP-4126).

In the general formula (1), the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ binds is an essential component in the present invention. Therefore, n, which is the number of substituent bonds in the R ₁ group, is an integer of 1 to 200. By providing many R ₁ groups, the content of the nucleic acid antimetabolite per molecule of the multi-branched carrier can be increased. On the other hand, taking into account the use of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention as a pharmaceutical, in consideration of optimization of water solubility and pharmacokinetic properties, n, the number of substituents bonded to the R ₁ group, ranges from 2 to 100. It is preferable that Particularly preferably, n is 5 to 50.

In the nucleic acid antimetabolite-bound hyperbranched compound of the present invention, a substituent containing a polyethylene glycol segment is bound. R ₂ in the general formula (1) is a substituent containing a polyethylene glycol segment. The polyethylene glycol segment is a segment having a repeating structure of an ethyleneoxy group; (CH ₂ CH ₂ O) unit. The degree of polymerization of ethyleneoxy group units is preferably 5 to 10,000 units, more preferably the degree of polymerization is 5 to 2,500 units, particularly preferably 10 to 1,000 units, and even more preferably 20 to 500 units. It is the segment structure containing the polyethyleneglycol chain of.
That is, the polyethylene glycol segment is preferably a segment having a molecular weight equivalent to polyethylene glycol of 0.2 kilodaltons or more and 500 kilodaltons or less, more preferably a molecular weight of 0.2 kilodaltons or more and 150 kilodaltons or less. It is a structural part, and the molecular weight is particularly preferably 0.5 kilodaltons or more and 50 kilodaltons or less. Particularly preferred is a polyethylene glycol segment having a molecular weight of 1 kilodalton or more and 20 kilodalton or less.
The molecular weight of the polyethylene glycol segment used in the present invention is determined by the GPC method based on the polyethylene glycol standard product of the polyethylene glycol segment structural compound used in preparing the nucleic acid antimetabolite-binding hyperbranched compound of the present invention. The average molecular weight calculated | required by the peak top molecular weight measured is employ | adopted.

One end group of the polyethylene glycol segment is a binding side with the multi-branched polymer carrier, and the other is a shell side of the nucleic acid antimetabolite-binding multi-branching compound of the present invention.
The terminal group on the bonding side with the multi-branched polymer carrier is not particularly limited, but an oxygen atom of an ethyleneoxy group; (CH ₂ CH ₂ O) unit is preferably the terminal group. In the substituent containing the polyethylene glycol segment according to R ₂ , the linking group with the terminal reactive functional group of the multi-branched polymer carrier is not particularly limited, and the terminal group of the polyethylene glycol segment and the multiple groups are not limited. It is preferably a carbon number (C1-C8) alkylene group which has a functional group capable of bonding to the terminal reactive functional group of the branched polymer carrier and may have a substituent.

On the other hand, the terminal group on the outer shell side of the polyethylene glycol segment is not particularly limited, and may be a hydrogen atom, a hydroxyl group, an optionally substituted alkoxy group having a carbon number (C1 to C8), or a substituent. An aralkyloxy group having a carbon number (C7 to C20) which may have a carbon atom and the like can be used.

Examples of the alkoxy group having a carbon number (C1 to C8) which may have a substituent in the terminal group include a linear, branched or cyclic alkoxy group having a carbon number (C1 to C8). For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, t-butoxy group, n-pentyloxy group, isopentyloxy group, 2-methylbutoxy group, neopentyloxy Group, 1-ethylpropoxy group, n-hexyloxy group, 4-methylpentyloxy group, 3-methylpentyloxy group, 2-methylpentyloxy group, 1-methylpentyloxy group, 3,3-dimethylbutoxy group, 2,2-dimethylbutoxy group, 1,1-dimethylbutoxy group, 1,2-dimethylbutoxy group, 1,3-dimethylbutoxy group, 2,3-dimethylbutoxy group, 2-ethylbutoxy group, cyclopropoxy group, Examples include a cyclopentyloxy group or a cyclohexyloxy group. Preferred is an alkoxy group having a carbon number (C1 to C4), such as a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an s-butoxy group, or a t-butoxy group. Particularly preferred is a methoxy group, an ethoxy group, an n-propoxy group or an isopropoxy group.
Examples of the substituent that may have include a hydroxyl group, an amino group, a formyl group, and a carboxy group.

The aralkyloxy group having a carbon number (C7 to C20) which may have a substituent in the terminal group is a linear or branched alkyl group in which any one hydrogen atom is substituted with an aryl group. is there. For example, benzyloxy group, 2-phenylethyloxy group, 4-phenylbutyloxy group, 3-phenylbutyloxy group, 5-phenylpentyloxy group, 6-phenylhexyloxy group, 8-phenyloctyloxy group, etc. It is done. A benzyloxy group, a 4-phenylbutyloxy group, and an 8-phenyloctyloxy group are preferable.
Examples of the substituent that may have include a hydroxyl group, an amino group, a formyl group, and a carboxy group.

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_１は末端官能基Ｆとの結合基を示す。］を用いることが好ましい。
　すなわち、エチレンオキシ基；（ＣＨ_２ＣＨ_２Ｏ）単位の繰り返し構造によるエチレンオキシ基単位重合度が５～２，５００ユニットのポリエチレングリコールセグメントであり、ポリエチレングリコール相当の平均分子量として０．２キロダルトン～１５０キロダルトンのセグメント部であることが好ましい。より好ましくは重合度が１０～１，０００ユニットであり、平均分子量として０．５キロダルトン～５０キロダルトンであり、更に好ましくは重合度が２０～５００ユニットで、平均分子量として１キロダルトン～２０キロダルトンであり、殊更好ましくは重合度が２０～３００ユニットで、平均分子量として１キロダルトン～１２キロダルトンのポリエチレングリコール鎖を含むセグメント構造である。 In the general formula (1), R ₂ represents the general formula (8).

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₁ represents a bonding group with the terminal functional group F. ] Is preferably used.
That is, it is a polyethylene glycol segment having an ethyleneoxy group; the degree of polymerization of the ethyleneoxy group unit having a repeating structure of (CH ₂ CH ₂ O) units of 5 to 2,500 units, and an average molecular weight equivalent to polyethylene glycol is 0.2 kilodalton. A segment of ~ 150 kilodaltons is preferred. More preferably, the degree of polymerization is from 10 to 1,000 units, and the average molecular weight is from 0.5 kilodaltons to 50 kilodaltons, and still more preferably, the degree of polymerization is from 20 to 500 units, and the average molecular weight is from 1 kilodaltons to 20 units. A segment structure having a degree of polymerization of 20 to 300 units and a polyethylene glycol chain having an average molecular weight of 1 to 12 kilodaltons is particularly preferred.

Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent in R ₈ include, for example, a methyl group, an ethyl group, and the like. Group, n-propyl group, n-butyl group, n-hexyl group, n-decyl group and the like. Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and an adamantyl group.
Examples of the substituent that may have include a hydroxyl group, an amino group, a formyl group, and a carboxy group.

X ₁ in the general formula (8) is a linking group that binds the substituent containing the polyethylene glycol segment according to R ₂ and the terminal reactive functional group [F] of the multi-branched polymer carrier. The linking group is particularly limited as long as it is a linking group having functional groups capable of binding to the oxygen atom of the polyethylene glycol segment end group and the terminal reactive functional group [F] at both ends. It is not a thing.

Binding groups according to the X ₁ has one end group, the terminal oxygen atoms and ether bond mode of the polyethylene glycol segment, an ester bond, have the binding functional group of a urethane bond or a carbonate bond, the other end group May have a substituent having a bonding functional group capable of forming an ester bond, an amide bond, a thioester bond, a urea bond or a urethane bond with the terminal reactive functional group [F] (C1˜ C8) is an alkylene group.

Examples of the linking group according to X ₁ include an ether bond with a polyethylene glycol segment, and a linking group having an amide bond, an ester bond or a thioester bond with a terminal reactive functional group [F], such as — (CH ₂ ) _x —NH -(X represents an integer of 1 to 8),-(CH ₂ ) _x -O- (x represents an integer of 1 to 8),-(CH ₂ ) _x -S- (x is 1 to 8) An integer).
As a linking group that ester bonds to a polyethylene glycol segment and bonds to a terminal reactive functional group [F] with an amide bond, an ester bond or a thioester bond, for example, —CO— (CH ₂ ) _x —NH— (where x is 1-8) -CO- (CH ₂ ) _x -O- (x represents an integer of 1 to 8), -CO- (CH ₂ ) _x -S- (x represents an integer of 1 to 8) Etc.
Examples of a linking group that is urethane-bonded to a polyethylene glycol segment and amide bond, ester bond, or thioester bond to the terminal reactive functional group [F] are, for example, —CONH— (CH ₂ ) _x —NH— (where x is 1 to 8). -CONH- (CH ₂ ) _x -O- (x represents an integer of 1 to 8), -CONH- (CH ₂ ) _x -S- (x represents an integer of 1 to 8) Etc.
Further, as a linking group that bonds to a polyethylene glycol segment and bonds to a terminal reactive functional group [F] with an amide bond, an ester bond or a thioester bond, for example, —COO— (CH ₂ ) _x —NH— (where x is 1 to 8 represents an integer of 8), —COO— (CH ₂ ) _x —O— (x represents an integer of 1 to 8), —COO— (CH ₂ ) _x —S— (x represents an integer of 1 to 8) For example).
X ₁ is preferably a linking group that is ether-bonded to a polyethylene glycol segment and amide-bonded to the terminal reactive functional group [F], and — (CH ₂ ) _x —NH— (x is an integer of 1 to 8). Show).

It is also possible to use an amino acid derivative as binding group according to the X _1. When the amino acid derivative is used as a linking group, the linking group is used in such a manner that the N-terminal amino group of the amino acid derivative is amide-bonded with the side chain carboxy group, and the C-terminal carboxy group is the terminal oxygen atom of the polyethylene glycol segment. And an ester bond.
When using an amino acid derivative as binding group according to the X _1, amino acids used may be natural amino acids or unnatural amino acids, L body, can be used without being limited particularly either D-form. For example, hydrocarbon amino acids such as glycine, β-alanine, alanine, leucine and phenylalanine, acidic amino acids such as aspartic acid and glutamic acid, basic amino acids such as lysine, arginine and histidine can be used.

The X ₁ may be a “bond”. The “bond” refers to an embodiment in which the terminal reactive functional group of the multi-branched polymer carrier and the terminal oxygen atom of the polyethylene glycol segment are directly ester-bonded without using a bonding group.

In the general formula (1), 0 to 199 units of the substituent containing the polyethylene glycol segment represented by R ₂ are bonded to the terminal reactive functional groups present in the multi-branched polymer carrier. That is, o, which is the number of substituents bonded to the R ₂ group, is an integer of 0 to 199.
The nucleic acid antimetabolite-binding hyperbranched compound of the present invention is used as a pharmaceutical product. For this reason, it is preferable because water solubility can be imparted by providing a polyethylene glycol segment. In the present invention, it is preferable that the multi-branched polymer carrier has a polyethylene glycol segment. The polyethylene glycol segment may be provided as R ₂ , or may be bonded as a substituent to the nucleic acid antimetabolite-binding succinic acid monoamide of R ₁ . Or it may be allowed to include a polyethylene glycol segment both R ₁ and R _2.
In the present invention, when R ₁ does not have a polyethylene glycol segment, the substituent containing the polyethylene glycol segment according to R ₂ is an essential substituent, and o which is the number of substituents bonded to the R ₂ group is It is an integer from 1 to 199. When the R ₂ group is provided with a polyethylene glycol segment, it is preferable to combine a plurality of units. Therefore, o which is the number of substituent bonds of the R ₂ group is more preferably an integer of 2 to 100, and particularly preferably 2 to 50.

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention has a substituent containing a succinic acid monoamide unit and a polyethylene glycol segment containing a nucleic acid antimetabolite bonded to the terminal reactive functional group of the multi-branched polymer carrier. Further, it may be a compound to which a substituent containing a succinic acid monoamide derivative residue and / or a succinimide residue is bonded.
That is, in the general formula (1), a substituent (R ₁ ) bound to a succinic acid monoamide unit bound to a nucleic acid antimetabolite is bound to a substituent (R ₂ ) containing a polyethylene glycol segment, and further succinic acid substituents containing monoamide derivative residue and / or succinimide residue (R ₃₎ may be provided with a.
The succinic acid monoamide derivative residue and / or succinimide residue according to R ₃ is dissociated from the substituent containing the succinic acid monoamide unit to which the nucleic acid metabolism antagonist according to R ₁ is bound. Residue.

［式中、Ｘは前記末端官能基Ｆとの結合基であり、Ｙは酸素原子又はＮ－Ｒ_４であり、Ｒ_９は水酸基及び／又は－Ｎ（Ｒ_１０）ＣＯＮＨ（Ｒ_１１）を示し、Ｒ_１０、Ｒ_１１は同一でも異なっていてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基を示し、Ｘ、Ｒ_４、Ｒ_５、Ｒ_６及びＲ_７は、一般式（１７）及び（１８）に記載した置換基と同義である。］からなる置換基群から選ばれる１種以上の基である。 R ₃ is preferably represented by the general formulas (20), (21) and (22).

[Wherein, X is a bonding group to the terminal functional group F, Y is an oxygen atom or N—R ₄ , and R ₉ is a hydroxyl group and / or —N (R ₁₀ ) CONH (R ₁₁ ). , R ₁₀ and R ₁₁ may be the same or different and each represents a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group, , R ₄ , R ₅ , R ₆ and R ₇ are synonymous with the substituents described in the general formulas (17) and (18). ] One or more groups selected from the substituent group consisting of

Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group in R ₁₀ and R ₁₁ include, for example, a methyl group, an ethyl group, n -Propyl group, isopropyl group, isobutyl group, sec-butyl group, t-butyl group, 1-methylbutyl group, 2-methylbutyl group, neopentyl group, cyclohexyl group, etc., preferably isopropyl group, cyclohexyl group Can be mentioned.
Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with the tertiary amino group include, for example, 2-dimethylaminoethyl group, 3-dimethylaminopropyl group , 5-dimethylaminopentyl group, 6-dimethylaminohexyl group and the like.
R ₁₀ and R ₁₁ are preferably an ethyl group, an isopropyl group, a cyclohexyl group, and a 3-dimethylaminopropyl group.

When R ₉ is a hydroxyl group, it represents a carboxylic acid embodiment. Moreover, the arbitrary salt aspect of the carboxylic acid may be sufficient.
R ₉ is a hydroxyl group and / or —N (R ₁₀ ) CONH (R ₁₁ ), but when it is only a hydroxyl group, a hydroxyl group and —N (R ₁₀ ) CONH (R ₁₁ ) coexist, or —N The mode in the case of (R ₁₀ ) CONH (R ₁₁ ) alone can be taken. The abundance ratio of the hydroxyl group to —N (R ₁₀ ) CONH (R ₁₁ ) may be arbitrarily set.

In the general formula (1), the substituent containing a succinic acid monoamide derivative residue and / or a succinimide residue represented by R ₃ is a succinic acid monoamide unit to which a nucleic acid antimetabolite according to R ₁ is bound. Since the nucleic acid antimetabolite is a dissociated residue, it is an optionally present group. P which is the number of substituents of the R ₃ group is an integer of 0 to 199.
The nucleic acid antimetabolite-binding hyperbranched compound of the present invention has a property of dissociating a nucleic acid antimetabolite over time in an aqueous solution. Accordingly, the substituent of the R ₁ is dissociated nucleic acid metabolism antagonist, because it is converted into a substituent of the R _3, number of bonds of R ₁ and R ₃ is accompanied by aging. The substituent according to R ₃ is used for dissociation of the nucleic acid antimetabolite during production of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention, during manufacture of a pharmaceutical preparation, during storage of a pharmaceutical preparation, or when used as a pharmaceutical. It can be generated at any time. P, which is the number of substituents bonded to the R ₃ group, is preferably an integer of 0 to 80, and more preferably 0 to 50.

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention comprises a substituent containing a succinic acid monoamide unit to which the nucleic acid antimetabolite is bound, a substituent containing the polyethylene glycol segment, and a succinic acid monoamide derivative residue and / or A terminal functional group to which a substituent containing a succinimide residue is not bonded may be included. In this case, these terminal functional groups are one or more terminal functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group represented by [F] in the general formula (1).

The terminal functional group may be selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group modified with a protecting group having an alkyl group having a carbon number (C1 to C6) which may have a substituent. One or more functional groups may be used. That is, [F] in the general formula (1) may remain as a terminal reactive functional group, or may be a protective group modified product of a terminal reactive functional group, and is a group in which these are mixed. Also good.
Examples of the alkyl group having the carbon number (C1 to C6) in the protecting group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, Examples thereof include n-pentyl group, cyclopentyl group, n-hexyl group, cyclohexyl group and the like.
The substituents that the protecting group may have include a hydroxyl group, an amino group, a halogen atom, an alkylcarbonylalkoxy group having carbon atoms (C1 to C4), an alkylcarbonylamide group having carbon atoms (C1 to C4), carbon An alkylcarbonylalkylamide group having a number (C1 to C4), an alkylaryl group having a carbon number (C1 to C8), an alkoxy group having a carbon number (C1 to C4), an alkylamino group having a carbon number (C1 to C4), a carbon number Examples thereof include (C1-C4) acylamide groups and carbon number (C1-C4) alkoxycarbonylamino groups. The protecting group is preferably a water-soluble protective group having a C1-C4 alkoxy group having no positive or negative charge as a substituent.

The protective group-modified product can be used without particular limitation as long as it is capable of binding to an amino group, a hydroxyl group, a carboxy group, or a mercapto group, which are terminal reactive functional groups. That is, an amide bond, an alkoxycarbonylamide bond, an ester bond, a carbonate bond, a thioester bond, an alkoxythiocarbonyl bond, and the like can be appropriately selected and used according to the corresponding terminal reactive functional group.

Preferred examples of the terminal reactive functional group-protecting group-modified product include an acetyl group, a propionyl group, a butyryl group, a trifluoroacetyl group, a trichloroacetyl group, when the terminal functional group is an amino group, a hydroxyl group, or a mercapto group. Examples include methoxycarbonyl group, trichloromethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group and the like.
When the terminal functional group is a carboxy group, an amide bond such as ethylamino group, methoxymethylamino group, methoxyethylamino group, methoxyethoxyethylamino group or the like, or ethoxy group, methoxymethoxy group, methoxyethoxy group, methoxyethoxymethoxy group An ester conjugate such as

M indicating the number of the terminal functional group [F] in the general formula (1) is an integer of 0 to 199. M is the remainder of the multi-branched polymer carrier to which R ₁ to R ₃ are bonded, and there is no reason why the number of functional groups should be specified, and it may be set as appropriate. Preferably, m is 0 to 150, and more preferably 0 to 100.
The protective group-modified product of the terminal reactive functional group may be present arbitrarily and may be 0 or more and 199 or less. The protective group-modified product can be provided because it can control the surface charge and the like of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention and can control physical properties such as water solubility and self-association. preferable. For this reason, it is preferable that the said protective group modification body is 4 groups or more and exists in 150 groups or less, and it is more preferable that it exists in 6 groups or more and 100 groups or less.

In the nucleic acid antimetabolite-binding hyperbranched compound of the present invention, the mass content of the nucleic acid antimetabolite and the mass content of the polyethylene glycol segment may affect the drug efficacy and side effects. Therefore, a method for calculating the mass content of these partial structures will be described.
As the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound, a calculated value obtained by adding the constituent molecular weights of the constituent parts of the hyperbranched compound is adopted as the “molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound”. That is, (1) the molecular weight of the multi-branched polymer carrier, (2) the total molecular weight of the polyethylene glycol segment obtained by multiplying the molecular weight of the polyethylene glycol segment by the number of bonds, and (3) the binding to the binding residue molecular weight of the nucleic acid antimetabolite. The total molecular weight of the nucleic acid antimetabolite multiplied by the number, and (4) a calculated value obtained by adding the total molecular weight of the succinic monoamide unit multiplied by the number of bonds to the molecular weight of the succinic monoamide unit for binding the nucleic acid antimetabolite. The molecular weight.

The molecular weight of the nucleic acid antimetabolite binding hyperbranched compound is required to be regulated by the accuracy in kilodalton units. Therefore, the analysis method of each component is particularly limited as long as it is an analysis method with sufficient accuracy in measuring the molecular weight of the nucleic acid antimetabolite-binding hyperbranched compound in kilodalton units (× 10 ³ order). Instead, various analysis methods may be selected as appropriate. Below, the preferable analysis method in each component is listed.

As the molecular weight of the (1) multi-branched polymer carrier, when a carrier having a clear chemical structure such as a dendrimer, a dendron and a hyperbranched polymer is used, a calculated molecular weight calculated from the chemical structural formula is adopted. In the case of using a multi-branched polymer carrier in which the terminal functional group of the used carrier is further chemically modified, the molecular weight of the modified compound residue is determined by the number of terminal functional groups and the introduction rate of the compound residue into the terminal functional group. By adding the multiplied value, the molecular weight of the multi-branched polymer carrier is adopted. As the introduction rate, a value calculated from a conversion rate calculated from an integral value of ¹ H-NMR can be used. When the multi-branched polymer carrier is modified with a single compound residue, the ¹ H-NMR analysis is simple and preferable, and it is calculated using the conversion rate calculated from the integrated value of ¹ H-NMR. It is preferable to do.

The total molecular weight of the (2) polyethylene glycol segment is a calculated value obtained by multiplying the molecular weight of the polyethylene glycol segment by the binding amount. As the molecular weight of the polyethylene glycol segment, an average molecular weight determined by the peak top molecular weight of the polyethylene glycol segment structural compound to be used, which is measured by a GPC method based on a polyethylene glycol standard product, is employed.
The amount of polyethylene glycol segment bound can be determined by cleaving the polyethylene glycol segment from the nucleic acid antimetabolite-bound hyperbranched compound and quantitatively analyzing the released polyethylene glycol segment, or by using the polyethylene for the multibranched polymer alone. In the reaction for introducing the glycol segment, a method of calculating from the consumption rate of the polyethylene glycol segment may be used.

The total molecular weight of the nucleic acid antimetabolite (3) is a calculated value obtained by multiplying the binding residue molecular weight of the nucleic acid antimetabolite by the number of bonds. The binding number of the nucleic acid antimetabolite is a value calculated by hydrolyzing the nucleic acid antimetabolite-bound hyperbranched compound and quantitatively analyzing the released nucleic acid antimetabolite by high performance liquid chromatography (HPLC). It is.
The total molecular weight of the succinic acid monoamide unit (4) is a calculated value obtained by multiplying the molecular weight of the succinic acid monoamide unit by the number of bonds. The number of bonds of the binding group is the same as the number of bonds of the nucleic acid antimetabolite described above, and can be calculated by using the value.

On the other hand, when the polymer of the succinic acid monoamide unit is used as the succinic acid monoamide unit, the total molecular weight of the succinic acid monoamide unit in (4) is obtained by multiplying the molecular weight of the succinic acid monoamide unit polymer by the number of bonds. Adopted values.
The molecular weight of the polymer of the succinic acid monoamide unit is a calculated value obtained by multiplying the molecular weight of the polymerization monomer unit by the number of polymerizations. The number of polymerizations is the number of polymerizations calculated from the integrated value of ¹ H-NMR in the product after the polymerization reaction, the number of polymerizations calculated by amino acid analysis, or the monomer in which the side chain carboxylic acid of aspartic acid is protected. When the polymerization is used, the number of polymerizations calculated by quantitatively analyzing the removed protecting group component generated when deprotecting the product of the reaction by high performance liquid chromatography (HPLC) is used. Can be used.

The mass content of the polyethylene glycol segment can be calculated from the content ratio of the total molecular weight of the polyethylene glycol segment of (2) above with respect to the molecular weight of the above-described nucleic acid antimetabolite-binding hyperbranched compound. That is, the mass content of the polyethylene glycol segment is calculated by the following formula.
Mass content of polyethylene glycol segment (%) = (total molecular weight of polyethylene glycol segment / nucleic acid antimetabolite binding hyperbranched compound) × 100
The mass molecular weight ratio of the polyethylene glycol segment is 20% by mass to 90% by mass, and preferably 40% by mass to 80% by mass. More preferably, it is 50 mass% or more and 80 mass% or less.
When the mass content of the polyethylene glycol segment is less than 20% by mass, myelosuppression tends to be strongly developed. In order to achieve sufficient medicinal effect and side effect reduction, it is preferable to set the mass content of the polyethylene glycol segment.

In the nucleic acid antimetabolite-binding multibranched compound of the present invention, the mass content of the nucleic acid antimetabolite in the multibranched compound is preferably 2% by mass or more and 60% by mass or less.
When the content of the nucleic acid antimetabolite is less than 2% by mass, the total amount of the multi-branched compound is increased in order to ensure an effective amount of the nucleic acid antimetabolite, which is not preferable. On the other hand, when the content of the nucleic acid antimetabolite is more than 60% by mass, myelosuppression tends to be strongly developed. It is preferable to set the content of the nucleic acid antimetabolite in order to ensure administration convenience and achieve sufficient drug efficacy and side effect reduction.
The mass content of the nucleic acid antimetabolite in the nucleic acid antimetabolite-bound hyperbranched compound is calculated based on the content ratio of the total molecular weight of the nucleic acid antimetabolite to the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound. Can do. A more preferable range of the content of the nucleic acid antimetabolite is 5% by mass or more and 40% by mass or less. It is particularly preferred that the content of the nucleic acid antimetabolite is 5% by mass or more and 20% by mass or less.

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention desirably has a molecular weight of 10 kilodaltons or more and 200 kilodaltons or less. More preferably, the molecular weight is 20 kilodaltons or more and 160 kilodaltons or less. As the molecular weight of the multi-branched compound, a calculated value obtained by adding the constituent molecular weights of the above-mentioned constituent parts is adopted as the “molecular weight of the nucleic acid antimetabolite-binding multi-branched compound”. That is, a calculated value obtained by adding the constituent molecular weights (1) to (4) is defined as the molecular weight.

The nucleic acid antimetabolite-binding multibranched compound of the present invention comprises a terminal reactive functional group of a multibranched polymer carrier; [F] having a nucleic acid antimetabolite-binding succinic acid monoamide unit having a function of releasing a nucleic acid antimetabolite. It is characterized by comprising. Furthermore, the nucleic acid antimetabolite binding hyperbranched compound preferably comprises a polyethylene glycol segment. That is, a preferred embodiment of the present invention is a nucleic acid antimetabolite-binding hyperbranched compound containing two types of functional substituents, a nucleic acid antimetabolite-binding succinic acid monoamide unit and a polyethylene glycol segment.
The embodiment of the present invention can be classified into the following two types based on the structure of the multi-branched polymer carrier depending on the binding mode of the succinic acid monoamide unit and the polyethylene glycol segment bound to the nucleic acid antimetabolite.
[Type 1]: An embodiment in which the succinic acid monoamide unit to which the nucleic acid antimetabolite is bonded and the polyethylene glycol segment are bonded to the terminal reactive functional group of the multi-branched polymer carrier as separate substituents.
[Type 2]: An embodiment in which the succinic acid monoamide unit to which the nucleic acid antimetabolite is bound and the polyethylene glycol segment linked together form a substituent that is bonded to the terminal reactive functional group of the multi-branched polymer carrier. . The succinic monoamide unit may be a polymer segment of a plurality of the succinic monoamide units.
In the above [Type 1], when the succinic acid monoamide unit bound to the nucleic acid antimetabolite is a succinic acid monoamide unit bound to the nucleic acid antimetabolite ([Type 1-1]), the nucleic acid antimetabolite And a polymer of succinic acid monoamide units bonded to each other ([Type 1-2]).

Specific examples of the present invention will be described below in each embodiment.
First, an embodiment [Type 1] in which the succinic acid monoamide unit to which the nucleic acid antimetabolite is bound and the polyethylene glycol segment are bonded to the terminal reactive functional group of the multi-branched polymer carrier as separate substituents. explain.

［式中、［Ｑ］は（ｍ＋ｎ＋ｏ＋ｐ）個の末端官能基化された多分岐高分子担体であり、（ｍ＋ｎ＋ｏ＋ｐ）は４～２００の整数であり、［Ｆ］は前記末端官能基であってアミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基、及び／又は置換基を有していても良い炭素数（Ｃ１～Ｃ６）のアルキル基を有する保護基で修飾された、アミノ基、水酸基、カルボキシ基及びメルカプト基からなる群から選択される１種以上の官能基であり、Ｆは前記末端官能基から水素原子又は水酸基が除かれた結合基であり、Ｒ_１は核酸代謝拮抗剤が結合したコハク酸モノアミドユニットを含む置換基であり、Ｒ_２はポリエチレングリコールセグメントを含む置換基であり、Ｒ_３はコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基であり、ｍは０～１９８の整数であり、ｎは１～１９９の整数であり、ｏは１～１９９の整数であり、ｐは０～１９８の整数である。］で示される核酸代謝拮抗剤結合多分岐化合物である。 The [type 1] includes a substituent (R ₁ ) containing a succinic acid monoamide unit to which a nucleic acid antimetabolite is bound and a substituent (R ₂ ) containing a polyethylene glycol segment in the general formula (1). An embodiment which is an essential substituent, which has the general formula (1)

[Wherein [Q] is (m + n + o + p) terminal functionalized multi-branched polymer carrier, (m + n + o + p) is an integer of 4 to 200, and [F] is the terminal functional group. A protecting group having one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and / or an optionally substituted alkyl group having a carbon number (C1 to C6). Is one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and F is a linking group obtained by removing a hydrogen atom or a hydroxyl group from the terminal functional group. , R ₁ is a substituent containing a succinic acid monoamide units nucleic acid antimetabolites are bonded, R ₂ is a substituent containing a polyethylene glycol segment, R ₃ is Zanmoto及acid monoamide derivative An / or succinimide residue, m is an integer of from 0 to 198, n is an integer of 1 ~ 199, o is an integer of 1 ~ 199, p is an integer of from 0 to 198. ] It is a nucleic acid antimetabolite binding hyperbranched compound shown by this.

In the embodiment of [Type 1], the content of the nucleic acid antimetabolite can be controlled by the binding amount (n) of the substituent containing the succinic acid monoamide unit to which the nucleic acid antimetabolite that is R ₁ is bound. . In addition, the content of polyethylene glycol can be controlled by the binding amount (o) of the substituent containing the polyethylene glycol segment which is R ₂ and the molecular weight of the polyethylene glycol segment used. This structure is advantageous in that the physical properties such as water solubility and self-association of the compound can be controlled.

In [Type 1], the polyethylene glycol segment of R ₂ is a segment having a repeating structure of an ethyleneoxy group; (CH ₂ CH ₂ O) unit. The degree of polymerization of ethyleneoxy group units is preferably 5 to 10,000 units, more preferably the degree of polymerization is 5 to 2,500 units, particularly preferably 10 to 1,000 units, and even more preferably 20 to 500 units. It is the segment structure containing the polyethyleneglycol chain of.
That is, the polyethylene glycol segment is preferably a segment part having an average molecular weight of 0.2 kilodaltons to 500 kilodaltons, more preferably a structural part having an average molecular weight of 0.2 kilodaltons to 150 kilodaltons. The average molecular weight is particularly preferably 0.5 to 50 kilodaltons. A polyethylene glycol segment having an average molecular weight of 1 kilodalton to 20 kilodalton is particularly preferred.
The average molecular weight of the polyethylene glycol segment used in the present invention is the GPC method based on the polyethylene glycol standard product of the polyethylene glycol segment structural compound used in preparing the nucleic acid antimetabolite-binding hyperbranched compound of the present invention. It is an average molecular weight calculated | required by the peak top molecular weight measured by (1).

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_１は末端官能基Ｆとの結合基を示す。］を用いることが好ましい。
　すなわち、エチレンオキシ基；（ＣＨ_２ＣＨ_２Ｏ）単位の繰り返し構造によるエチレンオキシ基単位重合度が５～２，５００ユニットのポリエチレングリコールセグメントであり、ポリエチレングリコール相当の平均分子量として０．２キロダルトン～１５０キロダルトンのセグメント部であることが好ましい。より好ましくは重合度が１０～１，０００ユニットであり、平均分子量として０．５キロダルトン～５０キロダルトンであり、更に好ましくは重合度が２０～５００ユニットで、平均分子量として１キロダルトン～２０キロダルトンであり、殊更好ましくは重合度が２０～３００ユニットで、平均分子量として１キロダルトン～１２キロダルトンのポリエチレングリコール鎖を含むセグメント構造である。
 
　なお、該Ｒ_８及び該Ｘ_１は、前述のＲ_２に係るポリエチレングリコールセグメントの記載と同義である。 R ₂ in the [type 1] is represented by the general formula (8)

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₁ represents a bonding group with the terminal functional group F. ] Is preferably used.
That is, it is a polyethylene glycol segment having an ethyleneoxy group; the degree of polymerization of the ethyleneoxy group unit having a repeating structure of (CH ₂ CH ₂ O) units of 5 to 2,500 units, and an average molecular weight equivalent to polyethylene glycol is 0.2 kilodalton. A segment of ~ 150 kilodaltons is preferred. More preferably, the degree of polymerization is from 10 to 1,000 units, and the average molecular weight is from 0.5 kilodaltons to 50 kilodaltons, and still more preferably, the degree of polymerization is from 20 to 500 units, and the average molecular weight is from 1 kilodaltons to 20 units. A segment structure having a degree of polymerization of 20 to 300 units and a polyethylene glycol chain having an average molecular weight of 1 to 12 kilodaltons is particularly preferred.

Note that the R ₈ and the X ₁ are as defined in the polyethylene glycol segment of the R ₂ above.

Next, [Type 1-1] in which the R ₁ of the general formula (1) is a nucleic acid antimetabolite-binding succinic acid monoamide unit in the above-mentioned [Type 1] nucleic acid antimetabolite-binding hyperbranched compound will be described.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｘ_４は前記末端官能基Ｆとの結合基であり、Ｒ_４、Ｒ_５及びＲ_６はそれぞれ独立して水素原子又は炭素数（Ｃ１～Ｃ８）のアルキル基であり、Ｒ_７は水素原子、置換基を有していても良い炭素数（Ｃ１～Ｃ２０）の直鎖状、分岐鎖状又は環状のアルキル基、置換基を有していても良い炭素数（Ｃ７～Ｃ２０）の直鎖状、分岐鎖状又は環状のアラルキル基、置換基を有していても良い芳香族基及びカルボキシ基が保護されたアミノ酸残基からなる群から選択される１種以上の基である。］で示される核酸代謝拮抗剤が結合したアスパラギン酸モノアミドユニットであることが好ましい。
　核酸代謝拮抗剤の結合様式は、アミド結合のみの場合、エステル結合のみの場合、又はアミド結合とエステル結合との混合体の場合の何れでも良い。用いる核酸代謝拮抗剤の結合性官能基に応じて、側鎖カルボキシ基への結合様式を適宜選択して良い。 As a preferred embodiment of the [Type 1-1], a succinic acid monoamide unit to which a nucleic acid antimetabolite represented by R ₁ is bonded is represented by the general formula (2) and / or (3).

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, X ₄ is a binding group to the terminal functional group F, and R ₄ , R ₅ and R ₆ are each independently a hydrogen atom or An alkyl group having a carbon number (C1 to C8), and R ₇ is a hydrogen atom, an optionally substituted linear (C1 to C20) linear, branched or cyclic alkyl group, substituted An amino acid residue in which a linear, branched or cyclic aralkyl group which may have a group (C7 to C20), an aromatic group which may have a substituent, and a carboxy group are protected. One or more groups selected from the group consisting of groups. It is preferably an aspartic acid monoamide unit to which a nucleic acid metabolism antagonist represented by
The binding mode of the nucleic acid antimetabolite may be any of an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite used, the mode of binding to the side chain carboxy group may be appropriately selected.

In the aspartic acid monoamide unit represented by the general formula (2) or (3), the alkyl group having carbon atoms (C1-C8) in R ₄ , R ₅ and R ₆ is linear or branched. Alternatively, it is a cyclic alkyl group (C1 to C8).
Examples of the linear alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-hexyl group.
Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like.
Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and the like.

In R _{7 according} to the general formula (2) or (3), the linear, branched or cyclic alkyl group having a carbon number (C1 to C20) which may have a substituent is, for example, methyl Group, ethyl group, isopropyl group, t-butyl group, cyclohexyl group, n-octyl group, dodecyl group and octadecyl group.
Examples of the linear, branched or cyclic aralkyl group having a carbon number (C7 to C20) which may have a substituent include, for example, benzyl group, 2-phenylethyl group, 4-phenylbutyl group, 8 -Phenyloctyl group and the like.
Examples of the aromatic group having a carbon number (C5 to C20) which may have a substituent include a phenyl group, a 4-methoxyphenyl group, a 4-dimethylaminophenyl group, and a 4-hydroxyphenyl group. .

R ₇ may be an amino acid residue in which a carboxy group is protected. Examples of amino acid residues in which the carboxyl group is protected include glycinyl-methyl ester group, alanyl-methyl ester group, leucinyl-methyl ester group, valinyl-methyl ester group, phenylalanyl-methyl ester group, alanyl-ethyl ester Group, leucinyl-ethyl ester group, alanyl-butyl ester group, leucinyl-butyl ester group and the like.

X ₄ is a linking group that binds the substituent containing the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ is bonded to the terminal reactive functional group [F] of the multi-branched polymer carrier. The X ₄ has the same meaning as X in the general formula (17) and / or (18).

The binding residue of the nucleic acid antimetabolite of [D] is an embodiment in which the above-described nucleic acid antimetabolite is bonded to the side chain carboxy group of the aspartic acid monoamide unit via an amide bond and / or an ester bond. And an amide bond residue and / or an ester bond residue of the nucleic acid antimetabolite. The binding mode of the nucleic acid antimetabolite may be any of an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite used, the mode of binding to the side chain carboxy group may be appropriately selected.

The nucleic acid antimetabolite of [D] has the same meaning as described above, and examples include pyrimidine antimetabolite, purine antimetabolite, triazine antimetabolite and the like. It is preferable to use a nucleic acid antimetabolite having an amino group and / or a hydroxyl group. More preferably, it is a nucleic acid antimetabolite having an amino group at the nucleoside base, and is preferably a nucleic acid antimetabolite capable of amide bonding to the carboxy group of the succinic acid monoamide unit through the amino group.

［式中、－Ｒｆは、式（１３）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基残基を示す。］。ここでＲ_２０における脂肪酸エステルのアシル基は、前述と同義である。 The nucleic acid antimetabolite is one or more selected from the following formula (12) for the nucleobase moiety, and any one selected from the following formula (13) for the group (Rf) bonded thereto: A nucleic acid antimetabolite that is a combination of the above is particularly preferred.

[Wherein, -Rf represents the formula (13):

R ₂₀ represents a hydrogen atom or an acyl group residue of a fatty acid ester. ]. Here, the acyl group of the fatty acid ester in R ₂₀ has the same meaning as described above.

As the nucleic acid antimetabolite, a cytidine antimetabolite is preferably used. The nucleobase moiety is a cytidine base represented by the following formula (14), and the group (Rf) bonded thereto is represented by the following formula (15). Particularly preferred is a nucleic acid antimetabolite that is a combination of any one or more selected from the group of substituents. Here, R ₂₀ is a compound represented by a hydroxyl group or an acyl group of a fatty acid ester.

［式中、－Ｒｆは、式（１５）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基を示す。］。Ｒ_２０における脂肪酸エステルのアシル基は、前述と同義である。

[Wherein, -Rf represents the formula (15):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. ]. The acyl group of the fatty acid ester in R ₂₀ has the same meaning as described above.

These cytidine antimetabolites are gemcitabine and its fatty acid ester derivative, cytarabine and its fatty acid ester derivative, and 3'-ethynylcytidine and its fatty acid ester derivative. Examples of fatty acid ester derivatives include cytarabine-5'-elaidic acid ester (CP-4055) and gemcitabine-5'-elaidic acid ester (CP-4126), which are preferably used in the present invention.

［式中、Ｘ_４は前記末端官能基Ｆとの結合基であり、Ｒ_９は水酸基及び／又は－Ｎ（Ｒ_１０）ＣＯＮＨ（Ｒ_１１）を示し、Ｒ_１０、Ｒ_１１は同一でも異なっていてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基を示し、Ｒ_４、Ｒ_５、Ｒ_６及びＲ_７は前述の［タイプ１－１］のＲ_１で用いられる一般式（２）及び（３）と同義である。］からなる置換基群から選ばれる１種以上の基であることが好ましい。ここで、Ｘ_４、Ｒ_１０及びＲ_１１は前述と同義である。
　前記Ｒ_３で表されるコハク酸モノアミド誘導体残基及び／又はコハク酸イミド残基は、前記Ｒ_１に係る核酸代謝拮抗剤が結合したコハク酸モノアミドユニットから核酸代謝拮抗剤が解離した残基であるため、任意に存在して良い基である。しかしながら、該Ｒ_３に係る置換基は、本発明の核酸代謝拮抗剤結合多分岐化合物の製造中や医薬品用製剤の製造中、並びに医薬製剤保存中や医薬品として使用時において、核酸代謝拮抗剤の解離に伴い、随時、生成し得ることになる。 In the above-mentioned [type 1-1] nucleic acid antimetabolite-binding hyperbranched compound, the succinic acid monoamide derivative residue and / or succinimide residue represented by R ₃ in the general formula (1) is represented by the general formula (9): , (10) and (11)

[Wherein, X ₄ is a linking group to the terminal functional group F, R ₉ represents a hydroxyl group and / or —N (R ₁₀ ) CONH (R ₁₁ ), and R ₁₀ and R ₁₁ are the same or different. Or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group, and R ₄ , R ₅ , R ₆ and R ₇ are It is synonymous with the general formulas (2) and (3) used for R ₁ of [Type 1-1] described above. It is preferably at least one group selected from the group of substituents consisting of Here, X ₄ , R ₁₀ and R ₁₁ are as defined above.
The succinic acid monoamide derivative residue and / or succinimide residue represented by R ₃ is a residue obtained by dissociating the nucleic acid antimetabolite from the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ is bound. Therefore, it is a group that may be present arbitrarily. However, the substituent according to R ₃ is used in the production of the nucleic acid antimetabolite-binding multibranched compound of the present invention, during the manufacture of pharmaceutical preparations, during storage of pharmaceutical preparations, and when used as pharmaceuticals. With dissociation, it can be generated at any time.

In nucleic acid metabolism antagonist binding multi-branched compound of the Type 1-1], the substituent which the nucleic acid antimetabolites comprises succinic acid monoamide units bound (R _1), a substituent containing the polyethylene glycol segment (R ₂ ), And a terminal functional group [F] to which the substituent (R ₃ ) containing the succinic monoamide derivative residue and / or succinimide residue is not bonded. [F] is one or more terminal functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group. The terminal functional group may be selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group modified with a protecting group having an alkyl group having a carbon number (C1 to C6) which may have a substituent. One or more functional groups may be used. That is, the [type 1-1] may be the terminal reactive functional group of [F] in the general formula (1), or may be a protective group modification of the terminal reactive functional group, A group in which these are mixed may be used.

In the protecting group for the terminal reactive functional group [F], the alkyl group having the carbon number (C1 to C6) has the same meaning as described above. Examples thereof include a butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, an n-pentyl group, a cyclopentyl group, an n-hexyl group, and a cyclohexyl group.
The substituents that the protecting group may have include a hydroxyl group, an amino group, a halogen atom, an alkylcarbonylalkoxy group having carbon atoms (C1 to C4), an alkylcarbonylamide group having carbon atoms (C1 to C4), carbon An alkylcarbonylalkylamide group having a number (C1 to C4), an alkylaryl group having a carbon number (C1 to C8), an alkoxy group having a carbon number (C1 to C4), an alkylamino group having a carbon number (C1 to C4), a carbon number Examples thereof include (C1-C4) acylamide groups and carbon number (C1-C4) alkoxycarbonylamino groups. The protecting group is preferably a water-soluble protective group having a C1-C4 alkoxy group having no positive or negative charge as a substituent.

Preferred examples of the terminal reactive functional group-protecting group-modified product include an acetyl group, a propionyl group, a butyryl group, a trifluoroacetyl group, a trichloroacetyl group, when the terminal functional group is an amino group, a hydroxyl group, or a mercapto group. Examples include methoxycarbonyl group, trichloromethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group and the like.
When the terminal functional group is a carboxy group, an amide bond such as ethylamino group, methoxymethylamino group, methoxyethylamino group, methoxyethoxyethylamino group or the like, or ethoxy group, methoxymethoxy group, methoxyethoxy group, methoxyethoxymethoxy group An ester conjugate such as

The protective group-modified product of the terminal reactive functional group may be present arbitrarily and may be 0 or more and 199 or less. The protective group-modified product can be provided because it can control the surface charge and the like of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention and can control physical properties such as water solubility and self-association. preferable. For this reason, it is preferable that the said protective group modification body is 4 groups or more and exists in 150 groups or less, and it is more preferable that it exists in 6 groups or more and 100 groups or less.

When the nucleic acid antimetabolite-binding hyperbranched compound of the present invention is [type 1-1], the number of substituents in each of the general formula (1) is m is an integer of 0 to 198, and n is 1 Is an integer from ˜199, o is an integer from 1 to 199, and p is an integer from 0 to 198. Preferably, m is an integer from 0 to 120, n is an integer from 2 to 100, o is an integer from 2 to 100, and p is an integer from 0 to 80. More preferably, m is an integer from 0 to 80, n is an integer from 5 to 50, o is an integer from 5 to 50, and p is an integer from 0 to 50. The total number of terminal substituents of the multi-branched polymer carrier (m + n + o + p) is an integer of 4 to 200. It is preferably 4 to 150, more preferably 8 to 100.
Bonding number of polyethylene glycol segment represented by the nucleic acid metabolism antagonist binding succinic acid monoamide units and R ₂ represented by R _1, and the pharmacokinetic properties of the nucleic acid metabolism antagonist binding hyperbranched compounds, the dissociation rate of nucleic acid metabolism antagonist It should be set as appropriate.

Another embodiment of [Type 1] according to the present invention includes [Type 1-2] wherein R ₁ in the general formula (1) is a polymer of a succinic acid monoamide unit bound with a nucleic acid antimetabolite. . Hereinafter, [Type 1-2] will be described.

[Type 1-2] refers to an embodiment in which R ₁ is a polymer of succinic acid monoamide units, and one or more molecules of a nucleic acid antimetabolite are bound to this side chain carboxy group. In the polymer of R ₁ succinic acid monoamide unit, it is preferable that two or more molecules of nucleic acid antimetabolite are bound. The [Type 1-2] is an advantageous structure in increasing the drug content because it can increase the number of binding of the nucleic acid antimetabolite per substituent.

The polymer of the succinic acid monoamide unit is preferably a polymer of a succinic acid monoamide unit having a polymerization number of 1 to 50. More preferably, the polymerization number is 2-30. The polymer of the succinic acid monoamide unit polymer may be a polymer based on α-amide bonds or a polymer based on β-amide bonds, and may be a polymer based on a mixture of α and β-amide bonds. Or either.

The nucleic acid antimetabolite that binds to the polymer of the succinic monoamide unit has the same meaning as described above, and examples thereof include a pyrimidine antimetabolite, a purine antimetabolite, and a triazine antimetabolite. It is preferable to use a nucleic acid antimetabolite having an amino group and / or a hydroxyl group. More preferably, it is a nucleic acid antimetabolite having an amino group in the nucleoside base, and is preferably a nucleic acid antimetabolite capable of amide bonding to the carboxy group of aspartic acid by the amino group.

R ₁ in [Type 1-2] is _one or more molecules, preferably 2 or more, preferably 2 to the side chain carboxy group of the polymer of the succinic acid monoamide unit via the amide bond and / or ester bond. Substituents that are bonded by multiple molecules. The binding mode of the nucleic acid antimetabolite may be any of an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite used, the mode of binding to the side chain carboxy group may be appropriately selected.
The nucleic acid metabolism antagonist may be bound to one or more side chain carboxy groups in the polymer of the succinic monoamide unit. A polymer of a succinic acid monoamide unit in which the nucleic acid antimetabolite is bonded to two or more side chain carboxy groups is preferred. That is, it is preferable that the binding rate of the nucleic acid antimetabolite to the total side chain carboxy group is 10 to 90%.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｒ_１２は水酸基及び／又は－Ｎ（Ｒ_１４）ＣＯＮＨ（Ｒ_１５）であり、該Ｒ_１４及び該Ｒ_１５は同一でも異なってもいてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、Ｒ_１３は水素原子、炭素数（Ｃ１～Ｃ８）のアシル基及び炭素数（Ｃ１～Ｃ８）のアルコキシカルボニル基からなる群から選択される基であり、Ｘ_２は末端官能基Ｆとの結合基であり、ａ、ｂ、ｃ、ｄ及びｅはそれぞれ独立して０～３０の整数を示し、（ａ＋ｂ）は１～３０の整数を示し、ポリアミノ酸誘導体の総重合数である（ａ＋ｂ＋ｃ＋ｄ＋ｅ）は１～３０であり、前記［Ｄ］が結合したアスパラギン酸単位、前記Ｒ_１２が結合したアスパラギン酸単位及び側鎖カルボキシ基が分子内環化型のアスパラギン酸単位が、それぞれ独立してランダムな配列である。］で示される置換基であることが好ましい。 R ₁ in the [type 1-2] is a polyaspartic acid derivative, and is represented by the general formula (4) or (5)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, R ₁₂ is a hydroxyl group and / or —N (R ₁₄ ) CONH (R ₁₅ ), and R ₁₄ and R ₁₅ are the same A linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be different or may be substituted with a tertiary amino group, R ₁₃ is a hydrogen atom, a carbon number ( A group selected from the group consisting of an acyl group of C1 to C8) and an alkoxycarbonyl group of carbon number (C1 to C8), X ₂ is a bonding group to the terminal functional group F, and a, b, c, d and e each independently represent an integer of 0 to 30, (a + b) represents an integer of 1 to 30, and the total number of polyamino acid derivatives (a + b + c + d + e) is 1 to 30, ], An aspartic acid unit bonded, and the R ₁₂ bonded The aspartic acid unit in which the sparagic acid unit and the side chain carboxy group are cyclized in the molecule are each independently a random sequence. It is preferable that it is a substituent shown by this.

[D] is a binding residue of a nucleic acid antimetabolite, and the nucleic acid antimetabolite binding residue is synonymous with the description of [D] in [Type 1-1].

R ₁₂ is a hydroxyl group and / or —N (R ₁₄ ) CONH (R ₁₅ ). Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group in R ₁₄ and R ₁₅ include, for example, methyl group, ethyl group N-propyl group, isopropyl group, isobutyl group, sec-butyl group, t-butyl group, 1-methylbutyl group, 2-methylbutyl group, neopentyl group, cyclohexyl group, etc., preferably isopropyl group, cyclohexyl group Groups.
Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with the tertiary amino group include, for example, 2-dimethylaminoethyl group, 3-dimethylaminopropyl group , 5-dimethylaminopentyl group, 6-dimethylaminohexyl group and the like.
R ₁₄ and R ₁₅ are preferably an ethyl group, an isopropyl group, a cyclohexyl group, and a 3-dimethylaminopropyl group.

When R ₁₂ is a hydroxyl group, it represents a carboxylic acid embodiment. Moreover, the arbitrary salt aspect of the carboxylic acid may be sufficient.
R ₁₂ is a hydroxyl group and / or —N (R ₁₄ ) CONH (R ₁₅ ), but when it is only a hydroxyl group, a hydroxyl group and —N (R ₁₄ ) CONH (R ₁₅ ) coexist, or —N A mode in the case of (R ₁₄ ) CONH (R ₁₅ ) alone can be taken. The abundance ratio of the hydroxyl group to —N (R ₁₄ ) CONH (R ₁₅ ) may be arbitrarily set.

The acyl group having carbon atoms (C1 to C8) in R ₁₃ is a linear, branched or cyclic acyl group having carbon atoms (C1 to C8). Examples include formyl group, acetyl group, propionyl group, butyroyl group, cyclopropylcarbonyl group, cyclopentanecarbonyl group and the like.
The alkoxycarbonyl group having 1 to 8 carbon atoms in R ₁₃ is a linear, branched or cyclic alkoxycarbonyl group having 1 to 8 carbon atoms (C1 to C8). For example, methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, isopropoxycarbonyl group, n-butoxycarbonyl group, t-butoxycarbonyl group, pentoxycarbonyl group, hexyloxycarbonyl group, cyclopropoxycarbonyl group, cyclopentyloxycarbonyl group Cyclohexyloxycarbonyl group, etc.

X ₂ is a bonding group between R ₁ represented by the general formula (4) or (5) and the terminal reactive functional group [F] of the multi-branched polymer carrier. The linking group X ₂ is not particularly limited as long as it is a linking group having functional groups capable of binding to the terminal group of R ₁ and the terminal reactive functional group [F] at both ends. is not.

X ₂ has _one end group bonded to the end group of R _{1 and the} other end group connected to the terminal reactive functional group [F] with an ester bond, an amide bond, a thioester bond, a urea bond, or a urethane. An alkylene group having a binding functional group that can be bonded and an optionally substituted carbon group (C1 to C8) is preferred.
The linking group related to X ₂ includes, for example, — (CH ₂ ) _y —NH— (where y is 0 to 8) as a linking group that bonds to the terminal reactive functional group [F] with an amide bond, an ester bond or a thioester bond. -(CH ₂ ) _y -O- (y is an integer from 0 to 8),-(CH ₂ ) _y -S- (y is an integer from 0 to 8),-(CH ₂ ) _y —CO— (y represents an integer of 0 to 8), —NH— (CH ₂ ) _y —CO— (y represents an integer of 0 to 8), and the like.

X ₂ may be a “bond”. The “bond” refers to an embodiment in which the terminal reactive functional group of the multi-branched polymer carrier and the terminal group related to R ₂ are directly bonded without using a bonding group.

The polyaspartic acid derivative substituent represented by the general formula (4) or (5) has a total polymerization number (a + b + c + d + e) of 1 to 30. A polyaspartic acid derivative substituent having a polymerization number of 4 to 30 is preferable, and a polymerization number of 5 to 25 is preferable.
A, b, c, d and e indicating the number of constituents of the aspartic acid derivative unit are each independently an integer of 0 to 30. However, the aspartic acid derivative unit to which the nucleic acid antimetabolite [D] is bound is an essential component, and (a + b) represents an integer of 1 to 30. (A + b) is preferably an integer of 4 to 25, and more preferably 5 to 20. In addition, the number of aspartic acid derivative units to which R ₁₂ which is a hydroxyl group and / or —N (R ₁₄ ) CONH (R ₁₅ ) is bonded (c + d) and the side chain carboxy group is an intramolecularly cyclized aspartic acid derivative unit The number e is an arbitrary configuration, and (c + d) and e are 0-29.

In addition, the polyaspartic acid derivative substituent represented by the general formula (4) or (5) is composed of an aspartic acid unit to which the [D] is bonded, an aspartic acid unit to which the R ₁₂ is bonded, and a side chain carboxy group. The internal cyclization type aspartic acid unit may be in the form of a localized sequence, or may be a polymer structure composed of a random sequence in which each structural unit has no regularity. This is a sequence with no particular regularity in the sequence order of the chain modifications.

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_１は末端官能基Ｆとの結合基を示す。］で示されるポリエチレングリコールセグメントであることが好ましい。ここで、Ｒ_８、ｋ及びＸ_１は前述と同義である。 R ₂ in the [Type 1-2] is a substituent containing a polyethylene glycol segment. The substituent containing the polyethylene glycol segment has the same meaning as described in the above [Type 1].
The R ₂ is represented by the general formula (8)

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₁ represents a bonding group with the terminal functional group F. It is preferable that it is a polyethyleneglycol segment shown by this. Here, R ₈ , k and X ₁ are as defined above.

In the nucleic acid antimetabolite-binding multibranched compound according to [Type 1-2], a polymer segment (R ₁ ) of a succinic acid monoamide unit to which a nucleic acid antimetabolite is bound and a substituent (R ₂ ) comprising a polyethylene glycol segment In addition, a substituent (R ₃ ) containing any succinic monoamide derivative residue and / or succinimide residue may be further bonded. These optional substituents are residues obtained by dissociating the nucleic acid antimetabolite from the polymer segment of the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₂ is bound.
When the polymer segment of the succinic acid monoamide unit to which the nucleic acid antimetabolite according to R ₁ of [Type 1-2] is bound is a polyaspartic acid derivative represented by the general formula (4) or (5), R ₃ is a polyaspartic acid derivative lacking an aspartic acid unit comprising the nucleic acid metabolism antagonist; [D] in the general formula (4) or (5). That is, in the general formula (4) or (5), a and b are 0, and R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , X ₂ , c, d and e are substituents as defined above. .

In the [Type 1-2] nucleic acid antimetabolite-binding hyperbranched compound, a polymer of a succinic acid monoamide unit (R ₁ ) bound to the nucleic acid antimetabolite, and a substituent (R ₂ ) containing the polyethylene glycol segment And a terminal functional group [F] to which the substituent (R ₃ ) containing the succinic monoamide derivative residue and / or succinimide residue is not bonded. [F] is one or more terminal functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group. The terminal functional group may be selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group modified with a protecting group having an alkyl group having a carbon number (C1 to C6) which may have a substituent. One or more functional groups may be used. That is, [F] in the general formula (1) may remain as a terminal reactive functional group, or may be a protective group modified product of a terminal reactive functional group, and is a group in which these are mixed. Also good. The terminal functional group according to [F] has the same meaning as in [Type 1-1].

Preferred examples of the terminal reactive functional group-protecting group-modified product include an acetyl group, a propionyl group, a butyryl group, a trifluoroacetyl group, a trichloroacetyl group, when the terminal functional group is an amino group, a hydroxyl group, or a mercapto group. Examples include methoxycarbonyl group, trichloromethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group and the like.
When the terminal functional group is a carboxy group, an amide bond such as ethylamino group, methoxymethylamino group, methoxyethylamino group, methoxyethoxyethylamino group or the like, or ethoxy group, methoxymethoxy group, methoxyethoxy group, methoxyethoxymethoxy group An ester conjugate such as

The protective group-modified product of the terminal reactive functional group may be present arbitrarily and may be 0 or more and 199 or less. The protective group-modified product can be provided because it can control the surface charge and the like of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention and can control physical properties such as water solubility and self-association. preferable. For this reason, it is preferable that the said protective group modification body is 4 groups or more and exists in 150 groups or less, and it is more preferable that it exists in 6 groups or more and 100 groups or less.

When the nucleic acid antimetabolite-bound hyperbranched compound of the present invention is [type 1-2], the number of substituents in each of the general formula (1) is m is an integer of 0 to 198, and n is 1 Is an integer from ˜199, o is an integer from 1 to 199, and p is an integer from 0 to 198. Preferably, m is an integer from 0 to 120, n is an integer from 2 to 100, o is an integer from 2 to 100, and p is an integer from 0 to 80. More preferably, m is an integer from 0 to 80, n is an integer from 5 to 50, o is an integer from 5 to 50, and p is an integer from 0 to 50. The total number of terminal substituents of the multi-branched polymer carrier (m + n + o + p) is an integer of 4 to 200. It is preferably 4 to 150, more preferably 8 to 100.
Bonding number of polyethylene glycol segment represented by the nucleic acid metabolism antagonist binding succinic acid monoamide units and R ₂ represented by R _1, and the pharmacokinetic properties of the nucleic acid metabolism antagonist binding hyperbranched compounds, the dissociation rate of nucleic acid metabolism antagonist It should be set as appropriate.
In the [type 1-2], since R ₁ uses a succinic acid monoamide polymer, a plurality of nucleic acid antimetabolites can be bonded to one substituent. For this reason, since the content of the nucleic acid antimetabolite can be increased, the number of R ₁ bonds; n can be decreased as compared with the above [Type 1-1].

Next, in another embodiment of the nucleic acid antimetabolite-binding hyperbranched compound according to the present invention, a substituent in which the polyethylene glycol segment and the succinic acid monoamide unit bound to the nucleic acid antimetabolite are linked together is an integral group. An embodiment of [Type 2] that binds to the terminal reactive functional group of the multi-branched polymer carrier will be described.
In the [type 2], R ₁ in the general formula (1) is a succinic monoamide unit binding type substituent in which a nucleic acid antimetabolite is bound to a polyethylene glycol segment and a side chain carboxy group.

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｙは多分岐高分子担体との結合基であって酸素原子又はＮ－Ｒ_４であり、前記Ｒ_４並びにＲ_５及びＲ_６はそれぞれ独立して水素原子又は炭素数（Ｃ１～Ｃ８）のアルキル基であり、Ｒ_１６はポリエチレングリコールセグメントである。］で示されるポリエチレングリコールセグメントと核酸代謝拮抗剤が結合したコハク酸モノアミドユニットの結合型置換基が好ましい。
　核酸代謝拮抗剤の結合様式は、アミド結合のみの場合、エステル結合のみの場合、又はアミド結合とエステル結合との混合体の場合の何れでも良い。用いる核酸代謝拮抗剤の結合性官能基に応じて、側鎖カルボキシ基への結合様式を適宜選択して良い。 In the above [Type 2], the succinic acid monoamide unit-bonded substituent of the polyethylene glycol segment and the side chain carboxy group is represented by the following general formula (25) and / or (26):

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, Y is a binding group to the multi-branched polymer carrier and is an oxygen atom or N—R ₄ , and R ₄ and R ₅ and R ₆ is each independently a hydrogen atom or an alkyl group having from 1 to 8 carbon atoms, and R ₁₆ is a polyethylene glycol segment. ] A succinic acid monoamide unit-bonded substituent in which a polyethylene glycol segment and a nucleic acid antimetabolite are bound is preferred.
The binding mode of the nucleic acid antimetabolite may be any of an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite used, the mode of binding to the side chain carboxy group may be appropriately selected.

The bonding type substituent of the polyethylene glycol segment and the succinic acid monoamide unit represented by the general formulas (25) and (26) may be an optically active substance or an arbitrary mixture thereof.
The nucleic acid metabolism antagonists according to R ₄ , R ₅ and R ₆ and [D] are as defined above.
In the substituents represented by the general formulas (25) and (26), Y is preferably NR ₄ . That is, it is preferably an aspartic acid monoamide unit in which a polyethylene glycol segment and a nucleic acid antimetabolite are bound. In this case, the R ₄ , R ₅ and R ₆ , and the nucleic acid antimetabolite according to [D] are also as defined above.

In [Type 2], the succinic monoamide unit to which the nucleic acid antimetabolite is bound may be a polymer segment of a plurality of the succinic monoamide units. In this case, the nucleic acid antimetabolite is bonded to the side chain carboxy group of the succinic acid monoamide unit polymer via an amide bond and / or an ester bond with one or more molecules, preferably two or more molecules. It is a substituent.
The binding mode of the nucleic acid antimetabolite may be any of an amide bond only, an ester bond only, or a mixture of an amide bond and an ester bond. Depending on the binding functional group of the nucleic acid antimetabolite used, the mode of binding to the side chain carboxy group may be appropriately selected.

The segment that is a polymer of succinic acid monoamide units to which a nucleic acid antimetabolite is bound in [Type 2] is preferably a polyaspartic acid segment to which a nucleic acid antimetabolite is bound. That is, a polyaspartic acid segment in which a nucleic acid antimetabolite is bonded to the side chain carboxy group through an amide bond and / or an ester bond is preferable. The polyaspartic acid may be an α-type polymer, a β-type polymer, or a polymer in which α-type and β-type are mixed.
The nucleic acid metabolism antagonist may be bound to one or more side chain carboxy groups in the polymer of the succinic acid monoamide unit. A polymer of a succinic acid monoamide unit in which the nucleic acid antimetabolite is bound to two or more side chain carboxy groups is preferred. That is, a polymer having a nucleic acid antimetabolite binding rate with respect to the total side chain carboxy group is preferably 10 to 90%.

The binding mode of the succinic acid monoamide unit in which the nucleic acid antimetabolite is bonded to the polyethylene glycol segment and the side chain carboxy group in [Type 2] is not particularly limited, and both substituents are directly bonded to each terminal group. Alternatively, they may be bonded via an appropriate bonding group. Substituents linked through a suitable linking group are preferred.
As the binding mode of the [type 2] polyethylene glycol segment and the multi-branched polymer carrier of the succinic acid monoamide unit bonded with the side chain carboxyl group to the side chain carboxy group, a nucleic acid metabolism antagonist is bound. It is preferable that the succinic acid monoamide unit and the terminal reactive functional group of the multi-branched polymer carrier are bonded to each other, and the polyethylene glycol segment has a structure forming an outer shell layer of the nucleic acid antimetabolite-binding multi-branched compound.

The polyethylene glycol segment represented by R ₁₆ in the general formulas (25) and (26) of [Type 2] is a segment having a repeating structure of an ethyleneoxy group; (CH ₂ CH ₂ O) unit. The degree of polymerization of ethyleneoxy group units is preferably 5 to 10,000 units, more preferably the degree of polymerization is 5 to 2,500 units, particularly preferably 10 to 1,000 units, and still more preferably 20 to 50 units. It is the segment structure containing the polyethyleneglycol chain of.
That is, the polyethylene glycol segment is preferably a segment part having an average molecular weight of 0.2 kilodaltons to 500 kilodaltons, more preferably a structural part having an average molecular weight of 0.2 kilodaltons to 150 kilodaltons. The average molecular weight is particularly preferably 0.5 to 50 kilodaltons. A polyethylene glycol segment having an average molecular weight of 1 kilodalton to 20 kilodalton is particularly preferred.

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_５は側鎖カルボキシ基に核酸代謝拮抗剤が結合したコハク酸モノアミドユニットとの結合基を示す。］を用いることが好ましい。
　すなわち、エチレンオキシ基；（ＣＨ_２ＣＨ_２Ｏ）単位の繰り返し構造によるエチレンオキシ基単位重合度が５～２，５００ユニットのポリエチレングリコールセグメントであり、ポリエチレングリコール相当の平均分子量として２００ダルトン～１５０キロダルトンのセグメント部であることが好ましい。より好ましくは重合度が２０～１，５００ユニットであり、平均分子量として１キロダルトン～５０キロダルトンのポリエチレングリコール鎖を含むセグメント構造である。なお、該Ｒ_８は前述と同義である。 The polyethylene glycol segment of R _{16 in} the general formulas (25) and (26) is represented by the general formula (23)

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₅ represents a linking group with a succinic acid monoamide unit in which a nucleic acid antimetabolite is bonded to a side chain carboxy group. ] Is preferably used.
That is, it is a polyethylene glycol segment having an ethyleneoxy group unit polymerization degree of 5 to 2,500 units due to a repeating structure of ethyleneoxy group; (CH ₂ CH ₂ O) units, and an average molecular weight equivalent to polyethylene glycol is 200 daltons to 150 kg. It is preferably a Dalton segment. More preferred is a segment structure containing a polyethylene glycol chain having a degree of polymerization of 20 to 1,500 units and an average molecular weight of 1 kilodalton to 50 kilodalton. The R ₈ has the same meaning as described above.

X ₅ in the general formula (23) represents a linking group with a succinic acid monoamide unit in which a nucleic acid antimetabolite is bonded to a side chain carboxy group. The binding site between the succinic acid monoamide unit and the polyethylene glycol segment is an embodiment in which an amide bond is formed with respect to one carboxy group of the succinic acid. Therefore, one of the terminal groups of the X ₅ is bonded to the amide bond, the other end group is an oxygen atom and an ether bond of the polyethylene glycol segment, an ester bond, the binding functional group capable urethane bond or carbonate bond Have. Therefore, X ₅ is preferably a carbon number (C1-C8) alkylene group which may have a substituent having the terminal group.

As the linking group according to X ₅ , — (CH ₂ ) _x — (x represents an integer of 1 to 8) is a linking group that is ether-bonded to a polyethylene glycol segment and linked to the amide bond of a succinic acid monoamide unit. Is mentioned. Examples of the linking group that is ester-bonded to the polyethylene glycol segment and linked to the amide bond of the succinic acid monoamide unit include —CO— (CH ₂ ) _x — (x represents an integer of 1 to 8). Examples of the linking group that is bonded to the polyethylene glycol segment by urethane and linked to the amide bond of the succinic acid monoamide unit include —CONH— (CH ₂ ) _x — (x represents an integer of 1 to 8).
In addition, examples of the bonding group that is carbonate-bonded to the polyethylene glycol segment and is linked to the amide bond of the succinic acid monoamide unit include —COO— (CH ₂ ) _x — (x represents an integer of 1 to 8).
X ₅ is preferably a linking group that is ether-bonded to a polyethylene glycol segment and linked to the amide bond of a succinic acid monoamide unit, and is — (CH ₂ ) _x — (x represents an integer of 1 to 8). .

［式中、［Ｄ］は核酸代謝拮抗剤の結合残基であり、Ｒ_１６はポリエチレングリコールセグメントであり、Ｒ_１７は水酸基及び／又は－Ｎ（Ｒ_１８）ＣＯＮＨ（Ｒ_１９）であり、該Ｒ_１８及び該Ｒ_１９は同一でも異なっていてもよく、三級アミノ基で置換されていても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、Ｘ_３は末端官能基Ｆとの結合基であり、ｆ、ｇ、ｈ、ｉ及びｊはそれぞれ独立して０～３０の整数を示し、（ｆ＋ｇ）は１～３０の整数を示し、ポリアミノ酸誘導体の総重合数である（ｆ＋ｇ＋ｈ＋ｉ＋ｊ）は１～３０であり、前記［Ｄ］が結合したアスパラギン酸単位、前記Ｒ_１７が結合したアスパラギン酸単位及び側鎖カルボキシ基が分子内環化型のアスパラギン酸単位が、それぞれ独立してランダムな配列である。］で示される置換基であることが好ましい。 In [Type 2], R ₁ in the general formula (1) is a block copolymer of a polyaspartic acid derivative segment in which a nucleic acid antimetabolite is bonded to a polyethylene glycol segment and a side chain carboxy group, and the general formula (6 Or (7)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, R ₁₆ is a polyethylene glycol segment, R ₁₇ is a hydroxyl group and / or —N (R ₁₈ ) CONH (R ₁₉ ), R ₁₈ and R ₁₉ may be the same or different and each is a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group; ₃ is a linking group to the terminal functional group F, f, g, h, i and j each independently represents an integer of 0 to 30, (f + g) represents an integer of 1 to 30, and a polyamino acid derivative (F + g + h + i + j) is 1 to 30, and the aspartic acid unit to which the [D] is bonded, the aspartic acid unit to which the R ₁₇ is bonded and the side chain carboxy group is an intramolecularly cyclized aspartic acid. Each unit is German It is a random sequence. It is preferable that it is a substituent shown by this.

The nucleic acid antimetabolite [D] has the same meaning as described above, and examples thereof include a pyrimidine antimetabolite, a purine antimetabolite, and a triazine antimetabolite. It is preferable to use a nucleic acid antimetabolite having an amino group and / or a hydroxyl group. More preferably, it is a nucleic acid antimetabolite that has an amino group at the nucleoside base, and is preferably a nucleic acid antimetabolite that can be amide-bonded to the carboxy group of the aspartic acid unit by the amino group.

［式中、－Ｒｆは、式（１３）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基残基を示す。］。ここでＲ_２０における脂肪酸エステルのアシル基は、前述と同義である。 The nucleic acid antimetabolite is one or more selected from the following formula (12) for the nucleobase moiety, and any one selected from the following formula (13) for the group (Rf) bonded thereto: A nucleic acid antimetabolite that is a combination of the above is particularly preferred.

[Wherein, -Rf represents the formula (13):

R ₂₀ represents a hydrogen atom or an acyl group residue of a fatty acid ester. ]. Here, the acyl group of the fatty acid ester in R ₂₀ has the same meaning as described above.

As the nucleic acid antimetabolite, a cytidine antimetabolite is preferably used. The nucleobase moiety is a cytidine base represented by the following formula (14), and the group (Rf) bonded thereto is represented by the following formula (15). Particularly preferred is a nucleic acid antimetabolite that is a combination of any one or more selected from the group of substituents. Here, R ₂₀ is a compound represented by a hydroxyl group or an acyl group of a fatty acid ester.

［式中、－Ｒｆは、式（１４）：

の置換基群より選ばれる基を示し、Ｒ_２０は水素原子又は脂肪酸エステルのアシル基を示す。］。Ｒ_２０における脂肪酸エステルのアシル基は、前述と同義である。

[Wherein, -Rf represents the formula (14):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. ]. The acyl group of the fatty acid ester in R ₂₀ has the same meaning as described above.

These cytidine antimetabolites are gemcitabine and its fatty acid ester derivative, cytarabine and its fatty acid ester derivative, and 3'-ethynylcytidine and its fatty acid ester derivative. Examples of fatty acid ester derivatives include cytarabine-5'-elaidic acid ester (CP-4055) and gemcitabine-5'-elaidic acid ester (CP-4126), which are preferably used in the present invention.

R ₁₇ is a hydroxyl group and / or —N (R ₁₈ ) CONH (R ₁₉ ). Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group in R ₁₈ and R ₁₉ include, for example, a methyl group, an ethyl group, Examples include n-propyl group, isopropyl group, isobutyl group, sec-butyl group, t-butyl group, 1-methylbutyl group, 2-methylbutyl group, neopentyl group, cyclohexyl group, etc., preferably isopropyl group, cyclohexyl group Is mentioned.
Examples of the linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with the tertiary amino group include, for example, 2-dimethylaminoethyl group, 3-dimethylaminopropyl group , 5-dimethylaminopentyl group, 6-dimethylaminohexyl group and the like.
R ₁₈ and R ₁₉ are preferably an ethyl group, an isopropyl group, a cyclohexyl group, and a 3-dimethylaminopropyl group.

When R ₁₇ is a hydroxyl group, it represents a carboxylic acid embodiment. Moreover, the arbitrary salt aspect of the carboxylic acid may be sufficient.
R ₁₇ is a hydroxyl group and / or —N (R ₁₈ ) CONH (R ₁₉ ), but when it is only a hydroxyl group, a hydroxyl group and —N (R ₁₈ ) CONH (R ₁₉ ) coexist, or —N An embodiment in which only (R ₁₈ ) CONH (R ₁₉ ) is employed can be employed. The abundance ratio of the hydroxyl group to —N (R ₁₈ ) CONH (R ₁₉ ) may be arbitrarily set.

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_５’は側鎖カルボキシ基に核酸代謝拮抗剤が結合したポリアスパラギン酸セグメントとの結合基を示す。］を用いることが好ましい。
　すなわち、エチレンオキシ基；（ＣＨ_２ＣＨ_２Ｏ）単位の繰り返し構造によるエチレンオキシ基単位重合度が５～２，５００ユニットのポリエチレングリコールセグメントであり、ポリエチレングリコール相当の平均分子量として０．２キロダルトン～１５０キロダルトンのセグメント部であることが好ましい。より好ましくは重合度が１０～１，０００ユニットであり、平均分子量として０．５キロダルトン～５０キロダルトンであり、更に好ましくは重合度が２０～５００ユニットで、平均分子量として１キロダルトン～２０キロダルトンであり、殊更好ましくは重合度が２０～３００ユニットで、平均分子量として１キロダルトン～１２キロダルトンのポリエチレングリコール鎖を含むセグメント構造である。なお、該Ｒ_８の詳細は前述と同義である。 The polyethylene glycol segment of R _{16 in} the general formulas (6) and (7) is represented by the general formula (24).

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X _{5 ′} represents a binding group to a polyaspartic acid segment in which a nucleic acid antimetabolite is bound to a side chain carboxy group. ] Is preferably used.
That is, it is a polyethylene glycol segment having an ethyleneoxy group; the degree of polymerization of the ethyleneoxy group unit having a repeating structure of (CH ₂ CH ₂ O) units of 5 to 2,500 units, and an average molecular weight equivalent to polyethylene glycol is 0.2 kilodalton. A segment of ~ 150 kilodaltons is preferred. More preferably, the degree of polymerization is from 10 to 1,000 units, and the average molecular weight is from 0.5 kilodaltons to 50 kilodaltons, and still more preferably, the degree of polymerization is from 20 to 500 units, and the average molecular weight is from 1 kilodaltons to 20 units. A segment structure having a degree of polymerization of 20 to 300 units and a polyethylene glycol chain having an average molecular weight of 1 to 12 kilodaltons is particularly preferred. The details of R ₈ are as defined above.

X _{5 ′} in the general formula (24) represents a binding group to a polyaspartic acid segment in which a nucleic acid antimetabolite is bound to a side chain carboxy group. The binding site of the polyaspartic acid segment and the polyethylene glycol segment is a nitrogen functional group, one end group of the X _{5 ′} is a binding functional group that can be linked to the nitrogen functional group, and the other end group is It has a binding functional group capable of forming an ether bond, an ester bond, a urethane bond or a carbonate bond with the oxygen atom of the polyethylene glycol segment. Therefore, X _{5 ′} is preferably a carbon number (C1-C8) alkylene group which may have a substituent having the terminal group.

Examples of the linking group related to X _{5 ′} include — (CH ₂ ) _x — (x represents an integer of 1 to 8) as a linking group that is ether-bonded to a polyethylene glycol segment and bonded to a polyaspartic acid segment. Can be mentioned. Examples of the linking group that bonds to the polyethylene glycol segment and bonds to the polyaspartic acid segment include —CO— (CH ₂ ) _x — (x represents an integer of 1 to 8). Examples of the linking group bonded to the polyethylene glycol segment and bonded to the polyaspartic acid segment include —CONH— (CH ₂ ) _x — (wherein x represents an integer of 1 to 8).
In addition, examples of the linking group bonded to the polyethylene glycol segment and bonded to the polyaspartic acid segment include —COO— (CH ₂ ) _x — (x represents an integer of 1 to 8).
X ₅ is preferably a linking group that is ether-bonded to a polyethylene glycol segment and bonded to a polyaspartic acid segment, and is — (CH ₂ ) _x — (x represents an integer of 1 to 8).

X ₃ is a bonding group between the polyaspartic acid derivative segment represented by the general formula (6) or (7) and the terminal reactive functional group [F] of the multi-branched polymer carrier. The linking group X ₃ is not particularly limited as long as it is a linking group having functional groups capable of binding to the terminal group of R ₁ and the terminal reactive functional group [F] at both ends. is not.

The X ₃ has one terminal group bonded to the terminal group of the polyaspartic acid derivative segment, and the other terminal group connected to the terminal reactive functional group [F], an ester bond, an amide bond, a thioester bond, a urea An alkylene group having a bonding functional group capable of bonding or urethane bonding, and optionally having a substituent (C1-C8).
The linking group related to X ₃ includes, for example, — (CH ₂ ) _y —NH— (where y is 0 to 8) as a linking group that bonds with the terminal reactive functional group [F] to an amide bond, an ester bond or a thioester bond. -(CH ₂ ) _y -O- (y is an integer from 0 to 8),-(CH ₂ ) _y -S- (y is an integer from 0 to 8),-(CH ₂ ) _y —CO— (y represents an integer of 0 to 8), —NH— (CH ₂ ) _y —CO— (y represents an integer of 0 to 8), and the like.

The X ₃ may be a “bond”. The “bond” refers to an embodiment in which the terminal reactive functional group of the multi-branched polymer carrier and the terminal group related to the polyaspartic acid derivative segment are directly bonded without using a bonding group.

The substituent which is a block copolymer of the polyethylene glycol segment and the polyaspartic acid derivative segment represented by the general formula (6) or (7) has a total polymerization number (f + g + h + i + j) of 1 to 30. A polyaspartic acid derivative segment having a polymerization number of 4 to 30 is preferable, and a segment structure having a polymerization number of 5 to 25 is preferable.
F, g, h, i, and j representing the number of constituents of the aspartic acid derivative unit are each independently an integer of 0 to 30. However, the aspartic acid derivative unit to which the nucleic acid antimetabolite [D] is bound is an essential component, and (f + g) represents an integer of 1 to 30. Preferably, (f + g) is an integer of 4 to 25, and more preferably 5 to 20. In addition, the number of aspartic acid derivative units to which R ₁₇ which is a hydroxyl group and / or —N (R ₁₈ ) CONH (R ₁₉ ) is bonded (h + i) and the side chain carboxy group is an intramolecular cyclized aspartic acid derivative unit The number j is an arbitrary configuration, and (h + i) and j are 0 to 29.
The substituent represented by the general formula (6) or (7) is an intramolecular cyclization type in which the aspartic acid unit to which the [D] is bonded, the aspartic acid unit to which the R ₁₇ is bonded, and a side chain carboxy group. The aspartic acid unit may be in the form of a localized sequence, or may be a polymer structure composed of a random sequence with no regularity in each structural unit, that is, the side chain modification product This is an array with no particular regularity in the array order.

［式中、Ｒ_８は水素原子又は置換基を有していても良い炭素数（Ｃ１～Ｃ８）の直鎖状、分岐鎖状又は環状のアルキル基であり、ｋは５～２，５００の整数であり、Ｘ_１は末端官能基Ｆとの結合基を示す。］で示されるポリエチレングリコールセグメントであることが好ましい。ここで、Ｒ_８、ｋ及びＸ_１は前述と同義である。 The nucleic acid antimetabolite-binding hyperbranched compound according to [Type 2] may have a substituent containing a polyethylene glycol segment represented by R ₂ in the general formula (1). The substituent containing the polyethylene glycol segment has the same meaning as described in the above [Type 1].
The R ₂ is represented by the general formula (8)

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₁ represents a bonding group with the terminal functional group F. It is preferable that it is a polyethyleneglycol segment shown by this. Here, R ₈ , k and X ₁ are as defined above.

In the nucleic acid antimetabolite-binding hyperbranched compound according to [Type 2], a substituent (R ₁ ) in which a polyethylene glycol segment and a nucleic acid antimetabolite-binding succinic acid monoamide unit are linked, and an optional polyethylene glycol segment-containing substituent A substituent (R ₃ ) containing (R ₂ ) and an arbitrary succinic monoamide derivative residue and / or succinimide residue may be bonded. These optional substituents are residues obtained by dissociating the nucleic acid antimetabolite from a substituent obtained by linking the polyethylene glycol segment according to R ₁ and the nucleic acid antimetabolite-binding succinic acid monoamide unit.
A polyethylene glycol segment and a polyaspartic acid derivative represented by the general formula (6) or (7), wherein the polyethylene glycol segment according to R ₁ of [Type 2] and the nucleic acid antimetabolite-binding succinic acid monoamide unit are linked. In the general formula (6) or (7), the R ₃ is a polyaspartic acid derivative lacking an aspartic acid unit comprising the nucleic acid antimetabolite; [D]. That is, in general formula (6) or (7), f and g are 0, and R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , X ₃ , h, i and j are substituents as defined above. .

In the [type 2] nucleic acid antimetabolite-binding hyperbranched compound, a substituent (R ₁ ) in which the polyethylene glycol segment and the nucleic acid antimetabolite-binding succinic acid monoamide unit are linked, and a substituent (R ₂ ), and a terminal functional group [F] to which the substituent (R ₃ ) containing the succinic monoamide derivative residue and / or the succinimide residue is not bonded.
[F] is one or more terminal functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group. The terminal functional group may be selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group modified with a protecting group having an alkyl group having a carbon number (C1 to C6) which may have a substituent. One or more functional groups may be used. That is, [F] in the general formula (1) may remain as a terminal reactive functional group, or may be a protective group modified product of a terminal reactive functional group, and is a group in which these are mixed. Also good. The terminal functional group according to [F] has the same meaning as in [Type 1-1].

Preferred examples of the terminal reactive functional group-protecting group-modified product include an acetyl group, a propionyl group, a butyryl group, a trifluoroacetyl group, a trichloroacetyl group, when the terminal functional group is an amino group, a hydroxyl group, or a mercapto group. Examples include methoxycarbonyl group, trichloromethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group and the like.
When the terminal functional group is a carboxy group, an amide bond such as ethylamino group, methoxymethylamino group, methoxyethylamino group, methoxyethoxyethylamino group or the like, or ethoxy group, methoxymethoxy group, methoxyethoxy group, methoxyethoxymethoxy group An ester conjugate such as

The protective group-modified product of the terminal reactive functional group may be present arbitrarily and may be 0 or more and 199 or less. The protective group-modified product can be provided because it can control the surface charge and the like of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention and can control physical properties such as water solubility and self-association. preferable. For this reason, it is preferable that the said protective group modification body is 4 groups or more and exists in 150 groups or less, and it is more preferable that it exists in 6 groups or more and 100 groups or less.

When the nucleic acid antimetabolite-binding hyperbranched compound of the present invention is the above [type 2], the number of each substituent bond in the general formula (1) is m is an integer of 0 to 199, and n is 1 to 200. O is an integer from 0 to 199, and p is an integer from 0 to 199. Preferably, m is an integer from 0 to 120, n is an integer from 2 to 100, o is an integer from 0 to 100, and p is an integer from 0 to 80. More preferably, m is an integer from 0 to 80, n is an integer from 5 to 50, o is an integer from 0 to 50, and p is an integer from 0 to 50. The total number of terminal substituents of the multi-branched polymer carrier (m + n + o + p) is an integer of 4 to 200. It is preferably 4 to 150, more preferably 8 to 100.
The number of substituents linked to the polyethylene glycol segment represented by R ₁ and the nucleic acid antimetabolite-bound succinic acid monoamide unit determines the pharmacokinetic properties of the nucleic acid antimetabolite-bound hyperbranched compound and the dissociation rate of the nucleic acid antimetabolite. It should be set as appropriate.
In the [Type 2], since the R ₁ uses a substituent in which two functional functional groups of a polyethylene glycol segment and a nucleic acid antimetabolite-binding succinic acid monoamide are integrated, in a multi-branched compound, the number of substituents is small. Desired physical properties can be obtained. Furthermore, by using a nucleic acid antimetabolite-binding succinic acid monoamide as the polymer, a plurality of nucleic acid antimetabolites can be bonded to one substituent. For this reason, the content of the nucleic acid metabolism antagonist can be increased, which is preferable. Therefore, the number of bonds of R ₁ ; n can be lowered.

The nucleic acid antimetabolite-binding multibranched compound of the present invention is preferably prepared by preparing an aqueous solution of the multibranched compound and administering it parenterally. The aqueous solution is prepared by dissolving in water, physiological saline, phosphate buffered saline (PBS solution), 5% glucose aqueous solution, or the like.

It is considered that the molecular weight of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention correlates with side effects such as drug efficacy and bone marrow suppression. Generally, when the molecular weight is 10 kilodaltons or less, excretion from the living body is rapidly performed after parenteral administration. In this form, it is considered that the drug is discharged out of the body before exhibiting the drug effect, and the desired drug effect cannot be obtained. On the other hand, when the molecular weight is 200 kilodaltons or more, it is considered that the retention time of the compound in the living body is excessively extended and the side effects of the nucleic acid antimetabolite are increased. Therefore, the hyperbranched compound of the present invention comprises a nucleic acid antimetabolite via a succinic acid monoamide, and has a molecular weight of 10 kilodaltons or more and 200 kilodaltons or less, while maintaining excellent medicinal effects, It is possible to provide a medicinal product with a small therapeutic effect. The molecular weight of the multi-branched compound is more preferably 20 kilodaltons or more and 160 kilodaltons or less.

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention preferably has physical properties that do not exhibit self-association in the aqueous solution. Here, the self-association property means that the hyperbranched compound is a physical property that forms an aggregate by self-associating with more than 10 molecules. Therefore, the “physical property not exhibiting self-association” in the present invention refers to an embodiment in which the multi-branched compound in an aqueous solution exists as a monomolecular body or forms a self-associating body of 10 molecules or less.
It is effective to use light scattering intensity using laser light as an index of the self-association property of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention. That is, the self-association property of the nucleic acid antimetabolite-binding hyperbranched compound in an aqueous solution can be confirmed using the laser light scattering intensity as an index. At that time, a method for confirming the self-association property of the nucleic acid antimetabolite-binding multibranched compound in an aqueous solution using toluene as a light scattering intensity standard sample and relative intensity with respect to toluene as an index is effective.
In the present invention, an aqueous solution in which the concentration of the nucleic acid antimetabolite binding hyperbranched compound is 1 mg / mL is measured with a laser light scattering photometer, and the light scattering intensity is 5 times or less as the relative intensity with respect to the light scattering intensity of toluene. In this case, the physical properties do not show self-association, and it is considered that the aqueous solution is dispersed in an aggregate of about a single molecule to several molecules. Preferably, the multi-branched compound has a light scattering intensity of 3 times or less as a relative intensity with respect to the light scattering intensity of toluene.

Examples of the laser light scattering photometer include a dynamic light scattering photometer DLS-8000DL manufactured by Otsuka Electronics Co., Ltd. (measurement temperature 25 ° C., measurement angle: 90 °, wavelength: 632.8 nm, ND filter: 5%, PH1: OPEN) , PH2: SLIT, sample concentration: 1 mg / mL), and measuring the light scattering intensity of an aqueous solution having a multibranched compound concentration of 1 mg / mL with a laser light scattering photometer. In addition, toluene used as a standard substance for light scattering intensity measurement is not particularly limited as long as it has a reagent level purity, and can be used. It is preferable to use toluene that has been subjected to pretreatment filtration, which is usually performed in sample preparation for light scattering analysis.
In the measurement method, the nucleic acid antimetabolite-binding hyperbranched compound preferably has a light scattering intensity of 5 times or less, more preferably 3 times or less as a relative intensity with respect to the light scattering intensity of toluene. In this case, the lower limit value is not particularly limited, and is a case where no clear light scattering intensity is exhibited, a state where no self-association property is exhibited in an aqueous solution, and almost monomolecular to several molecules in the aqueous solution. It shows that it is dispersed with a degree of aggregate.

The presence or absence of self-association in the aqueous solution of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention correlates with side effects such as bone marrow suppression. Nucleic acid metabolism antagonists have the problem that bone marrow suppression such as leukopenia occurs as a side effect, making it difficult to continue treatment using the therapeutic agent. For this reason, providing a therapeutic agent for a nucleic acid antimetabolite with low bone marrow suppression is very useful in a method for treating malignant tumors.
The nucleic acid antimetabolite-binding hyperbranched compound of the present invention uses a multi-branched polymer carrier having a small particle size as a physical property in an aqueous solution to prepare a nucleic acid antimetabolite prodrug and distribute a polyethylene glycol segment Thus, a polymerized prodrug with low physical properties is prepared. The derivative has a physical property that does not exhibit self-association in an aqueous solution, and as a result, can provide a pharmaceutical with a high therapeutic effect with little bone marrow suppression.

Next, the manufacturing method of the nucleic acid antimetabolite binding hyperbranched compound of this invention is demonstrated.
The nucleic acid antimetabolite-bound hyperbranched compound of the present invention uses a multi-branched polymer carrier having a terminal reactive functional group, and includes a polyethylene glycol segment and a succinic acid monoamide unit bound with a nucleic acid antimetabolite. It can be prepared by attaching groups.
Examples of the method for producing the multibranched compound include a method in which a polyethylene glycol segment and a succinic acid monoamide unit are simultaneously reacted with a multibranched polymer carrier, and then a nucleic acid antimetabolite is chemically bonded. Examples include a method in which a compound to which an antimetabolite is bound is prepared, and a compound of a succinic acid monoamide unit in which a polyethylene glycol segment and a previously prepared nucleic acid antimetabolite are bound to a multi-branched polymer carrier are reacted simultaneously. Alternatively, it can also be produced by a method in which a polyethylene glycol segment is reacted with a hyperbranched polymer carrier, and then a succinic acid monoamide unit is chemically bound thereto, and finally a nucleic acid antimetabolite is chemically bound. Alternatively, a method of reacting a polyethylene glycol segment with a hyperbranched polymer carrier and then reacting a compound containing a succinic acid monoamide unit to which a nucleic acid antimetabolite is bound, and the like can be mentioned. As the production method, since the amount of the polyethylene glycol segment introduced and the binding amount of the nucleic acid antimetabolite are easily controlled, it is preferable to use the two methods shown in the latter, in which two kinds of substituents are sequentially bonded.
After completion of the reaction, a purification step may be optionally performed, and a multi-branched compound in which a polyethylene glycol segment and a nucleic acid antimetabolite that can be applied as pharmaceuticals are introduced into a terminal functional group can be produced.

The following examples of the method for producing a nucleic acid antimetabolite-binding hyperbranched compound according to the present invention will be described for two types.
When preparing the above-mentioned [type 1] nucleic acid antimetabolite-bound hyperbranched compound, a polyethylene having a functional group capable of binding to the terminal reactive functional group with respect to the hyperbranched polymer carrier having the terminal reactive functional group By reacting a glycol segment compound and a compound containing a nucleic acid antimetabolite binding succinic acid monoamide unit having a functional group capable of binding to the terminal reactive functional group sequentially or simultaneously, the target [type 1] nucleic acid metabolism Antagonist-bound hyperbranched compounds can be prepared. The binding functional group of the compound including the polyethylene glycol segment compound and the nucleic acid antimetabolite-binding succinic acid monoamide unit may be the same type of functional group or a different type of functional group, It is preferable to use the same type of functional group.

For example, an amide condensation reaction between a polyethylene glycol segment compound having an amino group and a aspartic acid derivative in which a nucleic acid antimetabolite is bound to a carboxy group using a multi-branched polymer carrier having a carboxy group as a terminal reactive functional group By reacting under conditions, the target [type 1] nucleic acid antimetabolite-bound hyperbranched compound can be prepared. Under the present circumstances, the compound of desired polyethyleneglycol segment content and nucleic acid metabolism antagonist content can be prepared by controlling each reaction amount. After completion of the reaction, a purification step may optionally be performed, and the compound that can be applied as a pharmaceutical product can be produced.

On the other hand, when preparing the above-mentioned [type 2] nucleic acid antimetabolite-bound hyperbranched compound, a functional group capable of binding to the terminal reactive functional group is added to the multibranched polymer carrier having the terminal reactive functional group. This can be achieved by reacting a compound having a polyethylene glycol segment-nucleic acid antimetabolite binding (poly) aspartic acid linked.
For example, polyethylene glycol-polyaspartic acid is obtained by ring-opening polymerization of L-aspartic acid-N-carboxylic acid anhydride using a polyethylene glycol segment compound having an amino group as a reaction initiator. A polyethylene glycol segment-nucleic acid antimetabolite binding (poly) aspartic acid binding compound can be prepared by binding a nucleic acid antimetabolite to the aspartic acid side chain carboxy group. Preparation of the target [type 2] nucleic acid antimetabolite-bound hyperbranched compound by reacting this with a hyperbranched polymer carrier having a carboxy group at the terminal reactive functional group under amide condensation reaction conditions Can do. After completion of the reaction, a purification step may optionally be performed, and the compound that can be applied as a pharmaceutical product can be produced.

The nucleic acid antimetabolite-binding hyperbranched compound of the present invention has a property of gradually releasing a nucleic acid antimetabolite after administration in vivo, and has a use as a medicine containing the nucleic acid antimetabolite as an active ingredient.

The use of the nucleic acid antimetabolite-binding hyperbranched compound of the present invention as a pharmaceutical is not particularly limited as long as the nucleic acid antimetabolite has a therapeutic effect. For example, it is suitable for pharmaceuticals used for the treatment of malignant tumors, viral diseases and the like. Particularly preferred is a medicament for the treatment of malignant tumors. Examples of malignant tumors include non-small cell lung cancer, pancreatic cancer, gastric cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, bladder cancer, AIDS-related Kaposi's sarcoma and the like.

The medicament containing the nucleic acid antimetabolite-binding hyperbranched compound of the present invention may have other additives that are usually accepted as pharmaceuticals. Such additives include excipients, extenders, fillers, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives, flavoring agents. Agents, soothing agents, stabilizers, tonicity agents and the like.

The medicament containing the nucleic acid antimetabolite-binding hyperbranched compound of the present invention may be prepared as a therapeutic pharmaceutical preparation. The preparation can be administered by any method such as oral, injection, intrarectal administration, intraportal administration, mixing with organ perfusate, and local administration to the affected organ, preferably parenteral administration. More preferably, intravenous administration by injection, intraarterial administration, or local administration to an affected organ.

The dosage of the pharmaceutical containing the nucleic acid antimetabolite-binding hyperbranched compound of the present invention varies depending on the disease state, administration method, patient state, age, weight, etc., but is usually 1 mg per 1 m ² of body surface area in terms of nucleic acid antimetabolite. 5,000 mg, preferably 10 mg to 2,000 mg. As an administration method, it may be administered once or divided into several times a day. Although administration can be performed every day, repeated administration may be performed after several days to several months. As needed, administration methods, dosages, and administration schedules other than those described above can be used.

The present invention will be further described below with reference to examples. However, the present invention is not limited to these examples.
The “molecular weight of the nucleic acid antimetabolite-binding hyperbranched compound” in Examples 1 to 8 was calculated by the following formula.
[Molecular weight of nucleic acid antimetabolite-bound hyperbranched compound] = [Molecular weight of multi-branched polymer carrier] + [(polyethylene glycol segment + polyethylene glycol binding group residue) molecular weight x number of bonds] + [nucleic acid antimetabolite binding] Residual molecular weight x number of bonds] + [Aspartic acid monoamide bound molecular weight x number of bonds]

In this example, “polyethylene glycol segment” uses a polyethylene glycol segment compound in which propylene amine as a bonding group is integrated with a polyethylene glycol segment, and these are combined to obtain a polyethylene glycol segment molecular weight.
Therefore, the “molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound” in this example is the main component, “molecular weight of the hyperbranched polymer carrier”, “total molecular weight of (polyethylene glycol segment + the binding group residue)” "And" total molecular weight of the nucleic acid antimetabolite "and" total molecular weight of aspartic acid monoamide unit residues "were used together.

The molecular weight of the polyethylene glycol segment was the peak top molecular weight in GPC analysis based on the polyethylene glycol standard substance in the polyethylene glycol segment compound before the introduction reaction.

The number of bonds of the polyethylene glycol segment was calculated from the consumption rate in the reaction relative to the charged amount of the polyethylene glycol segment compound in the binding reaction between the multi-branched polymer carrier and the polyethylene glycol segment compound.
The consumption of the polyethylene glycol compound was calculated by the following calculation formula.
In the binding reaction between the multi-branched polymer carrier and the polyethylene glycol segment compound, 10 μL of the reaction solution before the start of the reaction (before addition of diisopropylcarbodiimide) was diluted with 90 μL of 1% phosphoric acid, and HPLC (use column: Superdex 75 10/300 GL, Analysis was carried out using a detector manufactured by GE Healthcare (a suggested refraction detection analyzer (RI)).
The peak area corresponding to the polyethylene glycol segment compound at this time is As, and 10 μL of the reaction solution at the end of the reaction is diluted with 90 μL of 1% phosphoric acid, and the peak area corresponding to the polyethylene glycol compound when analyzed by HPLC is At. It was.
And the consumption rate of the polyethylene glycol segment was computed with the following formula | equation.
[Consumption Rate of Polyethylene Glycol Segment Compound] = 1-At / As

The content and the number of binding of the nucleic acid antimetabolite are 10 mg of the obtained nucleic acid antimetabolite-bound hyperbranched compound of Examples and Comparative Examples, and 1 mL of acetonitrile is added and dissolved to obtain a 1 mol / L aqueous sodium hydroxide solution. 1 mL was added, mixed, and hydrolyzed by stirring for 30 minutes.
To this hydrolyzed solution, 1 mL of 1 mol / L hydrochloric acid was added, and a water / acetonitrile mixture (1: 1) was added to make exactly 10 mL. The content of the nucleic acid antimetabolite was calculated by quantitatively analyzing the nucleic acid antimetabolite released from this solution using HPLC.
The binding number of the nucleic acid antimetabolite was calculated from the molecular weight of the nucleic acid antimetabolite and the molecular weight of the multi-branched polymer carrier based on the content of the nucleic acid antimetabolite.
The total molecular weight of the nucleic acid antimetabolite binding was calculated from the following formula.
[Total molecular weight of nucleic acid antimetabolite binding] = [Number of binding of nucleic acid antimetabolite per molecule of nucleic acid antimetabolite binding hyperbranched compound] × [Molecular weight of nucleic acid antimetabolite]

“Polyethylene glycol segment content” in Examples and Comparative Examples was calculated by the following calculation formula.
[Polyethylene glycol segment content (%)] = [polyethylene glycol segment total molecular weight] / [nucleic acid antimetabolite binding hyperbranched compound molecular weight] × 100
As the “total molecular weight of the polyethylene glycol segment”, a value calculated by multiplying the molecular weight of the polyethylene glycol segment by the number of bonds of the polyethylene glycol segment was used.
The “nucleic acid antimetabolite-binding hyperbranched compound molecular weight” used herein was a value calculated from the sum of the molecular weights of the respective constituent parts.

The scattering intensity of the nucleic acid antimetabolite binding hyperbranched compounds of Examples and Comparative Examples was measured by a dynamic light scattering photometer DLS-8000DL (measurement temperature 25 ° C., measurement angle: 90 °, wavelength: 632.8 nm, manufactured by Otsuka Electronics Co., Ltd.) (ND filter: 5%, PH1: OPEN, PH2: SLIT).
As a measurement sample for the measurement of scattering intensity, a solution prepared by adding 5% glucose injection solution so that the concentration of the nucleic acid antimetabolite-binding hyperbranched compound was 1 mg / mL and irradiating with ultrasound for 3 minutes under ice cooling was used. .
Toluene (manufactured by Junsei Co., Ltd., special grade) used for measurement of light scattering intensity was used after being filtered three times with a 0.2 μm membrane filter.
The light scattering intensity of the toluene standard solution measured by the light scattering intensity meter was 12,934 cps.

The number of associated molecules in the measurement sample solution of the nucleic acid antimetabolite binding hyperbranched compounds of Examples and Comparative Examples was calculated by the following formula.
[Number of associated molecules] = [SEC-MALS measured molecular weight] / [Nucleic acid antimetabolite binding hyperbranched compound molecular weight]
The molecular weight measured by SEC-MALS was measured using DAWN EOS (light scattering detector) and Optilab rEX (RI detector) manufactured by Wyatt Technology, and dn / dc was calculated using the value of polyethylene glycol (0.135). .
Column used: Superdex 200 Increase 10/300 GL, manufactured by GE Healthcare Inc. For the measurement sample, 5% glucose injection solution was added so that the nucleic acid antimetabolite-binding hyperbranched compound was 1 mg / mL, and ultrasonication was performed under ice cooling. A solution prepared by irradiating for 3 minutes was used.

[Synthesis Example 1] Synthesis of a multi-branched polymer carrier (hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl) having 32 terminal functional groups (Compound 1)
Hyperbranched 2,2-bis (methylol) propionic acid polyester-32-hydroxyl (5.0 g, terminal functional group number 32, manufactured by Sigma-Aldrich), succinic anhydride (22.2 g), 4-dimethyl Aminopyridine (DMAP, 1.1 g) was dissolved in DMF (100 mL) and stirred at an external temperature of 90 ° C. for 8 hours. The temperature was lowered to room temperature, an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 50 mL) was added and stirred for 1 hour, and then the ion exchange resin was removed by filtration. The filtrate was dialyzed against 50% acetonitrile / purified water, and then the internal solution was concentrated under reduced pressure to remove acetonitrile and lyophilized to obtain the title compound (8.72 g) according to Compound 1.
¹ H-NMR (400 MHz, Methanol-d ₄ , ppm): 1.15 to 1.41 (br, 84H), 2.52-2.77 (br, 128H), 3.42 to 3.90 (br , 16H), 3.95-4.61 (br, 120H)
^From the area ratio of ¹ H-NMR, it was confirmed that all alcohols at the terminals of the multibranched polymer carrier as the raw material were converted to carboxylic acids. Therefore, the molecular weight of Compound 1 was calculated to be 6.8 kilodaltons according to the following formula.
[Molecular Weight of Compound 1] = [Molecular Weight of Multibranched Polymer Carrier Precursor] + [(Number of Terminal Functional Groups of Multibranched Polymer Carrier Precursor × Succinic Acid Molecular Weight)]

[Synthesis Example 2] Synthesis of multi-branched polymer carrier having 64 terminal functional groups (hyperbranched 2,2-bis (methylol) propionic acid polyester-64-carboxyl) (Compound 2)
Hyperbranched 2,2-bis (methylol) propionic acid polyester-64-hydroxyl (3.0 g, terminal functional group number 64, manufactured by Sigma-Aldrich), succinic anhydride (13.1 g), 4-dimethyl Aminopyridine (DMAP, 0.64 g) was dissolved in DMF (60 mL) and stirred at an external temperature of 90 ° C. for 8 hours. The temperature was lowered to room temperature, an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 30 mL) was added and stirred for 1 hour, and then the ion exchange resin was removed by filtration. The filtrate was dialyzed against 50% acetonitrile / purified water, and then the internal solution was concentrated under reduced pressure to remove acetonitrile and lyophilized to obtain the title compound (5.53 g) according to Compound 2.
¹ H-NMR (400 MHz, Methanol-d ₄ , ppm): 1.15 to 1.52 (br, 180H), 2.39 to 3.00 (br, 256H), 3.42 to 3.90 (br , 16H), 3.95-4.75 (br, 240H)
^From the area ratio of ¹ H-NMR, it was confirmed that all alcohols at the terminals of the multibranched polymer carrier as the raw material were converted to carboxylic acids. Therefore, the molecular weight of Compound 2 was calculated to be 13.7 kilodaltons according to the following formula.
[Molecular Weight of Compound 2] = [Molecular Weight of Multibranched Polymer Carrier Precursor] + [(Number of Terminal Functional Groups of Multibranched Polymer Carrier Precursor × Succinic Acid Molecular Weight)]

[Synthesis Example 3] Synthesis of Aspartic acid-1-alanine methyl ester-4-gemcitabine amide (Compound 3)
After dissolving N- (t-butoxycarbonyl) aspartic acid-4-benzyl ester (15.0 g) and L-alanine methyl ester (6.5 g) in DMF (160 mL), 1-ethyl-3- [3- (Dimethylamino) propyl] carbodiimide (WSCD) hydrochloride (13.3 g), 1-hydroxybenzotriazole (HOBt) (8.53 g), triethylamine (6.5 mL) were added, and the mixture was stirred for 4 hours in an ice bath. . Water was added to the reaction solution, extracted with ethyl acetate, and washed with 5% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine. After drying over magnesium sulfate, ethyl acetate was distilled off under reduced pressure, followed by vacuum drying to obtain an oil (13.9 g).
This oil (13.9 g) was dissolved in methanol (350 mL), 10% palladium carbon (moisture content 50%) (1.39 g) was added, and the system was replaced with hydrogen, followed by 3 hours at room temperature. Stir. 10% Palladium carbon was filtered and washed with methanol (50 mL), and then the methanol was distilled off under reduced pressure, followed by vacuum drying to obtain an oil (10.6 g).
This oil and gemcitabine (3.0 g, manufactured by SCINO PHARM) were dissolved in DMF (56 mL), then HOBt (2.1 g) and WSCD hydrochloride (3.3 g) were added, and the temperature was raised from 0 ° C. to room temperature. And stirred overnight. Purified water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (170 mL). The organic layer was washed twice with saturated brine and dried over sodium sulfate. After removing ethyl acetate by concentration under reduced pressure, purification by silica gel column chromatography was performed, followed by vacuum drying to obtain an oily substance (3.9 g).
This oil was dissolved in ethyl acetate (41 mL), 4N hydrochloric acid-ethyl acetate solution (31 mL) was added, and the mixture was stirred at room temperature for 2 hr. A mixed solvent of ethyl acetate (80 mL) and n-hexane (20 mL) was added to the reaction solution, and the precipitate was collected by filtration and dried in vacuo to give compound 3 (2.99 g).
¹ H-NMR (400 MHz, DMSO-d6, ppm): 1.20 (d, 3H), 2.95-3.10 (m, 2H), 3.63 (s, 3H), 3.67 (dd , 1H), 3.90-4.02 (m, 5H), 4.18-4.31 (m, 3H), 6.18 (dd, 1H), 7.22 (d, 1H), 8. 25 (s, 2H), 8.92 (d, 1H), 11.3 (br, 1H)

[Synthesis Example 4] Synthesis of aspartic acid-1-leucine methyl ester-4-gemcitabine amide (Compound 4)
N- (t-butoxycarbonyl) -L-aspartic acid-4-benzyl ester (4.9 g manufactured by Watanabe Chemical Industries) and L-leucine-methyl ester hydrochloride (2.7 g manufactured by Kokusan Chemical Co., Ltd.) were treated with DMF. (75 mL), HOBt (2.8 g), diisopropylethylamine (2.6 mL) and WSCD hydrochloride (4.3 g) were added, and the mixture was stirred at 0 ° C. for 2 hours. Purified water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (250 mL). The organic layer was washed with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over sodium sulfate. After removing ethyl acetate by concentration under reduced pressure, purification by silica gel column chromatography was performed, followed by vacuum drying to obtain an oily substance (6.8 g).
This oily substance was dissolved in methanol (150 mL), 10% by mass palladium carbon was added, and the mixture was stirred in a hydrogen atmosphere for 2 hours. The reaction solution was filtered, and the filtrate was vacuum-dried to obtain an oil (5.37 g).
This oil and gemcitabine (3.0 g, manufactured by SCINO PHARM) were dissolved in DMF (57 mL), HOBt (2.1 g) and WSCD hydrochloride (3.3 g) were added, and the temperature was raised from 0 ° C. to room temperature. And stirred overnight. Purified water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (170 mL). The organic layer was washed twice with saturated brine and dried over sodium sulfate. After removing ethyl acetate by concentration under reduced pressure, purification by silica gel column chromatography was performed, followed by vacuum drying to obtain an oily substance (4.9 g).
This oil was dissolved in ethyl acetate (44 mL), 4N ethyl acetate solution (33 mL) was added, and the mixture was stirred at room temperature for 2 hr. A mixed solvent of ethyl acetate (80 mL) and n-hexane (20 mL) was added to the reaction solution, and the precipitate was collected by filtration and dried in vacuo to give compound 4 (3.62 g).
¹ H-NMR (400 MHz, DMSO-d6, ppm): 0.89 (dd, 6H), 1.49-1.71 (m, 3H), 2.97-3.17 (m, 4H), 3 .63 (s, 3H), 3.66 (dd, 1H), 3.80-3.93 (m, 2H), 4.10-4.28 (m, 4H), 6.18 (dd, 1H) ), 7.22 (d, 1H), 8.32 (s, 2H), 8.91 (d, 1H), 11.3 (br, 1H)

Synthesis Example 5 Synthesis of aspartic acid-1-isoleucine methyl ester-4-gemcitabine amide (Compound 5)
N- (t-butoxycarbonyl) -L-aspartic acid-4-benzyl ester (made by Kokusan Chemical Co., Ltd., 4.9 g) and L-isoleucine-methyl ester hydrochloride (made by Tokyo Chemical Industry Co., Ltd., 2.7 g) were dissolved in dichloromethane. After being dissolved in (75 mL), HOBt (2.8 g), diisopropylethylamine (3.1 mL) and WSCD hydrochloride (4.3 g) were added, and the mixture was stirred at room temperature for 4 hours. Purified water was added to the reaction solution, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate, saturated aqueous ammonium chloride and saturated brine, and dried over sodium sulfate. Dichloromethane was removed by concentration under reduced pressure, followed by purification by silica gel column chromatography. After vacuum drying, an oil (7.0 g) was obtained.
This oily substance was dissolved in ethyl acetate (150 mL), 5% by mass palladium on carbon was added, and the mixture was stirred under a hydrogen atmosphere for 17 hours. The reaction solution was filtered, and the filtrate was vacuum-dried to obtain an oily product (5.42 g).
This oil and gemcitabine (3.0 g, manufactured by SCINO PHARM) were dissolved in DMF (57 mL), HOBt (2.1 g) and WSCD hydrochloride (3.3 g) were added, and the temperature was raised from 0 ° C. to room temperature. And stirred overnight. Purified water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (170 mL). The organic layer was washed twice with saturated brine and dried over sodium sulfate. After removing ethyl acetate by concentration under reduced pressure, purification by silica gel column chromatography was performed, followed by vacuum drying to obtain an oily substance (4.5 g).
The oil was dissolved in ethyl acetate (27 mL), 4N hydrochloric acid ethyl acetate solution (20 mL) was added, and the mixture was stirred at room temperature for 2 hr. A mixed solvent of ethyl acetate (48 mL) and n-hexane (12 mL) was added to the reaction mixture, and the precipitate was collected by filtration and dried in vacuo to give compound 5 (2.06 g).
¹ H-NMR (400 MHz, MeOD-d4, ppm): 0.89-1.01 (m, 6H), 1.27-1.36 (m, 1H), 1.44-1.55 (m, 1H), 1.91-2.00 (m, 1H), 3.08 (dd, 1H), 3.25 (dd, 1H), 3.73 (s, 3H), 3.84 (dd, 1H) ), 3.97-4.03 (m, 2H), 4.28-4.36 (m, 1H), 4.42 (dd, 1H), 4.47 (d, 1H), 6.29 ( dd, 1H), 7.29 (d, 1H), 8.45 (d, 1H)

[Synthesis Example 6] Synthesis of 3-aminopropyl-poly-α-aspartic acid (polymerization number: about 11) (Compound 6)
N-Boc-1,3-diaminopropane (80 mg, manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in DMSO (40 mL), and then γ-benzyl-L-aspartic acid-N-carboxylic acid anhydride (BLA-NCA, 3.0 g) was dissolved. And manufactured by ISOCHEM) and stirred at 32.5 ° C. overnight. The reaction solution was dropped into a mixed solvent of ethanol (100 mL) and diisopropyl ether (900 mL) over 15 minutes and stirred at room temperature for 3 hours. The precipitate was collected by filtration and dried in vacuo to obtain a solid.
This solid (2.01 g) was dissolved in DMI (40 mL), acetic anhydride (4 mL) was added, and the mixture was stirred at 20 ° C. overnight. The reaction solution was dropped into a mixed solvent of ethyl acetate (100 mL) and diisopropyl ether (900 mL) over 15 minutes and stirred at room temperature for 2 hours. The precipitate was collected by filtration and dried in vacuo to obtain a solid.
Dichloromethane (0.3 mL) and trifluoroacetic acid (1.2 mL) were added to the solid (1.43 g), and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (500 mL) was added to the reaction solution, and the precipitated solid was collected by filtration and dried in vacuo to give compound 6 (1.38 g).
The polymerization number of aspartic acid was calculated by quantifying the released benzyl alcohol by high performance liquid chromatography (HPLC) after alkaline hydrolysis. As a result, the benzyl alcohol content was 48% and the polymerization number was calculated to be 11.6.

[Synthesis Example 7] Hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl and 3-aminopropyl-poly-α-aspartic acid (with a polymerization number of about 11) and one terminal with an average molecular weight of 5 kilodaltons Synthesis of amide conjugate of polyethylene glycol with 3-aminopropyl group at one end of methoxy group (Compound 7)
Compound 1 obtained in Synthesis Example 1 (molecular weight 6.8 kilodalton, 0.69 g) and polyethylene glycol compound (5.1 g, SUNBRIGHT MEPA-50H, one terminal methoxy group and one terminal 3-aminopropyl group, Oil Co., Ltd., average molecular weight of 5 kilodaltons) was dissolved in DMF (32 mL) at 35 ° C., cooled to 25 ° C., and 1-hydroxybenzotriazole (HOBt) (0.48 g) and diisopropylcarbodiimide (DIPCI) ( 1.0 mL) was added and stirred for 3.5 hours. Thereafter, Compound 6 (1.50 g) obtained in Synthesis Example 6, DIPCI (0.50 mL) and diisopropylethylamine (0.25 mL) were added and stirred overnight. Then, DIPCI (0.50 mL) and 2-methoxyethyleneamine (0.28 mL) were added and stirred for 3 hours. Thereafter, ethyl acetate (43 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (430 mL) over 30 minutes, followed by stirring at room temperature for 25 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The obtained precipitate was dissolved in DMF (16 mL) and purified water (0.16 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 8 mL) was added. After stirring for 1.5 hours, the mixture was filtered and concentrated under reduced pressure to obtain a compound 7 precursor (7.50 g).

In the obtained compound 7 precursor, the binding amount of the polyethylene glycol compound was 10 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Therefore, the total polyethylene glycol segment molecular weight was 50 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier = 0.31 equivalent, and the consumption rate of the polyethylene glycol segment compound = 1.

The total content of aspartic acid units in the compound 7 precursor was calculated by alkaline hydrolysis of the compound 7 precursor, and then quantifying the released benzyl alcohol by high performance liquid chromatography (HPLC). As a result, the benzyl alcohol content was 9.9%, and the total number of aspartic acid units was calculated to be 64.9.
In addition, since the number of aspartic acids polymerized per molecule of compound 6 is 11.6, the number of compounds 6 bonded per molecule of the multi-branched polymer carrier is calculated as 5.6 molecules from the following formula. It was.
Number of compounds 6 bonded per molecule of the multi-branched polymer carrier = 64.9 / 11.6 = 5.6

The obtained compound 7 precursor was dissolved in DMF (65 mL), 10% palladium carbon (0.75 g, manufactured by Nacalai Tesque) was added, and the mixture was stirred overnight under a hydrogen atmosphere. After palladium carbon was filtered off, ethyl acetate (50 mL) was added for dilution, and the reaction mixture was added dropwise to diisopropyl ether (500 mL) over 30 minutes and stirred at room temperature for 1 hour. The precipitate was collected by filtration and dried in vacuo to give compound 7 (5.73 g).
The molecular weight of Compound 7 was calculated to be 64 kilodaltons from the following formula.
[Molecular Weight of Compound 7] = [Molecular Weight of Hyperbranched Polymer Carrier] + [Total Polyethylene Glycol Segment Molecular Weight] + [(Polyaspartic Acid Unit Molecular Weight) × (Total Number of Aspartic Acid Unit Bonds)]
In addition, 115.09 was used as the molecular weight of the polyaspartic acid unit molecule.

[Synthesis Example 8] Synthesis of a block copolymer of methoxypolyethylene glycol having an average molecular weight of 2 kilodaltons and poly-α-aspartic acid (polymerization number: about 5) (Compound 8)
A polyethylene glycol compound having one end methoxy group and one end 3-aminopropyl group (4.52 g, SUNBRIGHT MEPA-20H, manufactured by NOF Corporation, average molecular weight 2 kilodaltons) was dissolved in DMSO (40 mL), and γ-benzyl- L-Aspartic acid-N-carboxylic anhydride (BLA-NCA, 3.10 g, manufactured by ISOCHEM) was added, and the mixture was stirred at 30 ° C. overnight. The reaction solution was diluted with purified water (5 mL), dialyzed with external acetonitrile, the acetonitrile in the internal solution was removed under reduced pressure, and lyophilized to obtain Compound 8 (6.06 g).
The number of polymerization of aspartic acid of the obtained compound 8 was calculated by subjecting it to alkaline hydrolysis and then quantifying the released benzyl alcohol by high performance liquid chromatography (HPLC). As a result, the benzyl alcohol content was 16.7% and the polymerization number was calculated to be 4.5. Therefore, the number of aspartic acid polymerizations per molecule of Compound 8 is 4.5, and the molecular weight of the polyethylene glycol segment is 2 kilodaltons.

[Synthesis Example 9] of hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl and monomethoxypolyethylene glycol-block-poly-α-aspartic acid (polymerization number of about 5) having an average molecular weight of 2 kilodaltons Synthesis of amide conjugate (Compound 9)
Compound 1 obtained in Synthesis Example 1 (molecular weight 6.8 kilodalton, 0.86 g) and Compound 8 obtained in Synthesis Example 8 were dissolved in DMF (80 mL) at 35 ° C., and the temperature was lowered to 25 ° C. , 1-hydroxybenzotriazole (HOBt) (0.572 g) and diisopropylcarbodiimide (DIPCI) (1.0 mL) were added and stirred overnight. Then, DIPCI (0.42 mL) and 2-methoxyethyleneamine (0.24 mL) were added and stirred for 3 hours. Thereafter, ethyl acetate (160 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (1.6 L) over 30 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The obtained precipitate was dissolved in DMF (40 mL) and purified water (0.4 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 20 mL) was added. After stirring for 1 hour, ethyl acetate (80 mL) was added for dilution, and the reaction mixture was added dropwise to diisopropyl ether (800 mL) over 30 minutes and stirred at room temperature for 2 hours. The precipitate was collected by filtration and dried in vacuo to give Compound 9 precursor as a solid.
The total number of aspartic acid units bound in this compound 9 precursor was calculated by quantifying the released benzyl alcohol by high performance liquid chromatography (HPLC) after alkaline hydrolysis. As a result, the benzyl alcohol content was 15%, and the total number of aspartic acid units bound was calculated to be 72.
Moreover, since the number of polymerizations of aspartic acid per molecule of Compound 8 is 4.5, the number of Compound 8 bonded per molecule of Compound 9 precursor was calculated as 16 molecules from the following formula.
Number of compounds 8 bonded per molecule of multi-branched polymer carrier = 72 / 4.5 = 16

Compound 9 precursor was dissolved in DMF (40 mL), 10% palladium carbon (0.34 g, manufactured by Nacalai Tesque) was added, and the mixture was stirred overnight under a hydrogen atmosphere. After palladium carbon was filtered off, ethyl acetate (80 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (800 mL) over 30 minutes, followed by stirring at room temperature for 1 hour. The precipitate was collected by filtration and dried in vacuo to give compound 9 (2.99 g).
The molecular weight of Compound 9 was calculated to be 47 kilodaltons from the following formula.
[Molecular weight of compound 9] = [Molecular weight of multi-branched polymer carrier] + (Number of compounds 8 bonded per molecule of multi-branched polymer carrier) × [(Molecular weight of polyethylene glycol segment per molecule of compound 8) + ( Polyaspartic acid unit molecular weight) x (number of aspartic acid polymerizations per molecule of compound 8)]
In addition, 115.09 was used as the molecular weight of the polyaspartic acid unit molecule.

Synthesis Example 10 Synthesis of N- (t-butoxycarbonyl) -poly-β-L-aspartic acid benzyl ester (polymerization number 5) (Compound 10)
N- (t-butoxycarbonyl) -L-aspartic acid-1-benzyl ester (Watanabe Chemical Co., Ltd., 15.4 g) and 9-fluorenylmethanol (Tokyo Kasei Co., Ltd., 18.4 g) were mixed with DMF (95 2 mg), DMAP (1.16 g) and WSCD hydrochloride (13.6 g) were added, and the mixture was stirred at room temperature for 3 hours. Thereafter, purified water was added to the reaction solution, and the mixture was extracted with ethyl acetate (300 mL). The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate, saturated aqueous ammonium chloride and saturated brine, and dried over sodium sulfate. After removing ethyl acetate by concentration under reduced pressure, purification by silica gel column chromatography was performed, followed by vacuum drying to obtain a solid (8.4 g).
This solid (8.4 g) was dissolved in ethyl acetate (84 mL), 4N hydrochloric acid-ethyl acetate solution (84 mL) was added, and the mixture was stirred at room temperature for 2 hr. The solid matter precipitated in the reaction solution was filtered and vacuum-dried to obtain a solid matter (6.9 g).
This solid (6.7 g) and N- (t-butoxycarbonyl) -L-aspartate-1-benzyl (Watanabe Chemical Co., Ltd., 5.4 g) were dissolved in DMF, and then diisopropylethylamine (2.9 mL). , HOBt (2.8 g) and WSCD hydrochloride (4.8 g) were added, and the mixture was stirred at room temperature for 6 hours. Purified water was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate, saturated aqueous ammonium chloride and saturated brine, and dried over sodium sulfate. Dichloromethane was removed by vacuum concentration, followed by vacuum drying to obtain a solid (11.0 g).
Removal of the t-butoxycarbonyl group with the 4N ethyl acetate solution described above and N- (t-butoxycarbonyl) -L-- using WSCD hydrochloride as the condensing agent and HOBt as the condensing aid After repeating the condensation reaction with 1-benzyl aspartate three times, the obtained solid was dissolved in DMF (100 mL), piperidine (0.27 μL) was added, and the mixture was stirred at room temperature for 1 hour. Thereafter, the reaction solution was added dropwise to diisopropyl ether (1 L), and the deposited precipitate was collected by filtration and then vacuum-dried to obtain Compound 10 (2.7 g).
MS: m / z 1145 (M + H) ⁺ calculated as C60H66N5O18 (M + H) ⁺ 1145
Calculated as m / z 1143 (M−H) ⁻ C60H64N5O18 (M−H) ⁻

[Synthesis Example 11] Synthesis of amide conjugate of monomethoxypolyethylene glycol-block-poly-β-asparagine (polymerization number 5) having an average molecular weight of 5 kilodaltons (Compound 11)
Compound 10 (2.5 g) and one end methoxy group and one end 3-aminopropyl group polyethylene glycol compound (12.1 g, SUNBRIGHT MEPA-50H, manufactured by NOF Corporation, average molecular weight 5 kilodaltons) in DMF (50 mL) Then, HOBt (0.40 mg) and diisopropylcarbodiimide (DIPCI) (0.42 mL) were added, and the mixture was stirred at 30 ° C. for 9 hours. Thereafter, ethyl acetate (100 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (1 L) over 15 minutes and stirred overnight at room temperature. The precipitate was collected by filtration and washed with diisopropyl ether to obtain a solid (14.3 g).
The obtained solid (14.2 g) was dissolved in dichloromethane (9 mL), trifluoroacetic acid (12 mL) was added, and the mixture was stirred at room temperature for 2 hr. Thereafter, dichloromethane was removed under reduced pressure, and the residue was dropped into diisopropyl ether (1 L) over 30 minutes. After stirring at room temperature for 30 minutes, the precipitate was collected by filtration and washed with diisopropyl ether to obtain a solid. The obtained solid was dissolved in methanol (20 mL) and dialyzed with external liquid methanol. Thereafter, the internal solution was diluted with 300 mL of purified water, methanol was removed by concentration under reduced pressure, and freeze-dried to obtain Compound 11 (12.6 g).
In addition, the polyethylene glycol segment molecular weight per molecule of the compound 11 is 5 kilodaltons, and the number of aspartic acid polymerizations is 5.

[Synthesis Example 12] Hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl and amide of monomethoxypolyethylene glycol-block-poly-β-aspartic acid (polymerization number 5) having an average molecular weight of 5 kilodaltons Synthesis of conjugate (compound 12)
Compound 11 (10.2 g) obtained in Synthesis Example 11 and Compound 1 (molecular weight 6.8 kilodalton, 1.4 g) obtained in Synthesis Example 1 were dissolved in DMF (80 mL) at 35 ° C., and 30 The temperature was lowered to 1 ° C., 1-hydroxybenzotriazole (HOBt) (1.1 g), diisopropylcarbodiimide (DIPCI) (2.15 mL) and diisopropylethylamine (0.32 mL) were added, and the mixture was stirred overnight. Then, DIPCI (1.08 mL) and 2-methoxyethyleneamine (0.57 mL) were added and stirred for 3 hours. Thereafter, ethyl acetate (200 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (1.5 L) over 30 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The resulting precipitate was dissolved in DMF (50 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 25 mL) was added. After stirring for 1 hour, ethyl acetate (100 mL) was added for dilution, and the reaction mixture was added dropwise to diisopropyl ether (1 L) over 30 minutes and stirred at room temperature for 2 hours. The precipitate was collected by filtration and dried in vacuo to obtain a solid (9.02 g) as a compound 12 precursor.

The total number of aspartic acid units bound in this compound 12 precursor was calculated by quantifying the released benzyl alcohol by high performance liquid chromatography (HPLC) after alkaline hydrolysis. As a result, the benzyl alcohol content was 7.7%, and the total number of aspartic acid units was calculated to be 40.
Further, since the number of polymerizations of aspartic acid per molecule of compound 11 is 5, the number of compounds 11 bonded per molecule of the multi-branched polymer carrier was calculated as 8 molecules from the following formula.
Number of compounds 8 bonded per molecule of multi-branched polymer carrier = 40/5 = 8

This compound 12 precursor (9.0 g) was dissolved in DMF (90 mL), 10% palladium carbon (0.90 g, manufactured by Nacalai Tesque) was added, and the mixture was stirred overnight under a hydrogen atmosphere. After palladium carbon was filtered off, ethyl acetate (90 mL) was added for dilution, and the reaction mixture was added dropwise to diisopropyl ether (1.5 L) over 1 hour and stirred overnight at room temperature. The precipitate was collected by filtration and dried in vacuo to give compound 12 (5.01 g).
The molecular weight of Compound 12 was calculated to be 52 kilodaltons from the following formula.
[Molecular weight of compound 12] = [Molecular weight of multi-branched polymer carrier] + (Number of compounds 11 bonded per molecule of multi-branched polymer carrier) × [(Molecular weight of polyethylene glycol segment per molecule of compound 11) + ( Polyaspartic acid unit molecular weight) x (number of aspartic acid polymerizations per molecule of compound 11)]
In addition, 115.09 was used as the molecular weight of the polyaspartic acid unit molecule.

[Example 1] Multi-branched polymer carrier (terminal carboxylic acid number: 32), one-terminal methoxy group having one molecular weight of 5 kilodalton, one-terminal 3-aminopropyl group of polyethylene glycol and aspartic acid-1-alanine methyl ester-4- Synthesis of Amide Conjugate of Gemcitabine Amide Compound 1 obtained in Synthesis Example 1 (molecular weight: 6.8 kilodalton, 0.60 g) and a polyethylene glycol compound having a single terminal methoxy group and a single terminal 3-aminopropyl group (3. 5 g, SUNBRIGHT MEPA-50H (manufactured by NOF Corporation, average molecular weight 5 kilodaltons) was dissolved in DMF (28 mL) at 35 ° C., cooled to 20 ° C., and 1-hydroxybenzotriazole (HOBt) (0.47 g) And diisopropylcarbodiimide (DIPCI) (0.42 mL) was added and stirred for 4.5 hours. It was. Thereafter, Compound 3 (0.84 g) obtained in Synthesis Example 3, DIPCI (0.42 mL) and diisopropylethylamine (0.32 mL) were added and stirred overnight. Thereafter, ethyl acetate (54 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (540 mL) over 10 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The obtained precipitate was dissolved in purified water (106 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 53 mL) was added. After stirring for 30 minutes, the mixture was filtered and freeze-dried to obtain the title compound (4.69 g) according to Example 1.

After alkali hydrolysis of the compound of Example 1, the gemcitabine released was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in Example 1. As a result, the gemcitabine content in Example 1 was 7.4% by mass. Therefore, the gemcitabine binding rate with respect to the number of terminal carboxylic acids of the multi-branched polymer carrier was calculated to be 46.6%.
As a result, the total molecular weight of gemcitabine in Example 1 was calculated to be 3.9 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 1 was 8 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Accordingly, the total polyethylene glycol segment molecular weight was 40 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier = 0.25 equivalent, and the consumption rate of the polyethylene glycol segment compound = 1.

From the molecular weight of the compound 1, the bound polyethylene glycol segment molecular weight, the combined gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 1 was calculated to be 53 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 75% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 1 was measured by laser light scattering intensity. As a result, the light scattering intensity was 5,986 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 0.46 times. The molecular weight measured by SEC-MALS was 81,750, and the number of associated molecules was 1.5.

[Example 2] Multi-branched polymer carrier (number of terminal carboxylic acids: 32), one end methoxy group having an average molecular weight of 5 kilodaltons, one end 3-aminopropyl group of polyethylene glycol and aspartic acid-1-leucine methyl ester-4- Synthesis of Amide Conjugate of Gemcitabine Amide Compound 1 obtained in Synthesis Example 1 (molecular weight: 6.8 kilodalton, 0.59 g) and a polyethylene glycol compound having a single terminal methoxy group and a single terminal 3-aminopropyl group (3. 5 g, SUNBRIGHT MEPA-50H (manufactured by NOF Corporation, average molecular weight 5 kilodaltons) was dissolved in DMF (28 mL) at 35 ° C., cooled to 20 ° C., and 1-hydroxybenzotriazole (HOBt) (0.47 g) And diisopropylcarbodiimide (DIPCI) (0.41 mL) was added and stirred for 5 hours. . Thereafter, Compound 4 (0.90 g) obtained in Synthesis Example 4, DIPCI (0.41 mL) and diisopropylethylamine (0.32 mL) were added, and the mixture was stirred overnight. Thereafter, ethyl acetate (56 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (560 mL) over 10 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The resulting precipitate was dissolved in purified water (53 mL), and an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 53 mL) was added. After stirring for 30 minutes, filtration and lyophilization were performed to obtain the title compound (4.39 g) according to Example 2.

After alkali-hydrolyzing the compound of Example 2, the gemcitabine content in this compound was calculated | required by quantifying the liberated gemcitabine by a high performance liquid chromatography (HPLC). As a result, the gemcitabine content in Example 2 was 6.5% by mass. Therefore, the binding rate of the multi-branched polymer carrier with respect to the number of terminal carboxylic acids of 32 was calculated to be 40.9%.
As a result, the total molecular weight of gemcitabine in Example 2 was 3.4 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 2 was 7.9 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Accordingly, the total polyethylene glycol segment molecular weight was 40 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier was 0.25 equivalent, and the consumption rate of the polyethylene glycol segment compound was 0.993.

From the molecular weight of Compound 1 above, the bound polyethylene glycol segment molecular weight, the bound gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 2 was calculated to be 53 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 75% by mass.
Moreover, when the association degree of the nucleic acid antimetabolite binding multibranched compound of Example 2 was measured by laser light scattering intensity, the light scattering intensity was 7,030 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 0.54 times. The molecular weight measured by SEC-MALS was 85,410, and the number of associated molecules was 1.6.

[Example 3] Multi-branched polymer carrier (number of terminal carboxylic acids 32), one end methoxy group having an average molecular weight of 5 kilodaltons, one end 3-aminopropyl group of polyethylene glycol and aspartic acid-1-isoleucine methyl ester-4- Synthesis of Amide Conjugate of Gemcitabine Amide Compound 1 obtained in Synthesis Example 1 (molecular weight 6.8 kilodalton, 0.77 g) and a polyethylene glycol compound (4. 5 g, SUNBRIGHT MEPA-50H (manufactured by NOF Corporation, average molecular weight 5 kilodaltons) was dissolved in DMF (36 mL) at 35 ° C., cooled to 20 ° C., and 1-hydroxybenzotriazole (HOBt) (0.61 g) And diisopropylcarbodiimide (DIPCI) (0.54 mL) were added and stirred for 7 hours. It was. Thereafter, Compound 5 (1.17 g) obtained in Synthesis Example 5, DIPCI (0.54 mL) and diisopropylethylamine (0.69 mL) were added, and the mixture was stirred overnight. Thereafter, ethyl acetate (72 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (720 mL) over 15 minutes, followed by stirring at room temperature for 2 hours. The precipitate was collected by filtration and washed with diisopropyl ether (70 mL). The obtained precipitate was dissolved in purified water (64 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 64 mL) was added. After stirring for 30 minutes, the mixture was filtered and freeze-dried to obtain the title compound (5.73 g) according to Example 3.

After alkaline hydrolysis of the compound of Example 3, the released gemcitabine was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in this compound. As a result, the gemcitabine content in Example 3 was 7.7% by mass. Therefore, the binding rate of the multi-branched polymer carrier with respect to the number of terminal carboxylic acids of 32 was calculated to be 49.8%.
As a result, the total molecular weight of gemcitabine in Example 3 was 4.2 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 3 was 8 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Accordingly, the total polyethylene glycol segment molecular weight was 40 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier was 0.25 equivalent, and the consumption rate of the polyethylene glycol segment compound was 0.999.

From the molecular weight of Compound 1 above, the bound polyethylene glycol segment molecular weight, the combined gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 3 was calculated to be 54 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 74% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 3 was measured by the laser light scattering intensity. As a result, the light scattering intensity was 8,560 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 0.66 times.
Further, the molecular weight measured by SEC-MALS was 8,560, and the number of associated molecules was 2.6.

[Example 4] Multi-branched polymer carrier (number of terminal carboxylic acids: 64), one end methoxy group having an average molecular weight of 2 kilodaltons, one end 3-aminopropyl group of polyethylene glycol and aspartic acid-1-leucine methyl ester-4- Synthesis of Amide Conjugate of Gemcitabine Amide Compound 2 obtained in Synthesis Example 2 (molecular weight 13.7 kilodalton, 0.53 g) and a polyethylene glycol compound (1. 7 g, SUNBRIGHT MEPA-20H (manufactured by NOF Corporation, average molecular weight of 2 kilodaltons) was dissolved in DMF (20 mL) at 35 ° C., cooled to 20 ° C., and 1-hydroxybenzotriazole (HOBt) (0.31 g) And diisopropylcarbodiimide (DIPCI) (0.36 mL) were added and stirred for 4 hours. It was. Thereafter, Compound 4 (0.80 g) obtained in Synthesis Example 4, DIPCI (0.36 mL) and diisopropylethylamine (0.28 mL) were added and stirred overnight. Thereafter, ethyl acetate (20 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (500 mL) over 30 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The resulting precipitate was dissolved in purified water (60 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 40 mL) was added. After stirring for 20 minutes, the mixture was filtered and lyophilized to obtain the title compound (2.42 g) according to Example 4.

After alkaline hydrolysis of the compound of Example 4, the released gemcitabine was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in this compound. As a result, the gemcitabine content in Example 4 was 10.2% by mass. Therefore, the binding rate of the multi-branched polymer carrier with respect to the number of terminal carboxylic acids of 64 was calculated to be 37.5%.
As a result, the total molecular weight of gemcitabine in Example 4 was 6.3 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 4 was 18.3 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Therefore, the total polyethylene glycol segment molecular weight was 36.6 kilodaltons.
In this reaction, the charged equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier = 0.31 equivalent, and the consumption rate of the polyethylene glycol segment compound = 0.920.

From the molecular weight of the compound 2, the bound polyethylene glycol segment molecular weight, the combined gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 4 was calculated to be 62 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 59% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 4 was measured by the laser light scattering intensity, whereby the light scattering intensity was 5,746 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 0.44 times. Further, the molecular weight measured by SEC-MALS was 72,070, and the number of associated molecules was 1.2.

[Example 5] Multi-branched polymer carrier (number of terminal carboxylic acids 64), one end methoxy group having an average molecular weight of 5 kilodaltons, one end 3-aminopropyl group of polyethylene glycol and aspartic acid-1-alanine methyl ester-4- Synthesis of amide conjugate of gemcitabine amide Compound 2 obtained in Synthesis Example 2 (molecular weight: 13.7 kilodalton, 0.55 g) and a polyethylene glycol compound having one end methoxy group and one end 3-aminopropyl group ( 1.6 g, SUNBRIGHT MEPA-50H (manufactured by NOF Corporation, average molecular weight 5 kilodalton) was dissolved in DMF (20 mL) at 35 ° C., and the temperature was lowered to 20 ° C. to give 1-hydroxybenzotriazole (HOBt) (0. 32 g) and diisopropylcarbodiimide (DIPCI) (0.40 mL) It stirred. Thereafter, Compound 3 (1.15 g) obtained in Synthesis Example 3, DIPCI (0.40 mL) and diisopropylethylamine (0.45 mL) were added, and the mixture was stirred overnight. Thereafter, ethyl acetate (20 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (500 mL) over 10 minutes, followed by stirring at room temperature for 1 hour. The precipitate was collected by filtration and washed with diisopropyl ether (50 mL). The resulting precipitate was dissolved in purified water (120 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 50 mL) was added. After stirring for 20 minutes, the mixture was filtered and freeze-dried to obtain the title compound (2.86 g) according to Example 5.

After alkaline hydrolysis of the compound of Example 5, the released gemcitabine was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in this compound. As a result, the gemcitabine content in Example 5 was 15.7% by mass. Therefore, the binding rate of the multi-branched polymer carrier with respect to the number of terminal carboxylic acids of 64 was calculated to be 59.4%.
As a result, the total molecular weight of gemcitabine in Example 5 was 10.0 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 5 was 6.6 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Therefore, the total polyethylene glycol segment molecular weight was 33 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier was 0.125 equivalent, and the consumption rate of the polyethylene glycol segment compound was 0.825.

From the molecular weight of Compound 2 above, the bound polyethylene glycol segment molecular weight, the combined gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 5 was calculated to be 64 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 52% by mass.
Moreover, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 5 was measured by laser light scattering intensity. As a result, the light scattering intensity was 31,865 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 2.46 times. Further, the molecular weight measured by SEC-MALS was 220,500, and the number of associated molecules was 3.5.

[Example 6] Hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl and 3-aminopropyl-poly-α-aspartic acid (polymerization number of about 11) and one terminal with an average molecular weight of 5 kilodaltons Introduction of gemcitabine into the amide conjugate of polyethylene glycol having a 3-aminopropyl group at one end of methoxy group Compound 7 (5.7 g) obtained in Synthesis Example 7 and gemcitabine (1.2 g, manufactured by SCINO PHARM) were combined with DMF. (29 mL) was dissolved at 35 ° C., 1-hydroxybenzotriazole (HOBt) (0.96 g) and diisopropylcarbodiimide (DIPCI) (2.0 mL) were added at 25 ° C., and the mixture was stirred overnight. The reaction solution was diluted with ethyl acetate (45 mL), added dropwise to diisopropyl ether (500 mL) over 30 minutes, and stirred at room temperature for 1 hour. The precipitate was collected by filtration and washed with diisopropyl ether (10 mL). The obtained precipitate was dissolved in purified water (60 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 28 mL) was added. After stirring for 30 minutes, the mixture was filtered and freeze-dried to obtain the title compound (6.26 g) according to Example 6.

After alkaline hydrolysis of the compound of Example 6, the released gemcitabine was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in this compound. As a result, the gemcitabine content in Example 6 was 9.6% by mass. Therefore, the binding rate with respect to the total number of aspartic acid units of 64.9 was calculated to be 40% (number of bonds: 26).
As a result, the total molecular weight of gemcitabine in Example 6 was 6.8 kilodaltons.

The molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 6 was calculated to be 71 kilodaltons from the molecular weight of compound 7 and the total molecular weight of bound gemcitabine.
The polyethylene glycol segment content was calculated to be 70% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 6 was measured by laser light scattering intensity. As a result, the light scattering intensity was 17,740 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 1.37 times. The molecular weight measured by SEC-MALS was 179,600, and the number of associated molecules was 2.5.

[Example 7] Hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl and monomethoxypolyethylene glycol-block-poly-α-aspartic acid (polymerization number about 5) having an average molecular weight of 2 kilodaltons Introduction of gemcitabine into amide conjugate Compound 9 (3.0 g) obtained in Synthesis Example 9 and gemcitabine (0.97 g, manufactured by SCINO PHARM) were dissolved in DMF (31 mL) at 35 ° C., and 20 ° C. 1-hydroxybenzotriazole (HOBt) (0.85 g) and diisopropylcarbodiimide (DIPCI) (1.7 mL) were added and stirred overnight. The reaction solution was dropped into a mixed solvent of ethyl acetate (35 mL) and diisopropyl ether (350 mL) over 30 minutes, and stirred at room temperature for 1 hour. The precipitate was collected by filtration and washed with diisopropyl ether (10 mL). The resulting precipitate was dissolved in purified water (60 mL), and an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 30 mL) was added. After stirring for 30 minutes, filtration and lyophilization were performed to obtain the title compound (5.93 g) according to Example 7.

After alkaline hydrolysis of the compound of Example 7, the released gemcitabine was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in this compound. As a result, the gemcitabine content in Example 7 was 12.6% by mass. Therefore, the binding rate with respect to the total number 72 of aspartic acid units was calculated to be 36% (26 bindings).
As a result, the total molecular weight of gemcitabine in Example 7 was 6.8 kilodaltons.

The molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 7 was calculated to be 57 kilodaltons from the molecular weight of Compound 9 and the combined gemcitabine total molecular weight.
The polyethylene glycol segment content was calculated to be 56% by mass.
Moreover, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 7 was measured by laser light scattering intensity. As a result, the light scattering intensity was 8,917 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 0.69 times. Further, the molecular weight measured by SEC-MALS was 127,800, and the number of associated molecules was 2.2.

[Example 8] Hyperbranched 2,2-bis (methylol) propionic acid polyester-32-carboxyl and amide of monomethoxypolyethylene glycol-block-poly-β-aspartic acid (polymerization number 5) having an average molecular weight of 5 kilodaltons Introduction of gemcitabine into conjugate Compound 12 (4.9 g) obtained in Synthesis Example 12 and gemcitabine (0.79 g, manufactured by SCINO PHARM) were dissolved in DMF (25 mL) at 35 ° C. 1-hydroxybenzotriazole (HOBt) (0.63 g) and diisopropylcarbodiimide (DIPCI) (1.3 mL) were added, and the mixture was stirred overnight. The reaction solution was dropped into a mixed solvent of ethyl acetate (50 mL) and diisopropyl ether (1 L) over 1 hour, and stirred at room temperature for 1 hour. The precipitate was collected by filtration and washed with diisopropyl ether (20 mL). The resulting precipitate was dissolved in purified water (50 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 25 mL) was added. After stirring for 30 minutes, the mixture was filtered and lyophilized to obtain the title compound (4.3 g) according to Example 8.

After alkaline hydrolysis of the compound of Example 8, the released gemcitabine was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in this compound. As a result, the gemcitabine content in Example 8 was 8.7% by mass. Therefore, the binding rate with respect to the total number of bonds of aspartic acid units was calculated to be 47% (number of bonds 19).
As a result, the total molecular weight of gemcitabine in Example 8 was 4.9 kilodaltons.

The molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 8 was calculated to be 57 kilodaltons from the molecular weight of Compound 12 and the combined gemcitabine total molecular weight.
The polyethylene glycol segment content was calculated to be 71% by mass.
Moreover, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 8 was measured by the laser light scattering intensity. The light scattering intensity was 17,887 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 1.38 times. The molecular weight measured by SEC-MALS was 90,610 and the number of associated molecules was 1.6.

[Example 9] Multi-branched polymer carrier (number of terminal carboxylic acids 64) and one-terminal methoxy group having a mean molecular weight of 2 kilodaltons One-terminal 3-aminopropyl group of polyethylene glycol and aspartic acid-1-alanine methyl ester-4- Synthesis of amide conjugate of gemcitabine amide (polyethylene glycol segment mass content of 24%) Compound 2 obtained in Synthesis Example 2 (molecular weight 13.7 kilodalton, 0.60 g), one-end methoxy group and one-end A polyethylene glycol compound having a 3-aminopropyl group (0.37 g, SUNBRIGHT MEPA-20H, manufactured by NOF Corporation, average molecular weight of 2 kilodaltons) was dissolved in DMF (19 mL) at 35 ° C., and the temperature was lowered to 20 ° C. -Hydroxybenzotriazole (HOBt) (0.47 g) and diisopropylcarbo In addition imide (DIPCI) (0.41mL), and stirred for 7.0 hours. Thereafter, Compound 3 (0.78 g) obtained in Synthesis Example 3, DIPCI (0.41 mL) and diisopropylethylamine (0.39 mL) were added, and the mixture was stirred overnight. Thereafter, ethyl acetate (43 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (419 mL) over 10 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (250 mL). The obtained precipitate was dissolved in purified water (110 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 50 mL) was added. After stirring for 30 minutes, the mixture was filtered and lyophilized to obtain the title compound (1.69 g) according to Example 9.

After alkaline hydrolysis of the compound of Example 9, the gemcitabine released in Example 9 was determined by quantifying the liberated gemcitabine by high performance liquid chromatography (HPLC). As a result, the gemcitabine content in Example 9 was 20.3% by mass. Therefore, the gemcitabine binding ratio to the terminal carboxylic acid number 64 of the multi-branched polymer carrier was calculated to be 39.9%.
As a result, the total molecular weight of gemcitabine in Example 9 was calculated to be 6.7 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 9 was 4 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Accordingly, the total polyethylene glycol segment molecular weight was 8 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier was 0.063 equivalent, and the consumption rate of the polyethylene glycol segment compound was 1.

From the molecular weight of the compound 2, the combined polyethylene glycol segment molecular weight, the combined gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 9 was calculated to be 33 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 24% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 9 was measured by laser light scattering intensity, whereby the light scattering intensity was 1,306,438 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 101.01 times. The molecular weight measured by SEC-MALS was 9,917,000, and the number of associated molecules was 300.3.

[Example 10] Multi-branched polymer carrier (number of terminal carboxylic acids: 64), one-terminal methoxy group having an average molecular weight of 2 kilodaltons, one-terminal 3-aminopropyl group of polyethylene glycol and aspartic acid-1-alanine methyl ester-4- Synthesis of amide conjugate of gemcitabine amide (mass content of polyethylene glycol segment 44%) Compound 2 obtained in Synthesis Example 2 (molecular weight 13.7 kilodalton, 1.93 g), one-end methoxy group and one-end A polyethylene glycol compound having a 3-aminopropyl group (2.11 g, SUNBRIGHT MEPA-20H, manufactured by NOF Corporation, average molecular weight of 2 kilodaltons) is dissolved in DMF (81 mL) at 35 ° C., and the temperature is lowered to 20 ° C. -Hydroxybenzotriazole (HOBt) (1.51 g) and diisopropyl cal Added diimide (DIPCI) (1.34mL), and stirred for 4.5 hours. Thereafter, Compound 3 (0.91 g) obtained in Synthesis Example 3, DIPCI (1.34 mL) and diisopropylethylamine (0.78 mL) were added and stirred overnight. Thereafter, ethyl acetate (187 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (1827 mL) over 10 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (1000 mL). The resulting precipitate was dissolved in purified water (600 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 203 mL) was added. After stirring for 30 minutes, filtration and lyophilization were performed to obtain the title compound (4.35 g) according to Example 10.

After alkali-hydrolyzing the compound of Example 10, the gemcitabine content in Example 10 was calculated | required by quantifying the liberated gemcitabine by a high performance liquid chromatography (HPLC). As a result, the gemcitabine content in Example 10 was 8.3% by mass. Therefore, the gemcitabine binding ratio to the terminal carboxylic acid number 64 of the multi-branched polymer carrier was calculated to be 15.7%.
As a result, the total molecular weight of gemcitabine in Example 10 was calculated to be 2.7 kilodaltons.

The binding amount of the polyethylene glycol compound of Example 10 was 7 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Accordingly, the total polyethylene glycol segment molecular weight was 14 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier was 0.109 equivalent, and the consumption rate of the polyethylene glycol segment compound was 1.

From the molecular weight of Compound 2 above, the combined polyethylene glycol segment molecular weight, the combined gemcitabine total molecular weight, and the total molecular weight of the aspartic acid unit residue, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Example 10 was calculated to be 32 kilodaltons. It was.
The polyethylene glycol segment content was calculated to be 44% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Example 10 was measured by laser light scattering intensity. As a result, the light scattering intensity was 17,040 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 1.32. Further, the molecular weight measured by SEC-MALS was 209,000, and the number of associated molecules was 6.5.

[Comparative Example 1] Synthesis of a conjugate of a polybranched polymer carrier (number of terminal carboxylic acids 32) and a polyethylene glycol of one end methoxy group and one end 3-aminopropyl group having an average molecular weight of 5 kilodaltons and gemcitabine Compound 1 (molecular weight 6.8 kilodalton, 1.00 g) and a polyethylene glycol compound having one end methoxy group and one end 3-aminopropyl group (5.9 g, SUNBRIGHT MEPA-50H, manufactured by NOF Corporation, average A molecular weight of 5 kilodaltons) was dissolved in DMF (47 mL) at 35 ° C., and the temperature was lowered to 20 ° C., and N, N-dimethyl-4-aminopyridine (DMAP) (0.87 g), diisopropylcarbodiimide (DIPCI) (1 .74 mL) and gemcitabine (1.48 g, SCINO PHARM) were added and stirred for 32 hours . Thereafter, ethyl acetate (56 mL) was added for dilution, and the reaction solution was added dropwise to diisopropyl ether (1128 mL) over 30 minutes, followed by stirring at room temperature for 30 minutes. The precipitate was collected by filtration and washed with diisopropyl ether (564 mL). The resulting precipitate was dissolved in purified water (200 mL), and then an ion exchange resin (Dow Chemical Dowex 50 (H ⁺ ), 120 mL) was added. After stirring for 30 minutes, the mixture was filtered and freeze-dried to obtain the title compound (8.57 g) according to Comparative Example 1.

After alkaline hydrolysis of the compound of Comparative Example 1, the gemcitabine released was quantified by high performance liquid chromatography (HPLC) to determine the gemcitabine content in Comparative Example 1. As a result, the gemcitabine content in Comparative Example 1 was 6.8% by mass. Therefore, the gemcitabine binding ratio with respect to the number of terminal carboxylic acids of the multi-branched polymer carrier was calculated to be 40.2%.
As a result, the total molecular weight of gemcitabine in Comparative Example 1 was calculated to be 3.4 kilodaltons.

The binding amount of the polyethylene glycol compound of Comparative Example 1 was 8 molecules from the charged equivalent of the polyethylene glycol compound and the consumption rate of the polyethylene glycol compound. Accordingly, the total polyethylene glycol segment molecular weight was 40 kilodaltons.
In this reaction, the feed equivalent of the polyethylene glycol segment compound to the terminal carboxy group of the multi-branched polymer carrier = 0.25 equivalent, and the consumption rate of the polyethylene glycol segment compound = 1.

From the molecular weight of the compound 1, the bound polyethylene glycol segment molecular weight, and the combined gemcitabine total molecular weight, the molecular weight of the nucleic acid antimetabolite-bound hyperbranched compound of Comparative Example 1 was calculated to be 50 kilodaltons.
The polyethylene glycol segment content was calculated to be 80% by mass.
Further, the degree of association of the nucleic acid antimetabolite-bound hyperbranched compound of Comparative Example 1 was measured by laser light scattering intensity. As a result, the light scattering intensity was 6,199 cps. Therefore, the relative ratio with the light scattering intensity of toluene was 0.48 times. Further, the molecular weight measured by SEC-MALS was 77,270, and the number of associated molecules was 1.6.

[Test Example 1] Hematological toxicity evaluation for non-cancer-bearing mice
Hematological toxicity evaluation test for non-cancer-bearing mice (ICR mice (Crlj: CD-1)) using the compounds of Examples 1, 2, 5, 6, 8, 9 and Comparative Example 1 and gemcitabine hydrochloride as a control agent Carried out.
The compounds of Examples 1, 2, 5, 6, 8, 9 and Comparative Example 1 were prepared by dissolving in a 5% glucose injection solution. The dose was set below the maximum tolerated dose confirmed in advance for each compound. As a control drug, gemcitabine hydrochloride was prepared by dissolving in physiological saline. Each compound and control drug were each administered once intravenously. In addition, a group to which a solvent (5% glucose injection solution or physiological saline, 10 mL / kg) was administered was set, and the compounds of Examples 1, 2, 5, 6, 8, 9 and Comparative Example 1 were used. The 5% glucose injection solution administration group and the saline administration group for gemcitabine hydrochloride were the control group, respectively.
Blood was collected on the 7th or 5th day after administration, and the reticulocyte count was measured with a blood cell analyzer (XT-2000iV). The relative value of each compound administration group with respect to the reticulocyte count (100) of the solvent control group on the 7th or 5th day after administration was calculated. The results are shown in Tables 1 and 2.

As a result of Test Example 1, although the dose of the compound of Comparative Example 1 was low, the reticulocyte count was remarkably reduced as compared with gemcitabine hydrochloride as a control drug, and hematotoxicity was observed. . This phenomenon indicates that the recovery of the reticulocyte count is delayed even 5 days after administration, and is considered to be a prolongation of blood toxicity. In contrast, the compounds according to Examples 1, 2, 5, 6, 8, and 9 of the present invention have not been confirmed to decrease the reticulocyte count at 7 days after administration, and are similar to the target drug gemcitabine hydrochloride. Showed no hepatotoxicity.
The reason for this is considered that the compound of Comparative Example 1 stayed in the blood for a long period of time, so that the blood toxicity was prolonged. The compound of the present invention has a succinamide unit to which a nucleic acid antimetabolite is bound, whereas the compound of Comparative Example 1 does not have the succinamide unit. Therefore, the compounds according to Examples 1, 2, 5, 6, 8, and 9 are blood that is a side effect of gemcitabine hydrochloride by binding a nucleic acid antimetabolite to a multi-branched polymer carrier in an appropriate binding mode. The toxicity was similar, and it was confirmed that there was no manifestation of blood toxicity and the prolongation of concern caused by using a high molecular weight antimetabolite.

[Test Example 2] Antitumor effect test on nude mice transplanted with human pancreatic cancer Antitumor effect tests of the compounds of Examples 1, 2, 5, 6, 8, 9 and Comparative Example 1 were performed.
A tumor mass of human pancreatic cancer AsPC-1 subcultured subcutaneously in nude mice was made into blocks of about 3 mm square and transplanted subcutaneously on the dorsal side of nude mice using a trocar. The administration was started when the average tumor volume reached 300 mm ³ or more after tumor implantation.
The compounds of Examples 1, 2, 5, 6, 8, 9 and Comparative Example 1 were prepared by dissolving in a 5% glucose injection solution. The dose was set below the maximum tolerated dose (MTD) of each compound confirmed in advance. As a control drug, gemcitabine hydrochloride was prepared by dissolving in physiological saline. Each compound and control drug was administered into the tail vein 4 times at 3 day intervals.
The relative tumor volume was determined from the tumor volume on the administration start date and the evaluation date (16th day or 17th day after the start of administration) and used as an index of the antitumor effect. The tumor volume was calculated by the formula (L × W ² ) / 2 by measuring the major axis (L: mm) and minor axis (W: mm) of the tumor. The test was divided into four parts. The results are shown in Tables 3, 4, 5 and 6.

As a result of Test Example 2, the compounds according to Examples 1, 2, 5, 6, 8 and 9 of the present invention showed a strong antitumor effect and sustained effect compared to gemcitabine hydrochloride as a control drug. Admitted.

The nucleic acid antimetabolite-binding multibranched compound of the present invention suitably has a substituent containing a plurality of succinic monoamide units having a plurality of polyethylene glycol segments and a nucleic acid antimetabolite bonded to the terminal functional group of the multibranched polymer carrier. As a result, the bound nucleic acid antimetabolite was allowed to act with an appropriate release profile while staying in the blood and being distributed in the body. It was considered that the enhancement of the antitumor effect was achieved while avoiding prolongation.

Claims (14)

Nucleic acid antimetabolite binding hyperbranched compounds are represented by the general formula (1)

[Wherein [Q] is (m + n + o + p) terminal functionalized multi-branched polymer carrier, (m + n + o + p) is an integer of 4 to 200, and [F] is the terminal functional group. A protecting group having one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and / or an optionally substituted alkyl group having a carbon number (C1 to C6). Is one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carboxy group, and a mercapto group, and F is a linking group obtained by removing a hydrogen atom or a hydroxyl group from the terminal functional group. , R ₁ is a substituent containing a succinic acid monoamide units nucleic acid antimetabolites are bonded, R ₂ is a substituent containing a polyethylene glycol segment, R ₃ is Zanmoto及acid monoamide derivative And / or a substituent containing a succinimide residue, m is an integer from 0 to 199, n is an integer from 1 to 200, o is an integer from 0 to 199, and p is from 0 to 199. It is an integer. ] The nucleic acid antimetabolite binding hyperbranched compound shown by this. The nucleic acid antimetabolite is a nucleic acid antimetabolite having an amino group at a nucleobase, and the nucleic acid antimetabolite is bonded to a carboxy group of succinic acid monoamide via an amide bond by the amino group. The nucleic acid antimetabolite binding hyperbranched compound described. The nucleic acid antimetabolite-binding hyperbranched compound according to claim 1 or 2, wherein the mass content of the nucleic acid antimetabolite is 2 mass% or more and 60 mass% or less. The polyethylene glycol segment has a mass content of 20 mass% or more and 90 mass% or less in the nucleic acid antimetabolite-binding hyperbranched compound, and 2 to 100 units of polyethylene glycol segments are bonded. The nucleic acid antimetabolite-binding hyperbranched compound according to Item. A succinic acid monoamide unit to which a nucleic acid antimetabolite of R ₁ is bound is represented by the general formula (2) and / or (3)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, X ₄ is a binding group to the terminal functional group F, and R ₄ , R ₅ and R ₆ are each independently a hydrogen atom or carbon. An alkyl group having a number (C1 to C8), and R ₇ is a hydrogen atom, a linear, branched or cyclic alkyl group having a carbon number (C1 to C20) which may have a substituent, a substituent A linear, branched or cyclic aralkyl group which may have a carbon number (C7 to C20), an aromatic group which may have a substituent, and an amino acid residue in which a carboxy group is protected One or more groups selected from the group consisting of The nucleic acid antimetabolite-binding hyperbranched compound according to any one of claims 1 to 4. The nucleic acid metabolism antagonism according to any one of claims 1 to 5, wherein the succinic acid monoamide unit to which the nucleic acid antimetabolite of R ₁ is bound is a polyaspartic acid derivative having a side chain carboxy group bound to the nucleic acid antimetabolite. Agent-bound hyperbranched compound. The polyaspartic acid derivative of R ₁ is represented by the general formula (4) or (5)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, R ₁₂ is a hydroxyl group and / or —N (R ₁₄ ) CONH (R ₁₅ ), and R ₁₄ and R ₁₅ are the same A linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be different or may be substituted with a tertiary amino group, R ₁₃ is a hydrogen atom, a carbon number ( A group selected from the group consisting of an acyl group of C1 to C8) and an alkoxycarbonyl group of carbon number (C1 to C8), X ₂ is a bonding group to the terminal functional group F, and a, b, c, d and e each independently represent an integer of 0 to 30, (a + b) represents an integer of 1 to 30, and the total polymerization number of the polyaspartic acid derivative (a + b + c + d + e) is 1 to 30, D] is bound to an aspartic acid unit, and R ₁₂ is bound to The aspartic acid unit and the side chain carboxy group in the intramolecular cyclization type aspartic acid unit are each independently a random sequence. The nucleic acid antimetabolite-binding hyperbranched compound according to claim 6. 5. The succinic acid monoamide unit to which the nucleic acid antimetabolite of R ₁ is bonded is a linking succinic group of the succinic acid monoamide unit having a nucleic acid antimetabolite bonded to a polyethylene glycol segment and a side chain carboxy group. The nucleic acid antimetabolite-binding hyperbranched compound according to claim 1. The linking substituent of R ₁ is a block copolymer type substituent in which a polyaspartic acid segment in which a nucleic acid antimetabolite is bound to a polyethylene glycol segment and a side chain carboxy group is bound, 7)

[Wherein [D] is a binding residue of the nucleic acid antimetabolite, R ₁₆ is a polyethylene glycol segment, R ₁₇ is a hydroxyl group and / or —N (R ₁₈ ) CONH (R ₁₉ ), R ₁₈ and R ₁₉ may be the same or different and each is a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group; ₃ is a linking group to the terminal functional group F, f, g, h, i and j each independently represents an integer of 0 to 30, (f + g) represents an integer of 1 to 30, and a polyamino acid derivative (F + g + h + i + j) is 1 to 30, and the aspartic acid unit to which the [D] is bonded, the aspartic acid unit to which the R ₁₇ is bonded and the side chain carboxy group is an intramolecularly cyclized aspartic acid. Each unit is German It is a random sequence. The nucleic acid antimetabolite-binding hyperbranched compound according to claim 8. The polyethylene glycol segment of R ₂ has the general formula (8)

[Wherein R ₈ is a hydrogen atom or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may have a substituent, and k is 5 to 2,500. It is an integer, and X ₁ represents a bonding group with the terminal functional group F. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of claims 1 to 9. The succinic monoamide derivative residue and / or succinimide residue of R ₃ is represented by the general formulas (9), (10) and (11)

[Wherein, X ₄ is a bonding group to the terminal functional group F, R ₉ represents a hydroxyl group and / or —N (R ₁₀ ) CONH (R ₁₁ ), and R ₁₀ and R ₁₁ may be the same or different. Or a linear, branched or cyclic alkyl group having a carbon number (C1 to C8) which may be substituted with a tertiary amino group, wherein R ₄ , R ₅ , R ₆ and R ₇ are Is synonymous with. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of claims 1 to 10, which is at least one group selected from the group of substituents consisting of: The nucleic acid metabolism antagonist is represented by the formula (12):

[Wherein, -Rf represents the formula (13):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of claims 1 to 11, which is any one or more nucleic acid antimetabolite represented by the formula: The nucleic acid metabolism antagonist is represented by the formula (14):

[Wherein, -Rf represents the formula (15):

R ₂₀ represents a hydrogen atom or an acyl group of a fatty acid ester. The nucleic acid antimetabolite-binding hyperbranched compound according to any one of claims 1 to 12, which is any one or more nucleic acid antimetabolite represented by the formula: A medicament comprising the nucleic acid antimetabolite-binding hyperbranched compound according to any one of claims 1 to 13.