WO2019189419A1 - Anti-cancer agent-bonded alginic acid derivative - Google Patents

Anti-cancer agent-bonded alginic acid derivative Download PDF

Info

Publication number
WO2019189419A1
WO2019189419A1 PCT/JP2019/013286 JP2019013286W WO2019189419A1 WO 2019189419 A1 WO2019189419 A1 WO 2019189419A1 JP 2019013286 W JP2019013286 W JP 2019013286W WO 2019189419 A1 WO2019189419 A1 WO 2019189419A1
Authority
WO
WIPO (PCT)
Prior art keywords
alginic acid
group
compound
derivative
camptothecin
Prior art date
Application number
PCT/JP2019/013286
Other languages
French (fr)
Japanese (ja)
Inventor
智裕 鳴海
Original Assignee
持田製薬株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 持田製薬株式会社 filed Critical 持田製薬株式会社
Publication of WO2019189419A1 publication Critical patent/WO2019189419A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an alginic acid derivative in which alginic acid and a camptothecin derivative are covalently bonded via a linker, and a sustained-release pharmaceutical composition containing the same.
  • Alginic acid is a natural high molecular weight polysaccharide consisting of ⁇ -D-mannuronic acid and ⁇ -L-guluronic acid extracted from brown algae. It is not toxic and is difficult to be decomposed because there is no specific degrading enzyme in the body. It is biocompatible and non-immunogenic.
  • alginic acid has the property of causing physical property changes by ion exchange and is soluble in water in the presence of monovalent metal ions such as sodium, but gels by crosslinking in the presence of divalent metal ions such as calcium. Form. Utilizing such properties of alginic acid, it is widely used as industrial, food, and pharmaceutical additives.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2007-75425 (Patent Document 1)
  • cartilage disease treatment International Publication No. 2008/102855 (Patent Document 2)
  • rheumatoid arthritis International publication 2009/54181 (patent document 3)
  • intervertebral disc treatment use international publication 2017/163603 (patent document 4)
  • the property of physical property change due to ion exchange of alginic acid is useful, and it is possible to select an aqueous solution, sol or gel state depending on the indication disease of drug, administration route, dosage form, etc., as a pharmaceutical composition
  • the application range of is expected to expand.
  • Camptothecin is a kind of alkaloid having topoisomerase I inhibitory activity and is known to have anticancer activity.
  • Camptothecin since it exhibits poor solubility and serious side effects, various analog compounds thereof have been developed, It has been studied as an anticancer agent. SN-38 was found as one of these camptothecin analog compounds, but the prodrug irinotecan was developed as a pharmaceutical to overcome the poor solubility problem.
  • irinotecan is the metabolite SN-38, and a metabolic activation step in the living body such as the liver is necessary to bring about a strong anticancer effect.
  • a metabolic activation step in the living body such as the liver is necessary to bring about a strong anticancer effect.
  • cancerous peritonitis is a disease state in which cancer cells have metastasized mainly to the peritoneum in a disseminated manner, and is often observed in the state of progression of abdominal organ cancer such as ovarian cancer, stomach cancer, colon cancer, The prognosis is very bad.
  • intraperitoneal chemotherapy for peritoneal metastasis is one of the treatment methods
  • many water-soluble low-molecular-weight anticancer drugs are rapidly absorbed into the blood via capillaries and the residence time in the abdominal cavity is short. It is difficult to maintain an effective concentration.
  • macromolecular compounds and micelle-forming molecules are absorbed through the lymphatic system, absorption in the abdominal cavity is slow, and intraperitoneal chemotherapy using these properties has been studied, It has not yet been shown to be effective.
  • Patent Document 5 discloses a derivative obtained by introducing a compound such as doxorubicin into a polysaccharide having a carboxyl group, and an amino acid or peptide chain that can be dissociated by an enzyme.
  • Patent Document 6 discloses a derivative in which a specific camptothecin analog is introduced into a polysaccharide having a carboxy group via an amino acid or a peptide.
  • JP-A-8-24325 (Patent Document 7) describes providing a medical polymer gel capable of releasing a therapeutically effective amount of a drug only at a lesion site where an enzyme is produced. It is disclosed that a drug is bound via a degradable group (such as a peptide) whose main chain can be cleaved by an enzymatic reaction.
  • JP-A-8-502053 (Patent Document 8) discloses an alginate-bioactive agent combination connected through a biodegradable spacer bond that is stable at pH 7.4 but is unstable to acid. Specifically, a cis-aconityl group is shown as a biodegradable spacer.
  • Patent Document 9 discloses a derivative in which a carboxylic acid group of a polymer of polyethylene glycols and a polycarboxylic acid is bonded to a hydroxyl group of a phenolic camptothecin. The drug sustained release action until after time is shown.
  • Patent Document 10 discloses a treatment method using a micelle preparation formed from the derivative shown in Patent Document 9 for intraperitoneal administration.
  • Patent Document 11 discloses a derivative in which an anti-inflammatory compound is bound to hyaluronic acid via a biodegradable spacer.
  • Patent Document 12 a derivative in which a physiologically active substance is bound to a glycosaminoglycan via a spacer, and the spacer is selected according to the release rate of the physiologically active substance. And a method for controlling the release rate.
  • Patent Document 13 discloses a hyaluronic acid derivative in which a photoreactive group and a drug such as a non-steroidal anti-inflammatory agent are introduced and a photocrosslinked hyaluronic acid derivative gel. And providing a preparation with enhanced drug sustained release.
  • the object of the present invention is to use alginic acid as a base material that can be an option for a new base material, and use it in a sustained-release preparation that can stably release an active ingredient in vivo. It is to provide a compound that can.
  • the present inventor has found that an alginic acid derivative having a structure in which alginic acid or a salt thereof and a camptothecin derivative are covalently bonded with a specific linker can be provided.
  • the camptothecin derivative can be delivered to the affected area (tumor local area) stably and unexpectedly for a long period of time, and the present invention has been completed. That is, the present invention is configured as follows: Another aspect of the present invention may be as follows.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • L is a linker having a functional group that can be bonded to (A) by an amide bond and a functional group that can be bonded to (D) by an ester bond.
  • the alginic acid derivative according to [1], which has a structure represented by the following formula (2): (A) —NH— (CH 2 ) n1 — [X 1 ] n2 — (CR 1 R 2 ) n3 — [Y] n4 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 - (CH 2) n8 - ( CR 5 R 6) n9 -C ( O) - (D) (2) (Where (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • X 1 and X 2 represent a hetero atom which may have a substituent
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 each independently represents a hydrogen atom, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group.
  • R 1 and R 2 or R 3 and R 4 together represent ⁇ O
  • Y represents a cycloalkane ring, an aromatic ring or a heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group);
  • n1 represents any integer of 0 to 10
  • n2, n4 and n7 each independently represents 0 or 1
  • n3, n5, n6, n8 and n9 each independently represents any integer of 0 to 3. As shown, all of n1 to n9 are not 0).
  • the camptothecin derivative is camptothecin; 7-ethylcamptothecin; 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); irinotecan (CPT-11); 9- (dimethylamino) methylcamptothecin; Exatecan; T-2513 (10- (3-aminopropyloxy) -7-ethylcamptothecin); 10,11-methylenedioxycamptothecin; 7-ethyl-10,11-methylenedioxycamptothecin; 9-amino -10,11-methylenedioxycamptothecin; 9-chloro-10,11-methylenedioxycamptothecin; 7- (4-methyl-1-piperazinyl) methyl-10,11-methylenedioxycamptothecin; 10,11-eth Lurutetecan (7- (4-methyl-1-piperazinyl) methyl-10,11-methylenedioxycamptothecin;
  • the tertiary alcoholic hydroxyl group at position 20 of the camptothecin derivative is bonded to a linker, or the camptothecin derivative has a phenolic hydroxyl group, and the phenolic hydroxyl group is bonded to a linker.
  • a sustained-release pharmaceutical composition comprising the alginic acid derivative according to any one of [1] to [7] or the alginic acid derivative gel according to [8].
  • a sustained-release pharmaceutical composition comprising the alginic acid derivative according to any one of [1a], [7a], [7b] and [7c] or the alginic acid derivative gel according to [8a].
  • object. The sustained release pharmaceutical composition according to [9] above as an anticancer agent.
  • [11] Use of the alginic acid derivative according to any one of [1] to [7] or the alginic acid derivative gel according to [8] for the sustained release of a camptothecin derivative.
  • [11a] The alginic acid derivative according to any one of the above [1a], [7a], [7b] and [7c] or the alginic acid derivative according to [8a] for the sustained release of the camptothecin derivative Use of gel.
  • the present invention can provide a compound that can be used in a sustained-release preparation by stably releasing a camptothecin derivative stably. Moreover, the compound which can further improve the sustained-release property of the compound can be provided by making it gelatinize.
  • the present invention relates to an alginic acid derivative having a structure in which alginic acid or a salt thereof and a camptothecin derivative are covalently bonded via a linker.
  • the linker is preferably covalently bonded to the carboxyl group of alginic acid or a salt thereof and the hydroxyl group of the camptothecin derivative.
  • the binding mode is not particularly limited as long as the object of the present invention is achieved.
  • the bond between alginic acid and the linker is an amide bond
  • the bond between the camptothecin derivative and the linker is preferably an ester bond.
  • binding site functional group of alginic acid or a salt thereof
  • alginic acid or a salt thereof examples include a hydroxyl group or a carboxyl group, but a carboxyl group capable of forming an amide bond is more preferable.
  • the alginic acid derivative has a structure represented by the following formula (1): (A) -L- (D) (1)
  • (A) is a residue derived from alginic acid or a salt thereof, and is a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • L is a linker having a functional group that can be bonded to (A) by an amide bond and a functional group that can be bonded to (D) by an ester bond.
  • (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative, and the hydroxyl group of (D) is a tertiary alcoholic hydroxyl group at the 20th position of the camptothecin skeleton.
  • a hydroxyl group substituted and introduced at an arbitrary position preferably a tertiary alcoholic hydroxyl group at the 20th position or a phenolic hydroxyl group at an arbitrary position, more preferably a tertiary hydroxyl group at the 20th position.
  • It is an alcoholic hydroxyl group or a 10-position phenolic hydroxyl group, more preferably a 10-position phenolic hydroxyl group.
  • (A) is a residue derived from alginic acid or a salt thereof, and C ( ⁇ O) — of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • X 1 represents a residue having a group
  • (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • X 1 and X 2 represent a hetero atom which may have a substituent
  • R 1 Each of R 2 , R 3 , R 4 , R 5 and R 6 independently represents hydrogen, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group; Or R 1 and R 2 or R 3 and R 4 together represent ⁇ O
  • Y represents a cycloalkane ring, an aromatic ring or a heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring is a halogen in atomic or C 1-10 alkyl group may be substituted),
  • n1 is 0 Any one of 10 integers, n2, n4 and n7 independently represent 0 or 1,
  • alginic acid derivatives have structures represented by the following formulas (3) to (7), (7-1) and (7-2): (A) —NH— (CH 2 ) n1 — (CR 1 R 2 ) n3 — (CH 2 ) n5 —C ( ⁇ O) — (D) (3) (Where (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
  • R 1 and R 2 are either R 1 represents hydrogen or a halogen atom,
  • R 2 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or
  • R 1 and R 2 together represent ⁇ O.
  • Show n1, n3 and n5 each independently represents an integer of 0 to 3, but n1, n3 and n5 in total represent any integer of 1 to 4).
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • X 1 and X 2 each independently represent O or NH, preferably both O
  • R 1 and R 2 are those in which R 1 represents hydrogen and R 2 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 1 and R 2 together represent ⁇ O
  • R 1 is O
  • R 1 is preferably hydrogen
  • R 2 is preferably hydrogen, methyl group or ethyl group
  • R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ⁇ O
  • R 5 and R 6 are as follows:
  • R 5 represents hydrogen and R 2 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ⁇ O
  • (A) —NH— (CH 2 ) n1 — [Y] — (CH 2 ) n5 —C ( ⁇ O) — (D) (5)
  • (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • Y represents a cycloalkane ring, preferably cyclohexane
  • n1 and n5 independently represent any integer from 0 to 3, but n1 and n5 represent any integer from 1 to 4 in total).
  • (A) —NH— (CH 2 ) n1 — (CR 1 R 2 ) n3 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — (CH 2 ) n8 —C ( ⁇ O) — (D) ( 6)
  • (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • R 1 and R 2 are those in which R 1 represents hydrogen and R 2 represents hydrogen, methoxy group, ethoxy group, methoxycarbonyl group or ethoxycarbonyl group, or R 1 and R 2 together represent O
  • R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a methyl group or an ethyl group, or R 3 and R 4 together represent ⁇ O, n1, n3, n5, n6 and n8 independently represent any integer of 0 to 3, but n1, n3, n5, n6 and n8 represent any integer of 1 to 4 in total).
  • (A) —NH— (CH 2 ) n1 — [X 1 ] — [Y] — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 — (CH 2 ) n8 — (CR 5 R 6 ) n9 —C ( ⁇ O) — (D) (7)
  • (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
  • X 1 and X 2 each independently represent O, NH or NR 7 , preferably X 1 is O;
  • R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ⁇ O,
  • R 4 is preferably hydrogen, methyl group or ethyl group
  • R 4 is preferably hydrogen, a methyl group or an ethyl group, or R 3 and R 4 together are preferably ⁇ O
  • R 5 and R 6 are as follows:
  • R 5 represents hydrogen,
  • R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group,
  • R 7 represents a C 1-6 alkyl group, a C 2-7 alkanoyl group or
  • Y represents an aromatic ring or a heterocyclic ring (the aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group), preferably a benzene ring or a monocyclic heterocyclic ring.
  • n1 represents an integer of 1 to 10, preferably an integer of 1 to 3, more preferably 2, n7 represents 0 or 1, n5, n6, n8 and n9 independently represent an integer of 0 to 3, but n5, n6, n8 and n9 are preferably 0 to 3 in total, When n7 is 0, preferably n5 and n6 are 0, n8 and n9 are 0 to 3 in total, more preferably n5, n6 and n9 are 0, and n8 is 0 to 2 Yes, When n7 is 1 and X 2 is O, n5 and n6 are preferably 0 or 1 in total, more preferably both are 0, When n7 is 1 and X 2 is NH or NR 7 , n5 and n6 are preferably 1 or 2 in total, more preferably 1 in total, When n7 is 1, n8 and n9 preferably represent 1 to 3 in total, more
  • (A) -NH- (CH 2) n1 - [X 1] - [Y] - (CH 2) n8 - (CR 5 R 6) n9 -C ( O) - (D) (7-1) (Where (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C ( ⁇ O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid.
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
  • X 1 represents O or NH, preferably O
  • R 5 and R 6 are as follows:
  • R 5 represents hydrogen
  • R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group
  • Y represents an aromatic ring or a heterocyclic ring (the aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group), preferably a benzene ring or a monocyclic heterocyclic ring.
  • n1 represents an integer of 1 to 10, preferably an integer of 1 to 3, more preferably 2, n8 and n9 each independently represents an integer of 0 to 3, but n8 and n9 are preferably 0 to 3 in total, more preferably n8 is 0 to 2 and n9 is 0).
  • Show (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
  • X 1 and X 2 each independently represent O, NH or NR 7 , preferably X 1 is O;
  • R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ⁇ O,
  • R 4 is preferably hydrogen, methyl group or ethyl group
  • R 4 is preferably hydrogen, a methyl group or an ethyl group, or R 3 and R 4 together are preferably ⁇ O
  • R 5 and R 6 are as follows:
  • R 5 represents hydrogen,
  • R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group,
  • R 7 represents a C 1-6 alkyl group, a C 2-7 alkanoyl group or
  • Y represents an aromatic ring or a heterocyclic ring (the aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group), preferably a benzene ring or a monocyclic heterocyclic ring.
  • n1 represents an integer of 1 to 10, preferably an integer of 1 to 3, more preferably 2, n5, n6, n8 and n9 each independently represents an integer of 0 to 3,
  • n5 and n6 are preferably 0 or 1, and more preferably both are 0,
  • X 2 is NH or NR 7
  • n5 and n6 are preferably 1 or 2 in total, more preferably 1 in total
  • n8 and n9 are preferably 1 to 3 in total, more preferably n8 is 1 or 2
  • n9 is 0, and still more preferably n8 is 1 and n9 is 0.
  • Linker structure of the alginic acid derivative of the present invention will be described later in the section “Linker”.
  • a camptothecin derivative that can keep releasing a camptothecin derivative at a concentration that is less likely to cause side effects in the body and that can appropriately exhibit a pharmacological action, for example, a concentration that can suppress tumor reduction or growth.
  • the introduction rate is preferably.
  • the introduction rate (mol%) is preferably 0.2 mol% or more. More preferably, it is 0.5 mol% or more, More preferably, it is 1.0 mol% or more, Most preferably, it is 2.0 mol% or more.
  • the introduction rate of the camptothecin derivative can be increased to 10 mol%, more preferably 15 mol%.
  • the introduction rate (mol%) in the present invention refers to, for example, an introduction rate of 10 mol when a camptothecin derivative is introduced into the carboxyl group of L-guluronic acid or D-mannuronic acid constituting alginic acid via a linker.
  • % Indicates that one unit (piece) of monosaccharide of L-guluronic acid or D-mannuronic acid constituting alginic acid is introduced, and camptothecin derivative is introduced at a ratio of 10 to 100 monosaccharides. Therefore, a camptothecin derivative may be introduced to each carboxyl group of adjacent monosaccharides via a linker.
  • the type of linker and the introduction rate of the camptothecin derivative are determined depending on the final administration form (gel, sol, microbead, etc.) of the pharmaceutical composition containing the compound described later, or the affected part of the camptothecin derivative (tumor)
  • the amount may be adjusted as appropriate in consideration of the necessary amount in local) or sustained release efficiency.
  • the alginic acid derivative of the present invention is a polymer compound containing a camptothecin derivative, but is characterized by being water-soluble. That is, even when the introduction rate of the camptothecin derivative, which is generally known to be hydrophobic, in the alginic acid derivative is high, for example, 3 mol% or more, it can be dissolved in water. For example, when 0.1 part by mass of an alginic acid derivative is added to 100 parts by mass of water and shaken or stirred at room temperature, it is shown that it dissolves without becoming a gel within 24 hours. Is soluble in aqueous solvents at concentrations of 0.1% and above.
  • the water-soluble alginic acid derivative in the present invention has an advantage that it is easy to handle gelation or solification according to the use described later. Therefore, the solution of the alginic acid derivative of the present invention can be filtered, and dust removal, sterilization, and sterilization can be performed by filter filtration. In other words, dust removal and sterilization can be performed by passing through a 5 ⁇ m to 0.45 ⁇ m filter, and more preferably, sterilization can be performed by passing through a 0.22 ⁇ m filter.
  • the water-soluble alginic acid derivative of the present invention can be dissolved in water, an aqueous solution containing a pharmaceutically acceptable metal salt or a pH adjuster, or an aqueous solvent such as a buffer solution. Specifically, it can be dissolved in water for injection, phosphate buffered saline, physiological saline and the like.
  • the alginic acid derivative in the present invention alone does not bring about the strong anticancer effect of the camptothecin derivative, but when it is administered in vivo, for example, the camptothecin derivative is appropriately cleaved from the linker depending on the situation in the living body. By doing so, the camptothecin derivative is released and exerts an effect. Since the camptothecin derivative is continuously released in an amount necessary for tumor reduction and growth inhibition, an anticancer effect can be brought about at the affected area (tumor local area). In the alginic acid derivative, the sustained release rate of the camptothecin derivative can be adjusted to a desired mode depending on the structure of the linker which is a constituent component.
  • the alginic acid derivative in the present invention is less affected by the release rate of the camptothecin derivative and can stably and continuously release the active ingredient due to factors other than the cleavage of the linker site. .
  • the alginic acid derivative of the present invention by changing the binding mode of alginic acid or a salt thereof and a linker and the binding mode of a camptothecin derivative and a linker, degradability and degradation order in vivo can be changed.
  • the camptothecin derivative It is also possible to control the release rate and release rate. Specifically, it is known that ester bonds are more susceptible to hydrolysis than amide bonds in vivo.
  • the alginic acid derivative of the present invention may be decomposed in any order as long as the camptothecin derivative is finally released. However, it is preferable that the camptothecin derivative and the linker are first subjected to hydrolysis to release the camptothecin derivative.
  • alginic acid or a salt thereof and a linker are bonded by an amide bond, and the camptothecin derivative and the linker are bonded by an ester bond, whereby the ester bond is first hydrolyzed, and the camptothecin derivative is first released from the linker.
  • alginic acid does not adversely affect the applied organism, and since no specific receptor that binds to alginic acid in vivo has been identified, alginic acid or its salt after releasing the camptothecin derivative is toxic in the body. Without being disassembled.
  • the alginic acid derivative of the present invention is preferably released slowly in a situation where sustained release is expected under neutral conditions.
  • the camptothecin derivative has a liberation rate of 0.1% to 35% at pH 7.0, preferably 1 It is preferable to exhibit the behavior of being released at% to 30%, more preferably 4% to 25%.
  • the camptothecin derivative is released at a release rate of 0.1% to 60% at pH 7.0.
  • the alginic acid derivative of the present invention is preferably released more slowly in a situation where longer-term sustained release is expected under neutral conditions.
  • the alginic acid derivative of the present invention is prepared in a 0.01% by weight aqueous solution and incubated at 37 ° C. and pH 7.0, the camptothecin derivative is released at a release rate of 0.1% to 30% in 7 days. It is preferable to exhibit a behavior that is released at 1% to 25%, more preferably 3% to 20%.
  • the alginic acid derivative of the present invention is preferably released quickly in a situation where rapid release is expected under neutral conditions.
  • an alginic acid derivative of the present invention is prepared in a 0.01% by weight aqueous solution and incubated at 37 ° C. and pH 7.0
  • the camptothecin derivative is released at a release rate of 20% to 100% in one day. It is preferable to exhibit a behavior of being released at 30% to 100%, more preferably 50% to 100%, particularly preferably 70 to 100%.
  • a suitable alginate derivative of the present invention can be selected by measuring the liberation rate under conditions corresponding to the pH of the environment to be used. Moreover, it is possible to further enhance the sustained release effect by gelling the alginic acid derivative of the present invention with a crosslinking agent, and adjusting the release rate of the alginic acid derivative and the gelation state (gelation strength, form, etc.). By doing so, it is possible to adjust to a suitable release rate. In this way, it is possible to adjust the release period of the drug by adjusting the release rate and the like.
  • the alginic acid derivative of the present invention is administered as an anticancer agent by injection or the like to the affected area (tumor local area, for example, intraperitoneally)
  • the camptothecin derivative is continuously released for 7 days or more, preferably 15 days or more, more preferably 30 days or more, and further preferably 60 days or more.
  • the alginic acid derivative of the present invention is administered as an anticancer agent to an affected area by injection or the like, preferably 7 days or less, preferably Is expected to continue the sustained release of the camptothecin derivative in 3 days or less, more preferably 24 hours or less.
  • the release rate indicates the ratio of the amount of released camptothecin derivative to the total amount of camptothecin derivative contained in the alginic acid derivative.
  • the alginic acid, the linker, and the camptothecin derivative which are the respective constituent components, will be described, and then the alginic acid derivative gel and their uses will be described in detail.
  • alginic acid or a salt thereof is a monovalent metal such as Na + or K + , which is a “monovalent metal salt of alginic acid” and a hydrogen atom of carboxylic acid of D-mannuronic acid or L-guluronic acid of alginic acid.
  • Water-soluble salts produced by ion exchange with ions are preferred.
  • Specific examples of the monovalent metal salt of alginic acid include sodium alginate and potassium alginate, and sodium alginate is particularly preferable.
  • a solution of a monovalent metal salt of alginic acid can adjust the form of the alginic acid derivative of the present invention by utilizing the property of forming a gel when mixed with a crosslinking agent.
  • Alginic acid is a kind of natural polysaccharide that is produced by extracting and purifying from brown seaweed seaweed. Further, it is a polymer obtained by polymerizing D-mannuronic acid (M) and L-guluronic acid (G).
  • M D-mannuronic acid
  • G L-guluronic acid
  • the composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid of alginic acid varies depending on the type of organism mainly derived from seaweed, etc.
  • alginic acid it ranges from a high G type with an M / G ratio of about 0.4 to a high M type with an M / G ratio of about 5.
  • M / G ratio of alginic acid the arrangement of M and G, etc.
  • the physicochemical properties of alginic acid may differ, and the preferred application may differ.
  • the alginic acid or a salt thereof used in the present invention one having an appropriate M / G ratio may be used depending on the end use application.
  • Industrial methods for producing alginic acid include an acid method and a calcium method. In the present invention, those produced by any method can be used.
  • the range of 80 to 120% by mass of the quantitative value by HPLC method by purification is preferred, more preferably in the range of 90 to 110% by mass, and in the range of 95 to 105% by mass. Further preferred.
  • a substance whose quantitative value by HPLC method is included in the above range is referred to as high purity alginic acid.
  • the alginic acid or a salt thereof used in the present invention is preferably high-purity alginic acid.
  • a commercially available product for example, a product sold by Kimika Co., Ltd., preferably a high-purity food / pharmaceutical grade product can be purchased and used as the Kimika Argin series.
  • Commercially available products can be used after further appropriate purification.
  • a low endotoxin treatment is preferable.
  • the purification method and the low endotoxin treatment method for example, the method described in JP-A-2007-75425 (Patent Document 1) can be employed.
  • alginic acid or a salt thereof used in the present invention one having an appropriate weight average molecular weight may be used according to the end use application.
  • those having a weight average molecular weight of 10,000 to 10,000,000 are preferably used, more preferably 100,000 to 5,000,000, still more preferably 200,000 to 300,000. 10,000 or less.
  • alginic acid having a physical property such as a weight average molecular weight of 1.69 million Da ( ⁇ 20%) or 1.34 million Da ( ⁇ 20%) or a salt thereof.
  • a high molecular substance derived from a natural product is not a single molecular weight but an aggregate of molecules having various molecular weights, and thus is measured as a molecular weight distribution having a certain width.
  • a typical measurement technique is gel filtration chromatography.
  • Representative information on the molecular weight distribution obtained by gel filtration chromatography includes weight average molecular weight (Mw), number average molecular weight (Mn), and dispersion ratio (Mw / Mn).
  • Mw weight average molecular weight
  • Mn number average molecular weight
  • Mn dispersion ratio
  • the weight average molecular weight places importance on the contribution to the average molecular weight of a polymer having a large molecular weight and is represented by the following formula.
  • the number average molecular weight is calculated by dividing the total weight of the polymer by the total number of polymers.
  • W is the total weight of the polymer
  • Wi is the weight of the i-th polymer
  • Mi the molecular weight at the i-th elution time
  • Ni is the number of molecular weights Mi
  • Hi is the height at the i-th elution time.
  • the molecular weight of a high molecular weight substance derived from a natural product may vary depending on the measurement method (example of hyaluronic acid: Chikako YOMOTA et. Al. Bull. Natl. Health Sci., Vol. 117). , Pp135-139 (1999), Chikako YOMOTA et.al.Bull.Natl.Inst.Health Sci., Vol.121, pp30-33 (2003)).
  • the molecular weight by SEC-MALLS is used as a standard value on a catalog of alginic acid (FMC Biopolymer, PRONOVATM sodium alloys catalog).
  • FMC Biopolymer, PRONOVATM sodium alloys catalog a catalog of alginic acid
  • a measurement error of 10 to 20% can occur. For example, if it is 400,000, it is 32 to 480,000, and if it is 1 million, 80 to Value fluctuations can occur in the range of about 1.2 million.
  • the molecular weight of alginic acid or a salt thereof is specified, it is a weight average molecular weight calculated by gel filtration chromatography unless otherwise specified.
  • the gel filtration chromatography for example, the conditions of this example described later can be adopted.
  • alginic acid or its salt used by this invention it is good to use the thing of a suitable viscosity according to the end use application.
  • the alginic acid or a salt thereof used in the present invention it is preferable to use a combination of appropriate physical properties according to the end use application, specifically, an appropriate M / G ratio, an appropriate weight. It is preferable to use one having an average molecular weight, an appropriate viscosity and the like.
  • alginic acid or a salt thereof used in the present invention with a lowered endotoxin level.
  • the endotoxin value measured by the JP endotoxin test is preferably less than 100 EU / g, more preferably less than 75 EU / g, and even more preferably less than 50 EU / g.
  • “substantially free of endotoxin” means that the endotoxin value measured by the JP endotoxin test is in the above numerical range.
  • the linker of the alginic acid derivative of the present invention has a functional group that can be bonded to one residue derived from alginic acid or a salt thereof by an amide bond, and is bonded to one residue of the camptothecin derivative by an ester bond.
  • the linker of the alginic acid derivative of the present invention has a functional group that can be bonded to one residue derived from alginic acid or a salt thereof by an amide bond, and is bonded to one residue of the camptothecin derivative by an ester bond.
  • —NH— (CH 2 ) n1 — [X 1 ] n2 — (CR 1 R 2 ) n3 — [Y] n4 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 — (CH 2) n8 - (CR 5 R 6) n9 -C ( O) - (8)
  • —NH represents a terminal that forms an amide bond with one residue of alginic acid or a salt thereof
  • C ( ⁇ O) — represents a terminal that forms an ester bond with one residue of a camptothecin derivative. Show.
  • X 1 and X 2 each represents a hetero atom which may have a substituent, preferably any atom selected from O, S, NH and NR 7 (R 7 represents A C 1-6 alkyl group, a C 2-7 alkanoyl group or a C 1-6 alkylsulfonyl group, preferably a methyl group, an ethyl group, an acetyl group or a methylsulfonyl group, more preferably an acetyl group) More preferably O, NH or NR 7 , still more preferably X 1 represents O and X 2 represents O, NH or NR 7 .
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrogen, fluorine, C 1-6 alkyl group, C 1-6 alkoxy group or C 1-6 alkoxycarbonyl.
  • Y is a cycloalkane ring, aromatic ring or heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group).
  • a cycloalkane ring, an aromatic ring or a heterocyclic ring more preferably a cycloalkane ring, a benzene ring or a monocyclic heterocyclic ring, still more preferably a cyclohexane ring, a benzene ring or a pyridine ring
  • a benzene ring or a pyridine ring is shown.
  • n1 represents any integer of 0 to 10
  • n2, n4 and n7 independently represent 0 or 1
  • n3, n5, n6, n8 and n9 independently represent 0 to 3 Indicates one of the integers. However, all of n1 to n9 do not become zero.
  • n3, n5, n6, n8 and n9 are in total 1 to 12, more preferably 1 to 10.
  • N1 is preferably an integer of 1 to 3, more preferably 1 or 2, and still more preferably 2.
  • n3 is preferably an integer of 0 to 2, more preferably 0 or 1, and still more preferably 0.
  • n5 and n6 are preferably 0 to 2 in total, and more preferably 0 or 1.
  • n8 and n9 are preferably 0 to 2 in total, and more preferably 0 or 1.
  • examples of the “heteroatom” include O, S, and N or P.
  • examples of the “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • examples of the “C 1-6 alkyl group” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- Pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1-ethylpropyl, hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 1, 2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl,
  • C 1-10 alkyl group examples include heptyl, 1-methylhexyl, octyl, 2-ethylhexyl, 1,1-dimethylhexyl, nonyl, decyl, in addition to the groups listed as the above “C 1-6 alkyl group”. , Cycloheptyl, cyclohexylmethyl, 2-cyclohexylethyl, 4-methylcyclohexyl, 4,4-dimethylcyclohexyl, 3,3,5,5-tetramethylcyclohexyl and the like.
  • examples of the “C 1-6 alkoxy group” include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyl.
  • C 1-10 alkoxy group examples include heptyloxy, octyloxy, 2-ethylhexyloxy, nonyloxy, decyloxy, cycloheptyloxy, cyclohexylmethoxy, in addition to the groups listed as the above “C 1-6 alkoxy group”. -Cyclohexylethoxy, 4-methylcyclohexyloxy, 4,4-dimethylcyclohexyloxy, 3,3,5,5-tetramethylcyclohexyloxy and the like.
  • the “C 1-10 alkoxycarbonyl group” is a group represented by —C ( ⁇ O) —R (R is a C 1-10 alkoxy group).
  • the “C 2-7 alkanoyl group” is a group represented by —C ( ⁇ O) —R (R is a C 1-6 alkyl group). Examples include acetyl, propionyl, butyryl, isobutyryl and the like.
  • the “C 1-6 alkylsulfonyl group” is a group represented by —S ( ⁇ O) 2 —R (R is a C 1-6 alkyl group). For example, methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl and the like can be mentioned.
  • examples of the “cycloalkane ring” include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, and cyclodecane.
  • examples of the “aromatic ring” include a benzene ring, 1-naphthalene ring, 2-naphthalene ring, 2-, 3-, 4-biphenylanthrone ring, phenanthrene ring, and acenaphthene ring. Is mentioned.
  • heterocycle means a 3 to 14-membered monocyclic or condensed ring containing 1 to 5 heteroatoms of nitrogen, sulfur or oxygen. Means a ring.
  • examples of the “heterocycle” include “partially hydrogenated condensed ring heterocycle” and “non-aromatic heterocycle”.
  • the “heterocycle” is preferably one having 5 to 7 ring members, such as pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, 1 , 2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, frazal, 1,2 , 3-thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3 , 5-triazine, 2H-1,2,3-thiadiazine, 4H-1,2,4-thiadiazine, 6H-1,3,4-thiadiazine 1,4-d
  • the “condensed ring” preferably has 8 to 12 ring members.
  • the ester bond between the linker and the camptothecin derivative is hydrolyzed to release the camptothecin derivative.
  • the rate of hydrolysis of ester bonds varies depending on the surrounding environment. Therefore, there are some which can obtain a longer-term sustained release effect in the form of an ester bond. For example, by introducing an electron donating group or a bulky group in the vicinity of the C ( ⁇ O) terminal of the linker, or introducing it as a substituent, the rate of hydrolysis may be reduced.
  • an alkyl group, particularly A branched alkyl group can be mentioned.
  • the hydrolysis rate may be increased.
  • alginic acid or a salt thereof and a camptothecin derivative are bonded to any one of a group of linkers shown below.
  • linkers —NH— on the right side represents a terminal that forms an amide bond with one residue of alginic acid or a salt thereof, and —C ( ⁇ O) — on the left side represents a hydroxyl group and an ester bond of the camptothecin derivative. The end to be formed is indicated.
  • camptothecin derivative one having a camptothecin skeleton of the following formula in its chemical structure is used.
  • the camptothecin derivative may be in the form of a salt.
  • the camptothecin derivative in the present invention is not particularly limited, but among them, those having an anticancer activity are particularly desirable, and a camptothecin derivative having at least a hydroxyl group is preferable from the viewpoint of binding with a linker.
  • the position of the hydroxyl group is not particularly limited, and examples thereof include a tertiary alcoholic hydroxyl group at the 20th position of the camptothecin skeleton or a hydroxyl group substituted and introduced at an arbitrary position.
  • camptothecin derivative having a hydroxyl group substituted a camptothecin derivative having a phenolic hydroxyl group is more preferable, and a camptothecin derivative having a phenolic hydroxyl group at the 10-position of the camptothecin skeleton is more preferable. That is, in the alginic acid derivative of the present invention, the tertiary alcoholic hydroxyl group at the 20-position of the camptothecin derivative is bonded to the linker, or the phenolic hydroxyl group into which the substitution of the camptothecin derivative is introduced is bonded to the linker.
  • the alginic acid derivative of the present invention has a tertiary alcoholic hydroxyl group at the 20-position of the camptothecin derivative bonded to a linker.
  • camptothecin derivative having a hydroxyl group examples include camptothecin; 7-ethylcamptothecin; 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); irinotecan (CPT-11); 9- (dimethylamino) methylcamptothecin Exotecan; T-2513 (10- (3-aminopropyloxy) -7-ethylcamptothecin): 10,11-methylenedioxycamptothecin; 7-ethyl-10,11-methylenedioxycamptothecin; 9-amino-10,11-methylenedioxycamptothecin; 9-chloro-10,11-methylenedioxycamptothecin; 7- (4-methyl-1-piperazinyl) methyl-10,11-methylenedioxycamptothecin 10,11-ethylenedioxycamptothecin; luruthecan (7- (4-methyl-1-piperazinyl)
  • camptothecin derivative is SN-38 (7- ethyl-10-hydroxycamptothecin).
  • the camptothecin derivatives having a phenolic hydroxyl group include 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); topotecan (nogitecan); silathecan (7- (tert-butyldimethylsilyl) -10-hydroxycamptothecin SN-38 (7-ethyl-10-hydroxycamptothecin) or topotecan (nogitecan) is preferable, and SN-38 (7-ethyl-10-hydroxycamptothecin) is more preferable.
  • ⁇ Synthesis Method of Alginic Acid Derivative> In the synthesis of the alginic acid derivative, either the binding of the camptothecin derivative to the linker and the binding of alginic acid or a salt thereof to the linker may be performed first, but it is difficult to perform esterification in an aqueous solvent. It is preferable that the camptothecin derivative is first bound to the linker.
  • Examples of a method for achieving such bonding include a method using a condensing agent such as DCC, EDCI, and DMT-MM, a condensation reaction using a condensing agent such as HOSu and HOBt and the condensing agent, and a nucleophilic substitution reaction.
  • an active ester method an acid anhydride method, and the like. Bonding using a condensation reaction and a nucleophilic substitution reaction is preferable for the purpose of suppressing side reactions. More specifically, it can be synthesized by a method utilizing a condensation reaction (esterification reaction), for example, as in a scheme showing the following concept.
  • esterification reaction esterification reaction
  • the linker is interpreted as “CO-Linker-NH”
  • AL is a residue derived from alginic acid or a salt thereof
  • L-guluronic acid and D-mannuronic acid constituting alginic acid are included.
  • CPT is an abbreviation for a camptothecin derivative, but this is merely an example, and does not mean that the camptothecin derivative is limited to camptothecin in the present invention.
  • the introduction rate of the camptothecin derivative in the alginic acid derivative in the present invention can be adjusted by changing the input amount of the condensing agent, the condensation auxiliary agent, and the linker-bound camptothecin derivative in the synthesis step of the alginic acid derivative of the present invention.
  • the introduction rate can be measured by a method using absorbance measurement, HPLC, NMR, or the like.
  • the water solubility of the alginic acid derivative can be appropriately adjusted depending on the structure and introduction rate of the linker.
  • an amino compound used when binding a linker and a camptothecin derivative and then binding to alginic acid or a salt thereof is an amino compound represented by the following formula (9), a salt thereof, or a solvent thereof. Things.
  • (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative
  • X 1 and X 2 represent a hetero atom which may have a substituent
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 each independently represent a hydrogen atom, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group, or R 1 and R 2 or R 3 and R 4 together represent ⁇ O
  • Y represents a cycloxyl group
  • Preferred embodiments include the preferred embodiments described in the above formulas (1) to (8).
  • the alginic acid derivative of the present invention can form an alginic acid derivative gel by mixing with a substance generally used as a crosslinking agent for alginic acid.
  • a crosslinking agent is not particularly limited as long as it can fix the surface of the monovalent metal salt of alginic acid by crosslinking, but is not limited to Ca 2+ , Mg 2+ , Ba.
  • examples thereof include divalent or higher-valent metal ion compounds such as 2+ and Sr 2+, and crosslinkable reagents having 2 to 4 amino groups in the molecule. More specifically, CaCl 2 , MgCl 2 , CaSO 4 , BaCl 2, etc.
  • Diaminoalkanes that may have a lysyl group (—COCH (NH 2 ) — (CH 2 ) 4 —NH 2 ), ie, diaminoalkanes and their amino groups are substituted with lysyl groups to form lysylamino groups
  • specific examples include diaminoethane, diaminopropane, N- (lysyl) -diaminoethane, etc., but CaCl 2 solution is particularly preferred because of its availability and gel strength. Is preferable.
  • the alginic acid derivative gel obtained by crosslinking the alginic acid derivative of the present invention can be processed into a bead or sponge shape.
  • a beaded alginic acid derivative gel can be used as an anticancer agent for intraperitoneal administration or indwelling at the time of surgery.
  • the alginic acid derivative or alginic acid derivative gel of the present invention exhibits a behavior of sustained release of the camptothecin derivative in vivo, it can be used as a sustained-release pharmaceutical composition.
  • the sustained-release pharmaceutical composition of the present invention uses alginic acid or a salt thereof as the sustained-release base.
  • Alginic acid or a salt thereof is a high molecular compound, but it is generally known that a high molecular compound or micelle-forming molecule administered intraperitoneally is gently absorbed through the lymphatic system.
  • alginic acid or a salt thereof which is a polymer compound, is absorbed in the lymphatic system, which is an initial metastasis of peritoneal metastasis, and thus a targeting effect of alginic acid is expected. That is, the sustained-release pharmaceutical composition of the present invention is expected to have both the antitumor action of the sustained-release camptothecin derivative and the targeting action due to the lymph absorbability of alginic acid.
  • the target disease and administration route of the sustained-release pharmaceutical composition of the present invention are not particularly limited, it is preferably intended for the treatment, prevention or alleviation of peritoneal metastasis, and the administration route for direct injection into the abdominal cavity are preferably administered.
  • the sustained-release pharmaceutical composition of the present invention is used as an anticancer agent for intraperitoneal administration, it is administered for 7 days or more, preferably 15 days or more, more preferably when administered to an affected area (tumor local) by injection or the like. Is expected to stably release the camptothecin derivative stably for 30 days or longer, more preferably 60 days or longer.
  • an alginic acid derivative having a drug release period suitable for the aspect is selected, for example, the alginic acid derivative of the present invention is selected.
  • the camptothecin derivative When administered to the affected area (tumor local area) by injection or the like as an anticancer agent, it is expected that the camptothecin derivative will be sustainedly released in 7 days or less, preferably 3 days or less, more preferably 24 hours or less.
  • the dosage of the sustained-release pharmaceutical composition of the present invention depends on the amount of the camptothecin derivative contained, the administration route, the administration form, the purpose of use, the specific symptoms of the animal to be administered, the age, the body weight, etc. It is determined individually so that the therapeutic effect is most appropriately exhibited, and is not particularly limited.
  • the application site of the sustained-release pharmaceutical composition of the present invention is not particularly limited as long as it can be administered by parenteral administration, but administration to a tumor site is preferable, and intraperitoneal administration is more preferable. It is also preferable to use it for arterial chemoembolization, particularly hepatic arterial chemoembolization, and it is also preferable to administer a sustained-release pharmaceutical composition containing the alginic acid derivative gel of the present invention as an embolic substance into an artery.
  • an alginic acid derivative may be applied to the abdominal cavity that is an affected area (tumor local area) of peritoneal metastasis.
  • a crosslinking agent may be added to the surface of the applied derivative. Leakage from the abdominal cavity can be effectively prevented by gelling the surface of the derivative and solidifying the surface.
  • the application amount of the cross-linking agent is adjusted so as not to be excessive.
  • the application amount of the divalent or higher metal ion is not particularly limited as long as it can solidify the surface of the composition containing the monovalent metal salt of alginic acid.
  • the concentration of the cross-linking agent that promotes gelation is adjusted by changes in the environment such as time differences, temperature differences, or contact with calcium ions in vivo.
  • a composition that maintains a liquid state before administration and self-gelates after administration into a living body can be used.
  • examples of such a crosslinking agent include calcium gluconate, CaSO 4 , calcium alginate, and the like.
  • the method for adding a divalent or higher metal ion to a pharmaceutical composition containing an alginic acid derivative is not particularly limited.
  • a solution of a divalent or higher metal ion can be prepared with a syringe or a sprayer
  • Examples include a method of applying to the surface.
  • the timing of applying the cross-linking agent to the surface of the composition of the present invention may be after the composition of the present invention is applied to the affected area (tumor local) or simultaneously.
  • the sustained-release pharmaceutical composition containing the alginic acid derivative gel of the present invention may contain microbeads having an average particle size of less than 500 ⁇ m, for example.
  • the introduction rate (mol%) of the drug (camptothecin derivative) in the examples is 1 unit of monosaccharide of D-mannuronic acid or L-guluronic acid constituting alginic acid calculated from 1 H-NMR (D 2 O) ( Mol) and the ratio of the number of moles of the introduced drug to 100 units (mol) of monosaccharides constituting alginic acid.
  • Example 1 to 4 The alginate derivative solid according to the present invention obtained in the examples was weighed, added with 10 mmol / L phosphate buffer (pH 7.7), stirred and dissolved at room temperature for 1 hour or more, diluted, and 0.05% solution Was prepared. This solution was passed through a hydrophilic PVDF filtration filter (Mylex GV33 filter, Merck Millipore) having a pore size of 0.22 ⁇ m to remove insoluble materials, and then 200 ⁇ L of the solution was applied to a Superose 6 Increase 10/300 GL column (GE Healthcare). Filtration was performed.
  • a hydrophilic PVDF filtration filter Mylex GV33 filter, Merck Millipore
  • the molecular weight of the alginic acid derivative according to the present invention was determined by the following method. Blue dextran (molecular weight 2 million Da, SIGMA), thyroglobulin (molecular weight 66.9 million Da, GE Healthcare), ferritin (molecular weight 440,000 Da, GE Healthcare), conalbumin (molecular weight 75,000 Da) GE Healthcare) and ribonuclease A (molecular weight: 1.37,000 Da, GE Healthcare) were used as standard products, and gel filtration was performed under the same conditions as the sample to determine the amount of each solution.
  • Blue dextran molecular weight 2 million Da, SIGMA
  • thyroglobulin molecular weight 66.9 million Da, GE Healthcare
  • ferritin molecular weight 440,000 Da, GE Healthcare
  • conalbumin molecular weight 75,000 Da
  • ribonuclease A molecular weight: 1.37,000 Da, GE Healthcare
  • the amount of eluate of each component was plotted on the horizontal axis, and the logarithmic value of molecular weight was plotted on the vertical axis, followed by quadratic regression to create a calibration curve.
  • the molecular weight (Mi) at the elution time i in the chromatogram of the previously obtained sample was calculated.
  • the absorbance at the elution time i was read and taken as Hi. From these data, the weight average molecular weight (Mw) was determined from the following equation.
  • the molecular weight of the raw material alginic acid or a salt thereof was determined by the following method. Each alginic acid was weighed in consideration of loss on drying, and ultrapure water was added to prepare a 1% aqueous solution. Subsequently, it diluted with a 100 mmol / L phosphate buffer and ultrapure water so that it might become a final concentration of 10 mmol / L phosphate buffer (pH 7.7), and prepared 0.05% solution.
  • Insoluble matter was removed by a hydrophilic PVDF filter with a pore size of 0.22 ⁇ m (Mylex GV33 filter, Merck Millipore), 200 ⁇ L was subjected to gel filtration, and gel filtration was performed under the same conditions as the alginic acid derivative according to the present invention. . Detection was carried out with a differential refractometer, and the weight average molecular weight (Mw) was determined in the same manner as the alginic acid derivative according to the present invention.
  • Mw weight average molecular weight
  • Method B> The alginate derivative solid was weighed, 10 mmol / L phosphate buffer (pH 7 to 7.4) was added, stirred and dissolved at room temperature for 1 hour or more, and then diluted to prepare a 0.2 w / v% solution. This solution was passed through a 0.45 ⁇ m regenerated cellulose filtration filter (Sartorius Stedim) to remove insoluble matters, and then 200 ⁇ L was subjected to gel filtration in the same manner as in Method A, and the weight average molecular weight was determined.
  • Standard products include blue dextran (molecular weight 2 million Da, GE Healthcare), thyroglobulin (molecular weight 66.9 million Da, GE Healthcare), ferritin (molecular weight 440,000 Da, GE Healthcare), conalbumin (Molecular weight 75,000 Da, GE Healthcare), carbonic anhydrolase (molecular weight 23,000 Da, GE Healthcare) and aprotinin (molecular weight 6500 Da, GE Healthcare) were used.
  • the raw material alginic acid or salt thereof is diluted with 100 mmol / L phosphate buffer (pH 7) and ultrapure water so that the final concentration is 10 mmol / L phosphate buffer (pH 7 to 7.4).
  • a 0.2% solution was prepared. This solution was passed through a 0.45 ⁇ m regenerated cellulose filtration filter (Sartorius Stedim) to remove insoluble matters, and then 200 ⁇ L of the solution was subjected to gel filtration under the same conditions as the alginic acid derivative (Method B). Detection was carried out with a differential refractometer, and the weight average molecular weight was determined in the same manner as the alginic acid derivative (Method B).
  • A is a residue derived from alginic acid or a salt thereof, and is a monosaccharide —C (1) of one of L-guluronic acid and D-mannuronic acid constituting alginic acid.
  • O means a residue having a group.
  • the mixture was further stirred at room temperature for 19 hours.
  • the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 4-3 (91 mg).
  • the fraction containing Compound 4-3 (91 mg) was dissolved in 1,4-dioxane (1.8 mL). To this solution was added 4N-hydrogen chloride / 1,4-dioxane (1.8 mL) with stirring under ice cooling, the reaction mixture was stirred at room temperature for 15 hours, and diisopropyl ether (30 mL) was added.
  • Example 5 Drug Release Test 1 To 1 mg of each SN-38-conjugated alginate derivative prepared in Examples 1 to 4, 20 mM sodium phosphate buffer (pH 7.0) or 1N so that the concentration of each alginate derivative is 0.1% w / v. Aqueous sodium hydroxide was added, and the mixture was stirred for 6 hours using a magnetic stirrer (ASONE REMIX RS-6A, 750 rpm). After confirming that the gel was not formed, this solution was dispensed. Immediately after dissolution, the amount of free SN-38 present in each solution as an initial state (0 days of storage) was measured by LC-MS / MS. The other dispensed solutions were incubated at 37 ° C. for 3 days, and then the amount of free SN-38 was measured. At each time point, the release rate (%) was calculated using the ratio to the amount of free SN-38 by forced decomposition in 1N aqueous sodium hydroxide solution.
  • the sustained release rate can be adjusted depending on the structure of the linker, and a long-term sustainable anticancer effect can be expected by adjusting the linker type and the drug introduction rate together.
  • the compound 3-4 obtained in Example 3 has a release rate of 5% in 3 days, and theoretically, release of the drug over a sufficient period of 30 days or more can be expected.
  • reaction solution was filtered, and the filtrate was concentrated under reduced pressure.
  • residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound 6-5 (158 mg) as a pale yellow amorphous.
  • ⁇ Step 2> Synthesis of methyl 2- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) phenoxy) acetate (Compound 7-3) (Example 7)
  • Compound 7 obtained in ⁇ Step 1> -2 (520 mg), tert-butyl (2-bromoethyl) carbamate [CAS: 39684-80-5] (703.63 mg) and acetonitrile (5200 ⁇ L) were mixed with potassium carbonate (788.98 mg) under water-cooling and stirring. And stirred at 80 ° C. for 1 hour.
  • the aqueous solution was acidified with 1N hydrochloric acid (20 mL) and dissolved in ethyl acetate (20 mL). This solution was extracted twice with ethyl acetate (10 mL) and washed successively with water (10 mL) and saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 8-3 (0.55 g) as a white amorphous.
  • the reaction mixture was filtered and the filtrate was concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate, ethyl acetate to 10% methanol / ethyl acetate) to obtain the title compound 8-5 (62 mg) as a white amorphous. .
  • ⁇ Step 2> Synthesis of 5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinic acid (Compound 9-3) (Example 9)
  • Compound 9-2 obtained in ⁇ Step 1> (1. 237 g) and methanol (12.37 mL), sodium hydroxide (0.5 g) was added at room temperature, and the mixture was stirred at 60 ° C. for 30 minutes. After cooling to room temperature, the solvent was distilled off. Subsequently, water (20 mL) was added and extracted twice with methyl tert-butyl ether (10 mL).
  • the aqueous layer was acidified with 1N hydrochloric acid (15 mL) and extracted three times with ethyl acetate (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give the title compound 9-3 (0.92 g) as a white amorphous.
  • ⁇ Step 1> Synthesis of (5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinoyl) glycine (Compound 10-3) (Example 9)
  • Compound 10 synthesized in the same manner as in ⁇ Step 1> -1 (0.768 g) and methanol (6.97 mL) were added sodium hydroxide (0.28 g) at room temperature, stirred at the same temperature for 30 minutes, and stirred at 40 ° C. for 1 hour. After completion of the reaction, 1N hydrochloric acid (7 mL) was added, and the solution was concentrated under reduced pressure.
  • glycine methyl hydrochloride [CAS: 5680-79-5] (0.32 g), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyl Uronium hexafluorophosphate (0.98 g) and acetonitrile (6.97 mL) were added.
  • N, N-diisopropylethylamine (1.23 mL) was added dropwise with stirring under ice cooling, and the mixture was stirred at room temperature for 1 hour. Water (10 mL) was added to stop the reaction, and the mixture was extracted 3 times with ethyl acetate (10 mL).
  • the organic layer was washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. The dried organic layer was filtered and then concentrated under reduced pressure.
  • the crude product was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 10-2 (0.67 g).
  • sodium hydroxide (0.14 g) was added at room temperature, and the mixture was stirred at the same temperature for 17 hours.
  • Example 7 The same operation as in ⁇ Step 5> was performed to give the title compound 10-7 (110 mg) as a yellow solid.
  • the drug introduction rate was 1.46 mol%.
  • the molecular weight showed a broad elution peak from 2.25 million Da to 16,000 Da, and the weight average molecular weight was 13.3 million Da.
  • ⁇ Step 1> Synthesis of tert-butyl (2- (4-formylphenoxy) ethyl) carbamate (Compound 11-2) 4-hydroxybenzaldehyde [CAS: 123-08-0] (Compound 11-1, 1 g), Potassium carbonate at room temperature against a mixture of potassium iodide (1.36 g), tert-butyl (2-bromoethyl) carbamate [CAS: 39684-80-5] (2.2 g) and N-methylpyrrolidone (10 mL) (1.36 g) was added, and the mixture was stirred at 80 ° C. for 3 hours and 30 minutes.
  • ⁇ Step 2> Synthesis of (4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzyl) glycine hydrochloride (Compound 11-4) (Example 11) Compound 11- obtained in ⁇ Step 1> 2 (1.27 g), glycine methyl hydrochloride [CAS: 5680-79-5] (0.69 g) and methylene chloride (25.4 mL) were mixed with triethylamine (0.73 mL) under ice-cooling and stirring. The solution was added dropwise and stirred at the same temperature for 10 minutes.
  • sodium triacetoxyborohydride (1.52 g) was added little by little under ice-cooling and the reaction mixture was stirred at room temperature for 24 hours. Water (10 mL) was added to stop the reaction, and then the organic layer was separated and concentrated under reduced pressure. The resulting residue was dissolved in methyl tert-butyl ether (20 mL) and washed with 1N hydrochloric acid (10 mL). The aqueous layer was extracted twice with methyl tert-butyl ether (10 mL) and basified with 1N aqueous sodium hydroxide solution (10 mL).
  • the reaction mixture was diluted with ethyl acetate (20 mL), filtered, and the filtrate was concentrated under reduced pressure.
  • the crude product was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound 13-2 (142 mg) as a pale yellow amorphous.
  • Example 16 Drug Release Test 2 The release test of the SN-38-linked alginic acid derivative of the present application was carried out in the same manner as in Example 5. Specifically, various alginic acid derivatives were prepared to a concentration of 0.01 w / v% in 20 mM sodium phosphate buffer (pH 7.0) or 0.1 N sodium hydroxide aqueous solution, and the solution obtained by stirring and dissolving for 6 hours was dispensed. The release test was conducted at 37 ° C.
  • the sustained release rate can be adjusted according to the linker structure or the position of the linker in the drug, and long-term sustainability can be achieved by adjusting the linker type, drug binding position and drug introduction rate. It was found that anticancer activity can be expected. Specifically, a derivative in which a linker is bonded to the tertiary alcoholic hydroxyl group of SN-38 tends to have a slow drug release rate. For example, Compound 14-5 has a release rate of 15% in 7 days. Theoretically, release of the drug over 30 days or more can be expected.
  • Compound 7-8 which is a derivative bonded to the phenolic hydroxyl group of SN-38 via the same linker as Compound 14-5, is released in one day, and prompt drug release can be expected.
  • Compound 9-7 has a release rate of 47% in 7 days, and can be expected to release the drug over about 14 days.
  • Example 17 Cell Growth Inhibition Test
  • Compound 2-6 obtained in Example 2 (introduction rate: 3.0 mol% (5.5 w / w%)), Compound 6-7 obtained in Example 6 ( Introduction rate 6.5 mol% (10.9 w / w%)), cell growth inhibitory action of compound 9-7 obtained in Example 9 (introduction rate 3.2 mol% (5.8 w / w%))
  • MKN45 cells derived from human gastric cancer (National Institute of Biomedical Innovation, Health and Nutrition Research, JCRB Cell Bank) were seeded on a 96-well flat bottom plate at a concentration of 1,000 cells per well, and 24 hours later, test compounds A diluted solution of phosphate buffered saline (PBS) was added.
  • PBS phosphate buffered saline
  • the SN-38-conjugated alginic acid derivative suppresses cell growth of human gastric cancer cell lines and has the ability to inhibit survival of cancer cells.
  • the drug introduction rate in the compound used in the above test is about 5 to 10 w / w%.
  • the SN-38 concentration when SN-38 is completely released from a 0.000001% solution of the compound is 0.5. ⁇ 1 ng / mL.
  • the IC50 value of SN-38 in MKN45 cells has been reported to be 0.54 ng / mL (Topotecin Intravenous Drug Interview Form). Considering the SN-38 release rate in the drug release test of Example 16, this study The indicated cytostatic ability is believed to be a result of free SN-38.
  • Example 18 Intraperitoneal administration test Using the mouse peritoneal seeding model, the intraperitoneal sustained release effect of the SN-38-conjugated alginic acid derivative of the present application was examined.
  • a mouse peritoneal dissemination model was obtained by seeding 1.0 ⁇ 10 5 B16-F10 cells (ATCC), a mouse melanoma cell line, into the abdominal cavity of 8-week-old male C57BL / 6N mice (Charles River Japan). Produced.
  • Various alginic acid derivatives (0.1 w / v%) dissolved in 5% glucose solution were intraperitoneally administered to model mice 7 days after tumor seeding at 10 mL / kg.
  • SN-38 was detected in the intraperitoneal tumor tissue for 72 hours or longer in the animals once administered with the SN-38-conjugated alginic acid derivative. That is, it was found that the SN-38-binding alginic acid derivative of the present invention can retain SN-38 in the intraperitoneal tumor tissue for a long time and can expect a long-term sustainable anticancer effect in the intraperitoneal environment.
  • the SN-38 concentration in the tumor tissue was high (4.7 to 52 times) compared with the plasma concentration at all time points. Therefore, the conditions for reducing drug exposure to the whole body and reducing side effects The antiperitoneal seeding effect can be expected.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

A compound that can be used as a sustained-release preparation capable of releasing an active ingredient sustainably and stably in a living body is provided using alginic acid as a base material that can become a choice for a novel base material. The present invention relates to an alginic acid derivative having such a structure that alginic acid or a salt thereof is covalently bonded to a camptothecin derivative through a linker, preferably an alginic acid derivative having a structure represented by the formula: (A)-L-(D) (wherein (A) represents one residue that is derived from alginic acid or a salt thereof and has a C(=O)- group in either one monosaccharide selected from L-guluronic acid and D-mannuronic acid both constituting alginic acid; (D) represents a group formed by removing a hydrogen atom from a hydroxyl group in the camptothecin derivative; and L represents a linker that has a functional group capable of bonding to (A) through an amide bond and also has a functional group capable of bonding to (D) through an ester bond).

Description

抗癌剤結合アルギン酸誘導体Anticancer drug-bound alginate derivative
　本発明は、アルギン酸とカンプトテシン誘導体とがリンカーを介して共有結合されてなるアルギン酸誘導体等、及びこれを含む徐放性医薬組成物に関する。 The present invention relates to an alginic acid derivative in which alginic acid and a camptothecin derivative are covalently bonded via a linker, and a sustained-release pharmaceutical composition containing the same.
　アルギン酸は、褐藻類から抽出されるβ－Ｄ－マンヌロン酸とα－Ｌ－グルロン酸からなる天然の高分子多糖であり、毒性はなく、生体内に特定の分解酵素がないため分解されにくく、生体適合性があり、また非免疫原性である。さらにアルギン酸はイオン交換によって物性変化を起こす特性を有しており、ナトリウム等の１価金属イオン存在下では水に可溶であるが、カルシウム等の２価金属イオン存在下では架橋することによりゲルを形成する。このようなアルギン酸の性質を利用して、工業用や食品用、さらには医薬品添加物として広く利用されている。近年、さらに医薬の主剤として創傷被覆用途（特開２００７－７５４２５号公報（特許文献１））、軟骨疾患治療用途（国際公開第２００８／１０２８５５号公報（特許文献２））、関節リウマチ治療用途（国際公開第２００９／５４１８１号公報（特許文献３））及び椎間板治療用途（国際公開第２０１７／１６３６０３号公報（特許文献４））が提案されている。特に、アルギン酸のイオン交換による物性変化の特性は有用であり、医薬の適応疾患、投与経路や剤形等に応じて、水溶液、ゾル又はゲル状態を選択することが可能であり、医薬組成物としての応用範囲が広がることが期待されている。
　一方、悪性腫瘍の治療方法として抗癌剤化学療法が実施されているが、効果の増強は様々な副作用を引き起こすことがある。カンプトテシンはトポイソメラーゼＩ阻害作用を有するアルカロイドの一種であり、抗癌作用を有することが知られているが、溶解性の悪さや重篤な副作用を示すことから、様々なそのアナログ化合物が開発され、抗癌剤として検討されてきた。それらカンプトテシンアナログ化合物の一つとしてＳＮ－３８が見出されたが、難溶性の問題を克服するものとして、プロドラッグのイリノテカンが医薬品として開発された。イリノテカンの活性型は代謝物のＳＮ－３８であり、強力な抗癌作用をもたらすためには肝臓等の生体内における代謝活性化ステップが必要である。結果として、作用有効量を得るためには高用量の投与が必要となり、副作用の増大につながることから、使用に制限を受けることとなる。
　また、癌性腹膜炎（腹膜転移）は癌細胞が主に播種性に腹膜に転移した病態であり、卵巣癌、胃癌、大腸癌等の腹腔内臓器癌が進行した状態で認められることが多く、予後は極めて悪い。腹膜転移に対する腹腔内化学療法が治療方法の一つとして行なわれているが、多くの水溶性低分子抗癌剤は毛細血管を介して速やかに血中に吸収されてしまい、腹腔内の滞留時間が短く、有効な濃度を維持させることが困難である。一方で、高分子化合物やミセル形成分子はリンパ系を介して吸収されることから、腹腔内における吸収は緩徐であり、それらの特性を利用した腹腔内化学療法が検討されてきたが、充分な効果を示すには至っていない。 Alginic acid is a natural high molecular weight polysaccharide consisting of β-D-mannuronic acid and α-L-guluronic acid extracted from brown algae. It is not toxic and is difficult to be decomposed because there is no specific degrading enzyme in the body. It is biocompatible and non-immunogenic. In addition, alginic acid has the property of causing physical property changes by ion exchange and is soluble in water in the presence of monovalent metal ions such as sodium, but gels by crosslinking in the presence of divalent metal ions such as calcium. Form. Utilizing such properties of alginic acid, it is widely used as industrial, food, and pharmaceutical additives. In recent years, as a main pharmaceutical agent, it has been further used as a wound covering (Japanese Patent Application Laid-Open No. 2007-75425 (Patent Document 1)), cartilage disease treatment (International Publication No. 2008/102855 (Patent Document 2)), rheumatoid arthritis International publication 2009/54181 (patent document 3)) and intervertebral disc treatment use (international publication 2017/163603 (patent document 4)) have been proposed. In particular, the property of physical property change due to ion exchange of alginic acid is useful, and it is possible to select an aqueous solution, sol or gel state depending on the indication disease of drug, administration route, dosage form, etc., as a pharmaceutical composition The application range of is expected to expand.
On the other hand, although anticancer drug chemotherapy is performed as a treatment method for malignant tumors, the enhancement of the effect may cause various side effects. Camptothecin is a kind of alkaloid having topoisomerase I inhibitory activity and is known to have anticancer activity. However, since it exhibits poor solubility and serious side effects, various analog compounds thereof have been developed, It has been studied as an anticancer agent. SN-38 was found as one of these camptothecin analog compounds, but the prodrug irinotecan was developed as a pharmaceutical to overcome the poor solubility problem. The active form of irinotecan is the metabolite SN-38, and a metabolic activation step in the living body such as the liver is necessary to bring about a strong anticancer effect. As a result, in order to obtain an effective dose, it is necessary to administer a high dose, which leads to an increase in side effects, which limits use.
In addition, cancerous peritonitis (peritoneal metastasis) is a disease state in which cancer cells have metastasized mainly to the peritoneum in a disseminated manner, and is often observed in the state of progression of abdominal organ cancer such as ovarian cancer, stomach cancer, colon cancer, The prognosis is very bad. Although intraperitoneal chemotherapy for peritoneal metastasis is one of the treatment methods, many water-soluble low-molecular-weight anticancer drugs are rapidly absorbed into the blood via capillaries and the residence time in the abdominal cavity is short. It is difficult to maintain an effective concentration. On the other hand, since macromolecular compounds and micelle-forming molecules are absorbed through the lymphatic system, absorption in the abdominal cavity is slow, and intraperitoneal chemotherapy using these properties has been studied, It has not yet been shown to be effective.
　このような問題点を解決するために、近年、抗癌剤のドラッグ・デリバリー方法が検討されており、抗癌剤へ高分子物質を化学的に結合させる方法が試みられている。例えば、国際公開第９４／１９３７６号公報（特許文献５）には、カルボキシル基を有する多糖にドキソルビシン等の化合物を導入した誘導体が開示されており、結合部は酵素によって解離されうるアミノ酸又はペプチド鎖からなる。特開平１０－７２４６７号公報（特許文献６）には、カルボキシ基を有する多糖類に特定のカンプトテシンアナログをアミノ酸又はペプチドを介して導入した誘導体が開示されている。特開平８－２４３２５号公報（特許文献７）には、酵素が産生される病巣部位においてのみ、治療に有効な量の薬剤を放出させることが可能な医療用高分子ゲルを提供することが記載されており、薬剤は酵素反応で主鎖が切断され得る分解性基（ペプチド等）を介して結合されることが開示されている。特表平８－５０２０５３号公報（特許文献８）には、ｐＨ７．４では安定であるが酸に不安定な生物分解性スペーサー結合を介して接続されたアルジネート‐生物活性剤配合体が開示されており、具体的にはシス－アコニチル基が生物分解性スペーサーとして示されている。国際公開第２００４／０３９８６９号公報（特許文献９）には、ポリエチレングリコール類とポリカルボン酸との重合体のカルボン酸基とフェノール性カンプトテシン類の水酸基とが結合した誘導体が開示されており、２４時間後までの薬物徐放作用が示されている。さらに、国際公開第２０１１／０４９０４２号公報（特許文献１０）には、特許文献９で示された誘導体から形成されるミセル調製物を腹腔内投与に用いた治療法が開示されている。
　また、抗癌剤関連の開示ではないが、国際公開第２００５／０６６２１４号公報（特許文献１１）には、ヒアルロン酸に抗炎症化合物を生体内分解性スペーサーを介して結合させた誘導体が開示されており、関節症に伴う疼痛の抑制効果等が示され、持続的効果も示唆されている。国際公開第２０１５／００５４５８号公報（特許文献１２）には、グリコサミノグリカンに生理活性物質をスペーサーを介して結合させた誘導体において、生理活性物質の遊離速度に応じてスペーサーが選択される誘導体及びその遊離速度制御方法が示されている。また、国際公開第２００７／００４６７５号公報（特許文献１３）には、非ステロイド性抗炎症剤等の薬剤と光反応性基が導入されたヒアルロン酸誘導体及び光架橋されたヒアルロン酸誘導体ゲルが開示されており、薬剤徐放性を高めた製剤を提供することが記載されている。 In order to solve such problems, in recent years, drug delivery methods for anticancer agents have been studied, and methods for chemically binding a polymer substance to the anticancer agents have been attempted. For example, International Publication No. 94/19376 (Patent Document 5) discloses a derivative obtained by introducing a compound such as doxorubicin into a polysaccharide having a carboxyl group, and an amino acid or peptide chain that can be dissociated by an enzyme. Consists of. Japanese Patent Application Laid-Open No. 10-72467 (Patent Document 6) discloses a derivative in which a specific camptothecin analog is introduced into a polysaccharide having a carboxy group via an amino acid or a peptide. JP-A-8-24325 (Patent Document 7) describes providing a medical polymer gel capable of releasing a therapeutically effective amount of a drug only at a lesion site where an enzyme is produced. It is disclosed that a drug is bound via a degradable group (such as a peptide) whose main chain can be cleaved by an enzymatic reaction. JP-A-8-502053 (Patent Document 8) discloses an alginate-bioactive agent combination connected through a biodegradable spacer bond that is stable at pH 7.4 but is unstable to acid. Specifically, a cis-aconityl group is shown as a biodegradable spacer. International Publication No. 2004/039869 (Patent Document 9) discloses a derivative in which a carboxylic acid group of a polymer of polyethylene glycols and a polycarboxylic acid is bonded to a hydroxyl group of a phenolic camptothecin. The drug sustained release action until after time is shown. Furthermore, International Publication No. 2011/049042 (Patent Document 10) discloses a treatment method using a micelle preparation formed from the derivative shown in Patent Document 9 for intraperitoneal administration.
Further, although not related to anticancer agents, International Publication No. 2005/066214 (Patent Document 11) discloses a derivative in which an anti-inflammatory compound is bound to hyaluronic acid via a biodegradable spacer. In addition, the effect of suppressing pain associated with arthropathy has been shown, and a sustained effect has also been suggested. In WO 2015/005458 (Patent Document 12), a derivative in which a physiologically active substance is bound to a glycosaminoglycan via a spacer, and the spacer is selected according to the release rate of the physiologically active substance. And a method for controlling the release rate. Further, International Publication No. 2007/004675 (Patent Document 13) discloses a hyaluronic acid derivative in which a photoreactive group and a drug such as a non-steroidal anti-inflammatory agent are introduced and a photocrosslinked hyaluronic acid derivative gel. And providing a preparation with enhanced drug sustained release.
特開２００７－７５４２５号公報JP 2007-75425 A 国際公開第２００８／１０２８５５号公報International Publication No. 2008/102855 国際公開第２００９／５４１８１号公報International Publication No. 2009/54181 国際公開第２０１７／１６３６０３号公報International Publication No. 2017/163603 国際公開第９４／１９３７６号公報International Publication No. 94/19376 特開平１０－７２４６７号公報Japanese Patent Laid-Open No. 10-72467 特開平８－２４３２５号公報JP-A-8-24325 特表平８－５０２０５３号公報Japanese National Patent Publication No. 8-502053 国際公開第２００４／０３９８６９号公報International Publication No. 2004/039869 国際公開第２０１１／０４９０４２号公報International Publication No. 2011/049042 国際公開第２００５／０６６２１４号公報International Publication No. 2005/066214 国際公開第２０１５／００５４５８号公報International Publication No. 2015/005458 国際公開第２００７／００４６７５号公報International Publication No. 2007/004675
　このように、抗癌剤の開発において、様々な高分子物質を基材としたコンジュゲート化合物を用いたドラッグ・デリバリー方法が検討されてきており、薬物との結合部位であるリンカー構造の検討も進められてきたが、低分子化合物の徐放性製剤としては実用化にまで至っていない。例えば、ヒアルロン酸を基材として用いた徐放性製剤が提案されているが、ヒアルロン酸は生体内に存在する酵素（ヒアルロニダーゼ）によって速やかに分解されてしまい、その半減期の短さから薬剤の放出に影響を与える懸念がある。また、複雑な修飾を施した基材は生体内で異物として認識され、毒性や免疫原性等の懸念がある。患部（腫瘍局所）における薬物の放出は効果発現に重要なステップであるが、ペプチドリンカー等は生体内の酵素分布に依存した分解機構を要する。
　このような課題を踏まえて、本発明の目的は、新たな基材の選択肢となりうる基材としてアルギン酸を用いて、生体内において安定的に有効成分を持続放出しうる、徐放性製剤に使用し得る化合物を提供することである。 Thus, in the development of anticancer agents, drug delivery methods using conjugate compounds based on various polymer substances have been studied, and the linker structure that is the binding site with the drug has also been studied. However, it has not yet been put to practical use as a sustained-release preparation of a low-molecular compound. For example, a sustained-release preparation using hyaluronic acid as a base material has been proposed, but hyaluronic acid is rapidly degraded by an enzyme (hyaluronidase) present in the living body, and due to its short half-life, There are concerns that affect the release. Moreover, the base material which carried out the complicated modification | reformation is recognized as a foreign material in the living body, and there exists concern, such as toxicity and immunogenicity. Release of the drug in the affected area (tumor local) is an important step for the expression of the effect, but peptide linkers and the like require a degradation mechanism depending on the enzyme distribution in the living body.
In light of these problems, the object of the present invention is to use alginic acid as a base material that can be an option for a new base material, and use it in a sustained-release preparation that can stably release an active ingredient in vivo. It is to provide a compound that can.
　本発明者は、上記の課題を解決するために鋭意検討した結果、アルギン酸又はその塩とカンプトテシン誘導体とを特定のリンカーで共有結合させた構造を有するアルギン酸誘導体を提供できることを見出し、これを徐放性製剤として使用することで、予想外に長期間にわたり安定してカンプトテシン誘導体を患部（腫瘍局所）に届けることができることを見出し、本発明を完成させた。
　すなわち、本発明は以下のように構成される：
　本発明のまた別の態様は、以下のとおりであってもよい。
〔１〕アルギン酸又はその塩とカンプトテシン誘導体とがリンカーを介して共有結合されてなる構造を有する、アルギン酸誘導体。
〔１ａ〕リンカーが２価のリンカーである、前記〔１〕に記載のアルギン酸誘導体。
〔２〕下記式（１）で表される構造を有する、前記〔１〕に記載のアルギン酸誘導体：
（Ａ）－Ｌ－（Ｄ）　　　（１）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｌは、（Ａ）とアミド結合で結合しうる官能基を有し、かつ（Ｄ）とエステル結合で結合しうる官能基を有するリンカーである）。
〔３〕下記式（２）で表される構造を有する、前記〔１〕に記載のアルギン酸誘導体：
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］n2－（ＣＲ1Ｒ2）n3－［Ｙ］n4－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］n7－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（２）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｘ1及びＸ2は、置換基を有しても良いヘテロ原子を示し、
　Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5及びＲ6はそれぞれ独立して、水素、ハロゲン原子、Ｃ1-10アルキル基、Ｃ1-10アルコキシ基又はＣ1-10アルコキシカルボニル基を示すか、又は、Ｒ1及びＲ2若しくはＲ3及びＲ4が一緒になって＝Ｏを示し、
　Ｙは、シクロアルカン環、芳香族環又は複素環（前記シクロアルカン環、芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、
　ｎ１は０～１０のいずれかの整数を示し、ｎ２、ｎ４及びｎ７は独立して０または１を示し、ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ１～ｎ９の全てが０になることはない）。 As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that an alginic acid derivative having a structure in which alginic acid or a salt thereof and a camptothecin derivative are covalently bonded with a specific linker can be provided. As a result, it was found that the camptothecin derivative can be delivered to the affected area (tumor local area) stably and unexpectedly for a long period of time, and the present invention has been completed.
That is, the present invention is configured as follows:
Another aspect of the present invention may be as follows.
[1] An alginic acid derivative having a structure in which alginic acid or a salt thereof and a camptothecin derivative are covalently bonded via a linker.
[1a] The alginic acid derivative according to [1], wherein the linker is a divalent linker.
[2] The alginic acid derivative according to [1], which has a structure represented by the following formula (1):
(A) -L- (D) (1)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
L is a linker having a functional group that can be bonded to (A) by an amide bond and a functional group that can be bonded to (D) by an ester bond.
[3] The alginic acid derivative according to [1], which has a structure represented by the following formula (2):
(A) —NH— (CH 2 ) n1 — [X 1 ] n2 — (CR 1 R 2 ) n3 — [Y] n4 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 - (CH 2) n8 - ( CR 5 R 6) n9 -C (= O) - (D) (2)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
X 1 and X 2 represent a hetero atom which may have a substituent,
R 1 , R 2 , R 3 , R 4 , R 5 and R 6 each independently represents a hydrogen atom, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group. Or R 1 and R 2 or R 3 and R 4 together represent ═O,
Y represents a cycloalkane ring, an aromatic ring or a heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group);
n1 represents any integer of 0 to 10, n2, n4 and n7 each independently represents 0 or 1, n3, n5, n6, n8 and n9 each independently represents any integer of 0 to 3. As shown, all of n1 to n9 are not 0).
〔４〕カンプトテシン誘導体がカンプトテシン；７－エチルカンプトテシン；１０－ヒドロキシカンプトテシン；ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）；イリノテカン（ＣＰＴ－１１）；９－（ジメチルアミノ）メチルカンプトテシン；トポテカン（ノギテカン）；エキサテカン；Ｔ－２５１３（１０－（３－アミノプロピロキシ）－７－エチルカンプトテシン）；１０，１１－メチレンジオキシカンプトテシン；７－エチル－１０，１１－メチレンジオキシカンプトテシン；９－アミノ－１０，１１－メチレンジオキシカンプトテシン；９－クロロ－１０，１１－メチレンジオキシカンプトテシン；７－（４－メチル－１－ピペラジニル）メチル－１０，１１－メチレンジオキシカンプトテシン；１０，１１－エチレンジオキシカンプトテシン；ルルトテカン（７－（４－メチル－１－ピペラジニル）メチル－１０，１１－エチレンジオキシカンプトテシン）；ギマテカン（７－（ｔｅｒｔ－ブトキシイミノメチル）カンプトテシン）；９－アミノカンプトテシン；ルビテカン（９－ニトロカンプトテシン）；ベロテカン（７－（２－（Ｎ－イソプロピルアミノ）エチル）－カンプトテシン）；コシテカン（７－（２－（トリメチルシリル）エチル）－カンプトテシン）及びシラテカン（７－（ｔｅｒｔ－ブチルジメチルシリル）－１０－ヒドロキシカンプトテシン）からなる群から選ばれる、前記〔１〕～〔３〕のいずれか１項に記載のアルギン酸誘導体。
〔５〕カンプトテシン誘導体がＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）、イリノテカン（ＣＰＴ－１１）又はトポテカン（ノギテカン）である、前記〔４〕に記載のアルギン酸誘導体。
〔６〕カンプトテシン誘導体がＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）である、前記〔５〕に記載のアルギン酸誘導体。 [4] The camptothecin derivative is camptothecin; 7-ethylcamptothecin; 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); irinotecan (CPT-11); 9- (dimethylamino) methylcamptothecin; Exatecan; T-2513 (10- (3-aminopropyloxy) -7-ethylcamptothecin); 10,11-methylenedioxycamptothecin; 7-ethyl-10,11-methylenedioxycamptothecin; 9-amino -10,11-methylenedioxycamptothecin; 9-chloro-10,11-methylenedioxycamptothecin; 7- (4-methyl-1-piperazinyl) methyl-10,11-methylenedioxycamptothecin; 10,11-eth Lurutetecan (7- (4-methyl-1-piperazinyl) methyl-10,11-ethylenedioxycamptothecin); gimatecan (7- (tert-butoxyiminomethyl) camptothecin); 9-aminocamptothecin; rubitecan ( 9-nitrocamptothecin); beotecan (7- (2- (N-isopropylamino) ethyl) -camptothecin); cositecan (7- (2- (trimethylsilyl) ethyl) -camptothecin) and silathecan (7- (tert-butyldimethyl) The alginic acid derivative according to any one of [1] to [3], selected from the group consisting of (silyl) -10-hydroxycamptothecin).
[5] The alginic acid derivative according to [4], wherein the camptothecin derivative is SN-38 (7-ethyl-10-hydroxycamptothecin), irinotecan (CPT-11), or topotecan (nogitecan).
[6] The alginic acid derivative according to [5], wherein the camptothecin derivative is SN-38 (7-ethyl-10-hydroxycamptothecin).
〔７〕カンプトテシン誘導体の２０位の３級アルコール性水酸基がリンカーと結合されてなる、又は、カンプトテシン誘導体がフェノール性水酸基を有し、前記フェノール性水酸基がリンカーと結合されてなる、前記〔１〕～〔６〕のいずれか１項に記載のアルギン酸誘導体。
〔７ａ〕カンプトテシン誘導体の２０位の３級アルコール性水酸基がリンカーと結合されてなる、前記〔７〕に記載のアルギン酸誘導体。
〔７ｂ〕カンプトテシン誘導体がフェノール性水酸基を有し、前記フェノール性水酸基がリンカーと結合されてなる、前記〔７〕に記載のアルギン酸誘導体。
〔７ｃ〕カンプトテシン誘導体が１０位にフェノール性水酸基を有し、前記フェノール性水酸基がリンカーと結合されてなる、前記〔７ｂ〕に記載のアルギン酸誘導体。
〔８〕前記〔１〕～〔７〕のいずれか１項に記載のアルギン酸誘導体を架橋してなる、アルギン酸誘導体ゲル。
〔８ａ〕前記〔１ａ〕、〔７ａ〕、〔７ｂ〕及び〔７ｃ〕のいずれか１項に記載のアルギン酸誘導体を架橋してなる、アルギン酸誘導体ゲル。
〔９〕前記〔１〕～〔７〕のいずれか１項に記載のアルギン酸誘導体、又は、前記〔８〕に記載のアルギン酸誘導体ゲルを含む、徐放性医薬組成物。
〔９ａ〕前記〔１ａ〕、〔７ａ〕、〔７ｂ〕及び〔７ｃ〕のいずれか１項に記載のアルギン酸誘導体、又は、前記〔８ａ〕に記載のアルギン酸誘導体ゲルを含む、徐放性医薬組成物。
〔１０〕抗癌剤としての、前記〔９〕に記載の徐放性医薬組成物。
〔１０ａ〕抗癌剤としての、前記〔９ａ〕に記載の徐放性医薬組成物。
〔１１〕カンプトテシン誘導体を徐放するための、前記〔１〕～〔７〕のいずれか１項に記載のアルギン酸誘導体、又は、前記〔８〕に記載のアルギン酸誘導体ゲルの使用。
〔１１ａ〕カンプトテシン誘導体を徐放するための、前記〔１ａ〕、〔７ａ〕、〔７ｂ〕及び〔７ｃ〕のいずれか１項に記載のアルギン酸誘導体、又は、前記〔８ａ〕に記載のアルギン酸誘導体ゲルの使用。 [7] The tertiary alcoholic hydroxyl group at position 20 of the camptothecin derivative is bonded to a linker, or the camptothecin derivative has a phenolic hydroxyl group, and the phenolic hydroxyl group is bonded to a linker. [1] The alginic acid derivative according to any one of [6] to [6].
[7a] The alginic acid derivative according to [7] above, wherein the tertiary alcoholic hydroxyl group at the 20-position of the camptothecin derivative is bonded to a linker.
[7b] The alginic acid derivative according to [7], wherein the camptothecin derivative has a phenolic hydroxyl group, and the phenolic hydroxyl group is bonded to a linker.
[7c] The alginic acid derivative according to [7b] above, wherein the camptothecin derivative has a phenolic hydroxyl group at the 10-position, and the phenolic hydroxyl group is bonded to a linker.
[8] An alginic acid derivative gel obtained by crosslinking the alginic acid derivative according to any one of [1] to [7].
[8a] An alginic acid derivative gel obtained by crosslinking the alginic acid derivative according to any one of [1a], [7a], [7b] and [7c].
[9] A sustained-release pharmaceutical composition comprising the alginic acid derivative according to any one of [1] to [7] or the alginic acid derivative gel according to [8].
[9a] A sustained-release pharmaceutical composition comprising the alginic acid derivative according to any one of [1a], [7a], [7b] and [7c] or the alginic acid derivative gel according to [8a]. object.
[10] The sustained release pharmaceutical composition according to [9] above as an anticancer agent.
[10a] The sustained-release pharmaceutical composition according to [9a] as an anticancer agent.
[11] Use of the alginic acid derivative according to any one of [1] to [7] or the alginic acid derivative gel according to [8] for the sustained release of a camptothecin derivative.
[11a] The alginic acid derivative according to any one of the above [1a], [7a], [7b] and [7c] or the alginic acid derivative according to [8a] for the sustained release of the camptothecin derivative Use of gel.
　本発明は、安定的にカンプトテシン誘導体を持続放出することにより、徐放性製剤に使用し得る化合物を提供できる。また、ゲル化させることにより、その化合物の徐放性をさらに高めることが可能な化合物を提供できる。 The present invention can provide a compound that can be used in a sustained-release preparation by stably releasing a camptothecin derivative stably. Moreover, the compound which can further improve the sustained-release property of the compound can be provided by making it gelatinize.
　＜アルギン酸誘導体＞
　本発明は、アルギン酸又はその塩とカンプトテシン誘導体とがリンカーを介して共有結合されてなる構造を有する、アルギン酸誘導体に関する。リンカーは、アルギン酸又はその塩のカルボキシル基、及び、カンプトテシン誘導体の水酸基と、共有結合により結合されることが好ましい。結合様式は本発明の目的が達成される限り特に限定されないが、アルギン酸誘導体において、アルギン酸とリンカーとの結合はアミド結合であり、カンプトテシン誘導体とリンカーとの結合はエステル結合であることが好ましい。アルギン酸又はその塩における、リンカーとの結合部位（アルギン酸又はその塩の官能基）は、水酸基又はカルボキシル基が挙げられるが、アミド結合を形成しうるカルボキシル基がより好ましい。 <Alginic acid derivative>
The present invention relates to an alginic acid derivative having a structure in which alginic acid or a salt thereof and a camptothecin derivative are covalently bonded via a linker. The linker is preferably covalently bonded to the carboxyl group of alginic acid or a salt thereof and the hydroxyl group of the camptothecin derivative. The binding mode is not particularly limited as long as the object of the present invention is achieved. In the alginic acid derivative, the bond between alginic acid and the linker is an amide bond, and the bond between the camptothecin derivative and the linker is preferably an ester bond. Examples of the binding site (functional group of alginic acid or a salt thereof) in alginic acid or a salt thereof include a hydroxyl group or a carboxyl group, but a carboxyl group capable of forming an amide bond is more preferable.
　したがって、本発明の一形態としては、アルギン酸誘導体は、下記式（１）で表される構造を有する：
（Ａ）－Ｌ－（Ｄ）　　　（１）
　式（１）中、（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示す。また、式（１）中、Ｌは、（Ａ）とアミド結合で結合しうる官能基を有し、かつ（Ｄ）とエステル結合で結合しうる官能基を有するリンカーである。また、式（１）中、（Ｄ）は、カンプトテシン誘導体の水酸基から水素原子を除いた基を示すが、当該（Ｄ）の水酸基は、カンプトテシン骨格の２０位の３級アルコール性水酸基であってもよいし、任意の位置に置換導入されている水酸基であってもよく、好ましくは、２０位の３級アルコール性水酸基又は任意の位置のフェノール性水酸基であり、より好ましくは２０位の３級アルコール性水酸基又は１０位のフェノール性水酸基であり、更に好ましくは１０位のフェノール性水酸基である。 Therefore, as one embodiment of the present invention, the alginic acid derivative has a structure represented by the following formula (1):
(A) -L- (D) (1)
In formula (1), (A) is a residue derived from alginic acid or a salt thereof, and is a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. 1 residue having In Formula (1), L is a linker having a functional group that can be bonded to (A) by an amide bond and a functional group that can be bonded to (D) by an ester bond. In formula (1), (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative, and the hydroxyl group of (D) is a tertiary alcoholic hydroxyl group at the 20th position of the camptothecin skeleton. Or a hydroxyl group substituted and introduced at an arbitrary position, preferably a tertiary alcoholic hydroxyl group at the 20th position or a phenolic hydroxyl group at an arbitrary position, more preferably a tertiary hydroxyl group at the 20th position. It is an alcoholic hydroxyl group or a 10-position phenolic hydroxyl group, more preferably a 10-position phenolic hydroxyl group.
　さらに、本発明の一形態としては、アルギン酸誘導体は、下記式（２）で表される構造を有する：
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］n2－（ＣＲ1Ｒ2）n3－［Ｙ］n4－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］n7－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（２）
　式（２）中では、（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、Ｘ1及びＸ2は、置換基を有しても良いヘテロ原子を示し、Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5及びＲ6はそれぞれ独立して、水素、ハロゲン原子、Ｃ1-10アルキル基、Ｃ1-10アルコキシ基又はＣ1-10アルコキシカルボニル基を示すか、又は、Ｒ1及びＲ2若しくはＲ3及びＲ4が一緒になって＝Ｏを示し、Ｙは、シクロアルカン環、芳香族環又は複素環（前記シクロアルカン環、芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、ｎ１は０～１０のいずれかの整数を示し、ｎ２、ｎ４及びｎ７は独立して０または１を示し、ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ１～ｎ９の全てが０になることはない。ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は合計して１～１２が好ましく、１～１０がより好ましい。 Furthermore, as one embodiment of the present invention, the alginic acid derivative has a structure represented by the following formula (2):
(A) —NH— (CH 2 ) n1 — [X 1 ] n2 — (CR 1 R 2 ) n3 — [Y] n4 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 - (CH 2) n8 - ( CR 5 R 6) n9 -C (= O) - (D) (2)
In the formula (2), (A) is a residue derived from alginic acid or a salt thereof, and C (═O) — of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. 1 represents a residue having a group, (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative, X 1 and X 2 represent a hetero atom which may have a substituent, R 1 , Each of R 2 , R 3 , R 4 , R 5 and R 6 independently represents hydrogen, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group; Or R 1 and R 2 or R 3 and R 4 together represent ═O, Y represents a cycloalkane ring, an aromatic ring or a heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring is a halogen in atomic or C 1-10 alkyl group may be substituted), n1 is 0 Any one of 10 integers, n2, n4 and n7 independently represent 0 or 1, n3, n5, n6, n8 and n9 independently represent any integer of 0 to 3, All of .about.n9 are never 0. n3, n5, n6, n8 and n9 in total are preferably 1 to 12, and more preferably 1 to 10.
　式（２）のうち、好ましいアルギン酸誘導体は、下記式（３）～（７）、（７－１）及び（７－２）で表される構造を有する：
（Ａ）－ＮＨ－（ＣＨ2）n1－（ＣＲ1Ｒ2）n3－（ＣＨ2）n5－Ｃ（＝Ｏ）－（Ｄ）　　　（３）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｒ1及びＲ2は、Ｒ1が水素若しくはハロゲン原子を示し、Ｒ2が、水素、ハロゲン原子、メチル基若しくはエチル基を示すか、或いは、Ｒ1及びＲ2が一緒になって＝Ｏを示し、
　ｎ１、ｎ３及びｎ５は独立して０～３のいずれかの整数を示すが、ｎ１、ｎ３及びｎ５は合計して１～４のいずれかの整数を示す）。 Of the formula (2), preferred alginic acid derivatives have structures represented by the following formulas (3) to (7), (7-1) and (7-2):
(A) —NH— (CH 2 ) n1 — (CR 1 R 2 ) n3 — (CH 2 ) n5 —C (═O) — (D) (3)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
R 1 and R 2 are either R 1 represents hydrogen or a halogen atom, R 2 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 1 and R 2 together represent ═O. Show
n1, n3 and n5 each independently represents an integer of 0 to 3, but n1, n3 and n5 in total represent any integer of 1 to 4).
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］－（ＣＲ1Ｒ2）n3－（ＣＲ3Ｒ4）n6－［Ｘ2］－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（４）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｘ1及びＸ2は、それぞれ独立してＯまたはＮＨを示し、好ましくは共にＯであり、
　Ｒ1及びＲ2は、Ｒ1が水素を示し、Ｒ2が、水素、ハロゲン原子、メチル基若しくはエチル基を示すか、或いは、Ｒ1及びＲ2が一緒になって＝Ｏを示し、
　Ｘ1がＯの場合は、Ｒ1は水素が好ましく、Ｒ2は水素、メチル基又はエチル基が好ましく、
　Ｘ1がＮＨの場合は、Ｒ1は水素が好ましく、Ｒ2は水素、メチル基又はエチル基が好ましく、または、Ｒ1及びＲ2が一緒になって＝Ｏを示すのが好ましく、
　Ｒ3及びＲ4は、Ｒ3が水素を示し、Ｒ4が、水素、ハロゲン原子、メチル基若しくはエチル基を示すか、或いは、Ｒ3及びＲ4が一緒になって＝Ｏを示し、
　Ｒ5及びＲ6は、Ｒ5が水素を示し、Ｒ6が、水素、ハロゲン原子、メチル基若しくはエチル基を示し、
　ｎ１は１～３のいずれかの整数を示し、ｎ３、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ３及びｎ６は合計して１～３のいずれかの整数を示し、ｎ８及びｎ９は合計して１～３のいずれかの整数を示し、好ましくはｎ１は２、ｎ３及びｎ６は合計して２、ｎ８及びｎ９は合計して１である）。 (A) —NH— (CH 2 ) n1 — [X 1 ] — (CR 1 R 2 ) n3 — (CR 3 R 4 ) n6 — [X 2 ] — (CH 2 ) n8 — (CR 5 R 6 ) n9 -C (= O)-(D) (4)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
X 1 and X 2 each independently represent O or NH, preferably both O,
R 1 and R 2 are those in which R 1 represents hydrogen and R 2 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 1 and R 2 together represent ═O,
When X 1 is O, R 1 is preferably hydrogen, R 2 is preferably hydrogen, methyl group or ethyl group,
When X 1 is NH, R 1 is preferably hydrogen, R 2 is preferably hydrogen, a methyl group or an ethyl group, or R 1 and R 2 together are preferably ═O,
R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ═O,
R 5 and R 6 are as follows: R 5 represents hydrogen, R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group,
n1 represents any integer of 1 to 3, n3, n6, n8 and n9 independently represent any integer of 0 to 3, but n3 and n6 are any one of 1 to 3 in total And n8 and n9 represent any integer of 1 to 3, and preferably n1 is 2, n3 and n6 are 2, and n8 and n9 are 1).
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｙ］－（ＣＨ2）n5－Ｃ（＝Ｏ）－（Ｄ）　　　（５）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｙは、シクロアルカン環を示し、好ましくはシクロヘキサンであり、
　ｎ１及びｎ５は独立して０～３のいずれかの整数を示すが、ｎ１及びｎ５は合計して１～４のいずれかの整数を示す）。 (A) —NH— (CH 2 ) n1 — [Y] — (CH 2 ) n5 —C (═O) — (D) (5)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
Y represents a cycloalkane ring, preferably cyclohexane,
n1 and n5 independently represent any integer from 0 to 3, but n1 and n5 represent any integer from 1 to 4 in total).
（Ａ）－ＮＨ－（ＣＨ2）n1－（ＣＲ1Ｒ2）n3－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－（ＣＨ2）n8－Ｃ（＝Ｏ）－（Ｄ）　　　（６）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｒ1及びＲ2は、Ｒ1が水素を示し、Ｒ2が、水素、メトキシ基、エトキシ基、メトキシカルボニル基またはエトキシカルボニル基を示すか、或いは、Ｒ1及びＲ2が一緒になって＝Ｏを示し、
　Ｒ3及びＲ4は、Ｒ3が水素を示し、Ｒ4が、水素、メチル基若しくはエチル基を示すか、或いは、Ｒ3及びＲ4が一緒になって＝Ｏを示し、
　ｎ１、ｎ３、ｎ５、ｎ６及びｎ８は独立して０～３のいずれかの整数を示すが、ｎ１、ｎ３、ｎ５、ｎ６及びｎ８は合計して１～４のいずれかの整数を示す）。 (A) —NH— (CH 2 ) n1 — (CR 1 R 2 ) n3 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — (CH 2 ) n8 —C (═O) — (D) ( 6)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
R 1 and R 2 are those in which R 1 represents hydrogen and R 2 represents hydrogen, methoxy group, ethoxy group, methoxycarbonyl group or ethoxycarbonyl group, or R 1 and R 2 together represent O
R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a methyl group or an ethyl group, or R 3 and R 4 together represent ═O,
n1, n3, n5, n6 and n8 independently represent any integer of 0 to 3, but n1, n3, n5, n6 and n8 represent any integer of 1 to 4 in total).
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］－［Ｙ］－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］n7－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（７）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｘ1及びＸ2は、それぞれ独立してＯ、ＮＨまたはＮＲ7を示し、好ましくはＸ1はＯであり、
　Ｒ3及びＲ4は、Ｒ3が水素を示し、Ｒ4が、水素、ハロゲン原子、メチル基若しくはエチル基を示すか、或いは、Ｒ3及びＲ4が一緒になって＝Ｏを示し、
　Ｘ2がＯの場合は、Ｒ4は水素、メチル基又はエチル基が好ましく、
　Ｘ2がＮＨまたはＮＲ7の場合は、Ｒ4は水素、メチル基又はエチル基が好ましく、または、Ｒ3及びＲ4が一緒になって＝Ｏを示すのが好ましく、
　Ｒ5及びＲ6は、Ｒ5が水素を示し、Ｒ6が、水素、ハロゲン原子、メチル基若しくはエチル基を示し、
　Ｒ7は、Ｃ1-6アルキル基、Ｃ2-7アルカノイル基又はＣ1-6アルキルスルホニル基を示し、好ましくは、メチル基、エチル基、アセチル基又はメチルスルホニル基であり、より好ましくはアセチル基であり、
　Ｙは、芳香族環又は複素環（前記芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、好ましくはベンゼン環又は単環式の複素環であり、より好ましくはベンゼン環又はピリジン環であり、
　ｎ１は１～１０のいずれかの整数を示し、好ましくは１～３のいずれかの整数であり、より好ましくは２であり、
　ｎ７は０または１を示し、
　ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ５、ｎ６、ｎ８及びｎ９は合計して０～３が好ましく、
　ｎ７が０の場合は、好ましくは、ｎ５及びｎ６が０であり、ｎ８及びｎ９は合計して０～３であり、より好ましくはｎ５、ｎ６及びｎ９が０であり、ｎ８が０～２であり、
　ｎ７が１であり、Ｘ2がＯの場合は、ｎ５及びｎ６は合計して０又は１が好ましく、より好ましくは共に０であり、
　ｎ７が１であり、Ｘ2がＮＨまたはＮＲ7の場合は、ｎ５及びｎ６は合計して１又は２が好ましく、より好ましくは合計して１であり、
　ｎ７が１の場合は、ｎ８及びｎ９は合計して１～３を示すのが好ましく、より好ましくはｎ８が１又は２、ｎ９が０であり、更に好ましくはｎ８が１、ｎ９が０である）。 (A) —NH— (CH 2 ) n1 — [X 1 ] — [Y] — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 — (CH 2 ) n8 — (CR 5 R 6 ) n9 —C (═O) — (D) (7)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
X 1 and X 2 each independently represent O, NH or NR 7 , preferably X 1 is O;
R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ═O,
When X 2 is O, R 4 is preferably hydrogen, methyl group or ethyl group,
When X 2 is NH or NR 7 , R 4 is preferably hydrogen, a methyl group or an ethyl group, or R 3 and R 4 together are preferably ═O,
R 5 and R 6 are as follows: R 5 represents hydrogen, R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group,
R 7 represents a C 1-6 alkyl group, a C 2-7 alkanoyl group or a C 1-6 alkylsulfonyl group, preferably a methyl group, an ethyl group, an acetyl group or a methylsulfonyl group, more preferably an acetyl group. Group,
Y represents an aromatic ring or a heterocyclic ring (the aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group), preferably a benzene ring or a monocyclic heterocyclic ring. More preferably a benzene ring or a pyridine ring,
n1 represents an integer of 1 to 10, preferably an integer of 1 to 3, more preferably 2,
n7 represents 0 or 1,
n5, n6, n8 and n9 independently represent an integer of 0 to 3, but n5, n6, n8 and n9 are preferably 0 to 3 in total,
When n7 is 0, preferably n5 and n6 are 0, n8 and n9 are 0 to 3 in total, more preferably n5, n6 and n9 are 0, and n8 is 0 to 2 Yes,
When n7 is 1 and X 2 is O, n5 and n6 are preferably 0 or 1 in total, more preferably both are 0,
When n7 is 1 and X 2 is NH or NR 7 , n5 and n6 are preferably 1 or 2 in total, more preferably 1 in total,
When n7 is 1, n8 and n9 preferably represent 1 to 3 in total, more preferably n8 is 1 or 2, n9 is 0, and still more preferably n8 is 1 and n9 is 0. ).
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］－［Ｙ］－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（７－１）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｘ1は、ＯまたはＮＨを示し、好ましくはＯであり、
　Ｒ5及びＲ6は、Ｒ5が水素を示し、Ｒ6が、水素、ハロゲン原子、メチル基若しくはエチル基を示し、
　Ｙは、芳香族環又は複素環（前記芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、好ましくはベンゼン環又は単環式の複素環であり、より好ましくはベンゼン環又はピリジン環であり、
　ｎ１は１～１０のいずれかの整数を示し、好ましくは１～３のいずれかの整数であり、より好ましくは２であり、
　ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ８及びｎ９は合計して０～３が好ましく、より好ましくはｎ８が０～２であり、ｎ９が０である）。 (A) -NH- (CH 2) n1 - [X 1] - [Y] - (CH 2) n8 - (CR 5 R 6) n9 -C (= O) - (D) (7-1)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
X 1 represents O or NH, preferably O,
R 5 and R 6 are as follows: R 5 represents hydrogen, R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group,
Y represents an aromatic ring or a heterocyclic ring (the aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group), preferably a benzene ring or a monocyclic heterocyclic ring. More preferably a benzene ring or a pyridine ring,
n1 represents an integer of 1 to 10, preferably an integer of 1 to 3, more preferably 2,
n8 and n9 each independently represents an integer of 0 to 3, but n8 and n9 are preferably 0 to 3 in total, more preferably n8 is 0 to 2 and n9 is 0).
（Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］－［Ｙ］－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（７－２）
（式中、
　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
　Ｘ1及びＸ2は、それぞれ独立してＯ、ＮＨまたはＮＲ7を示し、好ましくはＸ1はＯであり、
　Ｒ3及びＲ4は、Ｒ3が水素を示し、Ｒ4が、水素、ハロゲン原子、メチル基若しくはエチル基を示すか、或いは、Ｒ3及びＲ4が一緒になって＝Ｏを示し、
　Ｘ2がＯの場合は、Ｒ4は水素、メチル基又はエチル基が好ましく、
　Ｘ2がＮＨまたはＮＲ7の場合は、Ｒ4は水素、メチル基又はエチル基が好ましく、または、Ｒ3及びＲ4が一緒になって＝Ｏを示すのが好ましく、
　Ｒ5及びＲ6は、Ｒ5が水素を示し、Ｒ6が、水素、ハロゲン原子、メチル基若しくはエチル基を示し、
　Ｒ7は、Ｃ1-6アルキル基、Ｃ2-7アルカノイル基又はＣ1-6アルキルスルホニル基を示し、好ましくは、メチル基、エチル基、アセチル基又はメチルスルホニル基であり、より好ましくはアセチル基であり、
　Ｙは、芳香族環又は複素環（前記芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、好ましくはベンゼン環又は単環式の複素環であり、より好ましくはベンゼン環又はピリジン環であり、
　ｎ１は１～１０のいずれかの整数を示し、好ましくは１～３のいずれかの整数であり、より好ましくは２であり、
　ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示し、
　Ｘ2がＯの場合は、ｎ５及びｎ６は合計して０又は１が好ましく、より好ましくは共に０であり、
　Ｘ2がＮＨまたはＮＲ7の場合は、ｎ５及びｎ６は合計して１又は２が好ましく、より好ましくは合計して１であり、
　ｎ８及びｎ９は合計して１～３が好ましく、より好ましくはｎ８が１又は２、ｎ９が０であり、更に好ましくはｎ８が１、ｎ９が０である）。
　なお、本発明のアルギン酸誘導体のリンカー構造については、≪リンカー≫の項で後述する。 (A) -NH- (CH 2) n1 - [X 1] - [Y] - (CH 2) n5 - (CR 3 R 4) n6 - [X 2] - (CH 2) n8 - (CR 5 R 6 ) n9 -C (= O)-(D) (7-2)
(Where
(A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
(D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
X 1 and X 2 each independently represent O, NH or NR 7 , preferably X 1 is O;
R 3 and R 4 are those in which R 3 represents hydrogen and R 4 represents hydrogen, a halogen atom, a methyl group or an ethyl group, or R 3 and R 4 together represent ═O,
When X 2 is O, R 4 is preferably hydrogen, methyl group or ethyl group,
When X 2 is NH or NR 7 , R 4 is preferably hydrogen, a methyl group or an ethyl group, or R 3 and R 4 together are preferably ═O,
R 5 and R 6 are as follows: R 5 represents hydrogen, R 6 represents hydrogen, a halogen atom, a methyl group or an ethyl group,
R 7 represents a C 1-6 alkyl group, a C 2-7 alkanoyl group or a C 1-6 alkylsulfonyl group, preferably a methyl group, an ethyl group, an acetyl group or a methylsulfonyl group, more preferably an acetyl group. Group,
Y represents an aromatic ring or a heterocyclic ring (the aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group), preferably a benzene ring or a monocyclic heterocyclic ring. More preferably a benzene ring or a pyridine ring,
n1 represents an integer of 1 to 10, preferably an integer of 1 to 3, more preferably 2,
n5, n6, n8 and n9 each independently represents an integer of 0 to 3,
When X 2 is O, n5 and n6 are preferably 0 or 1, and more preferably both are 0,
When X 2 is NH or NR 7 , n5 and n6 are preferably 1 or 2 in total, more preferably 1 in total,
n8 and n9 are preferably 1 to 3 in total, more preferably n8 is 1 or 2, n9 is 0, and still more preferably n8 is 1 and n9 is 0.
The linker structure of the alginic acid derivative of the present invention will be described later in the section “Linker”.
　また、本発明のアルギン酸誘導体において、体内において副作用を生じにくく、適切に薬理作用を発現し得る濃度で、例えば腫瘍の減少や増殖抑制し得る濃度で、カンプトテシン誘導体を放出させ続けることができるカンプトテシン誘導体の導入率であることが好ましい。例えば導入率（モル％）が、０．２モル％以上であることが好ましい。より好ましくは０．５モル％以上、さらに好ましくは１．０モル％以上、特に好ましくは２．０モル％以上である。本発明のアルギン酸誘導体においては、当該カンプトテシン誘導体の導入率を１０モル％にまで、より好ましくは１５モル％にまで高めることができる。ここで、本発明における導入率（モル％）とは、例えば、アルギン酸を構成するＬ－グルロン酸又はＤ－マンヌロン酸のカルボキシル基にリンカーを介してカンプトテシン誘導体を導入する場合において、導入率１０モル％とは、アルギン酸を構成するＬ－グルロン酸又はＤ－マンヌロン酸の単糖を１単位（個）とし、単糖１００個にカンプトテシン誘導体が１０個の割合で導入されていることを示す。したがって、隣り合う単糖各々のカルボキシル基にそれぞれカンプトテシン誘導体がリンカーを介して導入されていてもかまわない。
　リンカーの種類およびカンプトテシン誘導体の導入率は、後述する当該化合物を含む医薬組成物の最終投与形態（ゲル状、ゾル状、マイクロビーズ状など）、あるいは生体に投与するときのカンプトテシン誘導体の患部（腫瘍局所）における必要量あるいは徐放効率などを考慮して適宜調整されうる。 Further, in the alginic acid derivative of the present invention, a camptothecin derivative that can keep releasing a camptothecin derivative at a concentration that is less likely to cause side effects in the body and that can appropriately exhibit a pharmacological action, for example, a concentration that can suppress tumor reduction or growth. The introduction rate is preferably. For example, the introduction rate (mol%) is preferably 0.2 mol% or more. More preferably, it is 0.5 mol% or more, More preferably, it is 1.0 mol% or more, Most preferably, it is 2.0 mol% or more. In the alginic acid derivative of the present invention, the introduction rate of the camptothecin derivative can be increased to 10 mol%, more preferably 15 mol%. Here, the introduction rate (mol%) in the present invention refers to, for example, an introduction rate of 10 mol when a camptothecin derivative is introduced into the carboxyl group of L-guluronic acid or D-mannuronic acid constituting alginic acid via a linker. % Indicates that one unit (piece) of monosaccharide of L-guluronic acid or D-mannuronic acid constituting alginic acid is introduced, and camptothecin derivative is introduced at a ratio of 10 to 100 monosaccharides. Therefore, a camptothecin derivative may be introduced to each carboxyl group of adjacent monosaccharides via a linker.
The type of linker and the introduction rate of the camptothecin derivative are determined depending on the final administration form (gel, sol, microbead, etc.) of the pharmaceutical composition containing the compound described later, or the affected part of the camptothecin derivative (tumor) The amount may be adjusted as appropriate in consideration of the necessary amount in local) or sustained release efficiency.
　ここで、本発明のアルギン酸誘導体は、カンプトテシン誘導体を含む高分子化合物であるが、水溶性であることに特徴を有する。すなわち、一般に疎水性であると知られているカンプトテシン誘導体のアルギン酸誘導体における導入率が高い場合であっても、例えば３モル％以上であっても、水に溶解可能である。例えば、水１００質量部に対し、アルギン酸誘導体を０．１質量部添加し、室温にて振とう又は撹拌した場合、２４時間以内にゲル状にならず溶解することが示され、すなわち、アルギン酸誘導体は０．１％以上の濃度で水性溶媒に溶解することが示される。
　また、本発明における水溶性であるアルギン酸誘導体は、後述する用途に応じたゲル化又はゾル化のハンドリングが容易であるとの利点がある。よって、本発明のアルギン酸誘導体の溶液はフィルター濾過が可能であり、フィルター濾過による除塵、除菌、滅菌が可能となる。すなわち、５μｍ～０．４５μｍのフィルターを通過させることにより除塵、除菌が可能となり、更に望ましくは０．２２μｍのフィルターを通過させることにより滅菌することも可能となる。
　なお、本発明の水溶性であるアルギン酸誘導体は、水、薬学的に許容される金属塩若しくはｐＨ調整剤等を含む水溶液、緩衝液等の水性溶媒に溶解可能である。具体的には注射用水、リン酸緩衝生理食塩水、生理食塩水等に溶解可能である。 Here, the alginic acid derivative of the present invention is a polymer compound containing a camptothecin derivative, but is characterized by being water-soluble. That is, even when the introduction rate of the camptothecin derivative, which is generally known to be hydrophobic, in the alginic acid derivative is high, for example, 3 mol% or more, it can be dissolved in water. For example, when 0.1 part by mass of an alginic acid derivative is added to 100 parts by mass of water and shaken or stirred at room temperature, it is shown that it dissolves without becoming a gel within 24 hours. Is soluble in aqueous solvents at concentrations of 0.1% and above.
Further, the water-soluble alginic acid derivative in the present invention has an advantage that it is easy to handle gelation or solification according to the use described later. Therefore, the solution of the alginic acid derivative of the present invention can be filtered, and dust removal, sterilization, and sterilization can be performed by filter filtration. In other words, dust removal and sterilization can be performed by passing through a 5 μm to 0.45 μm filter, and more preferably, sterilization can be performed by passing through a 0.22 μm filter.
The water-soluble alginic acid derivative of the present invention can be dissolved in water, an aqueous solution containing a pharmaceutically acceptable metal salt or a pH adjuster, or an aqueous solvent such as a buffer solution. Specifically, it can be dissolved in water for injection, phosphate buffered saline, physiological saline and the like.
　また、本発明におけるアルギン酸誘導体は、単独ではカンプトテシン誘導体が有する強力な抗癌効果をもたらさないが、例えばそれを生体内に投与した場合において、生体内の状況に応じてリンカーからカンプトテシン誘導体が適宜切断されることにより、カンプトテシン誘導体が放出され、効果を発揮する。カンプトテシン誘導体が腫瘍の減少や増殖抑制するのに必要な量が放出し続けるため、結果として患部（腫瘍局所）で抗癌効果をもたらすことができる。アルギン酸誘導体は、その構成成分であるリンカーの構造によって、カンプトテシン誘導体の徐放速度を所望の態様に調整することができる。そのような調整に加え、求める効果に応じて、カンプトテシン誘導体の導入率とリンカーの種類との組み合わせを最適化することで、生体内に注射した場合、例えば腹腔内注射した場合、代謝・排泄の影響を受けにくくなり、腹腔内にて長期持続可能な抗癌作用をもたらしうる。
　また、アルギン酸は生体内の酵素によって分解されないため、本発明におけるアルギン酸誘導体は、リンカー部位の切断以外の要因では、カンプトテシン誘導体の放出速度に影響を受けにくく、安定的に有効成分を持続放出しうる。 In addition, the alginic acid derivative in the present invention alone does not bring about the strong anticancer effect of the camptothecin derivative, but when it is administered in vivo, for example, the camptothecin derivative is appropriately cleaved from the linker depending on the situation in the living body. By doing so, the camptothecin derivative is released and exerts an effect. Since the camptothecin derivative is continuously released in an amount necessary for tumor reduction and growth inhibition, an anticancer effect can be brought about at the affected area (tumor local area). In the alginic acid derivative, the sustained release rate of the camptothecin derivative can be adjusted to a desired mode depending on the structure of the linker which is a constituent component. In addition to such adjustments, by optimizing the combination of the camptothecin derivative introduction rate and the type of linker according to the desired effect, when injected into a living body, for example, when injected intraperitoneally, metabolism / excretion Can be less affected and can have long-lasting anticancer effects in the abdominal cavity.
In addition, since alginic acid is not decomposed by enzymes in the living body, the alginic acid derivative in the present invention is less affected by the release rate of the camptothecin derivative and can stably and continuously release the active ingredient due to factors other than the cleavage of the linker site. .
　本発明のアルギン酸誘導体において、アルギン酸又はその塩とリンカーとの結合様式とカンプトテシン誘導体とリンカーとの結合様式を変えることにより、生体内における分解性や分解順序を変えることができ、その結果、カンプトテシン誘導体の遊離率や遊離速度を制御することも可能となる。具体的には、生体内において、アミド結合よりもエステル結合は加水分解を受けやすいことが知られている。本発明のアルギン酸誘導体は、最終的にカンプトテシン誘導体が遊離されれば、その分解順序は問わないが、カンプトテシン誘導体とリンカーの結合が先に加水分解を受け、カンプトテシン誘導体が遊離することが好ましい。具体的には、アルギン酸又はその塩とリンカーはアミド結合で結合し、カンプトテシン誘導体とリンカーはエステル結合で結合することで、エステル結合が先に加水分解を受け、カンプトテシン誘導体がリンカーから先に遊離する。
　また、アルギン酸は、適用した生体に悪影響を及ぼさず、生体内においてアルギン酸と結合する特定の受容体は同定されていないことから、カンプトテシン誘導体を放出した後のアルギン酸又はその塩は体内において毒性をもたらさずに分解される。 In the alginic acid derivative of the present invention, by changing the binding mode of alginic acid or a salt thereof and a linker and the binding mode of a camptothecin derivative and a linker, degradability and degradation order in vivo can be changed. As a result, the camptothecin derivative It is also possible to control the release rate and release rate. Specifically, it is known that ester bonds are more susceptible to hydrolysis than amide bonds in vivo. The alginic acid derivative of the present invention may be decomposed in any order as long as the camptothecin derivative is finally released. However, it is preferable that the camptothecin derivative and the linker are first subjected to hydrolysis to release the camptothecin derivative. Specifically, alginic acid or a salt thereof and a linker are bonded by an amide bond, and the camptothecin derivative and the linker are bonded by an ester bond, whereby the ester bond is first hydrolyzed, and the camptothecin derivative is first released from the linker. .
In addition, alginic acid does not adversely affect the applied organism, and since no specific receptor that binds to alginic acid in vivo has been identified, alginic acid or its salt after releasing the camptothecin derivative is toxic in the body. Without being disassembled.
　本発明のアルギン酸誘導体は、中性の条件下で徐放が期待される局面では、ゆっくり遊離されることが好ましい。例えば、本発明のアルギン酸誘導体を０．１重量％濃度水溶液に調製し、３７℃で３日間インキュベートした場合において、カンプトテシン誘導体が、ｐＨ７．０では遊離率０．１％～３５％、好ましくは１％～３０％、より好ましくは４％～２５％で放出される挙動を示すことが好ましい。あるいは、本発明のアルギン酸誘導体を０．０１重量％濃度水溶液に調製し、３７℃で３日間インキュベートした場合において、カンプトテシン誘導体が、ｐＨ７．０では遊離率０．１％～６０％で放出される挙動を示すことが好ましく、より好ましくは１％～４０％、更に好ましくは３％～３５％、特に好ましくは５～３０％で放出される挙動を示すことが好ましい。
　また、本発明のアルギン酸誘導体は、中性の条件下でさらに長期の徐放が期待される局面では、さらにゆっくりと遊離されることが好ましい。例えば、本発明のアルギン酸誘導体を０．０１重量％濃度水溶液に調製し、３７℃、ｐＨ７．０でインキュベートした場合において、カンプトテシン誘導体が、７日間で遊離率０．１％～３０％で放出される挙動を示すことが好ましく、より好ましくは１％～２５％、更に好ましくは３％～２０％で放出される挙動を示すことが好ましい。
　一方で、本発明のアルギン酸誘導体は、中性の条件下で速やかな放出が期待される局面では、迅速に遊離されることが好ましい。例えば、本発明のアルギン酸誘導体を０．０１重量％濃度水溶液に調製し、３７℃、ｐＨ７．０でインキュベートした場合において、カンプトテシン誘導体が、１日間で遊離率２０％～１００％で放出される挙動を示すことが好ましく、より好ましくは３０％～１００％、更に好ましくは５０％～１００％、特に好ましくは７０～１００％で放出される挙動を示すことが好ましい。 The alginic acid derivative of the present invention is preferably released slowly in a situation where sustained release is expected under neutral conditions. For example, when the alginic acid derivative of the present invention is prepared in a 0.1% by weight aqueous solution and incubated at 37 ° C. for 3 days, the camptothecin derivative has a liberation rate of 0.1% to 35% at pH 7.0, preferably 1 It is preferable to exhibit the behavior of being released at% to 30%, more preferably 4% to 25%. Alternatively, when the alginic acid derivative of the present invention is prepared in a 0.01% by weight aqueous solution and incubated at 37 ° C. for 3 days, the camptothecin derivative is released at a release rate of 0.1% to 60% at pH 7.0. It is preferable to exhibit a behavior, more preferably 1% to 40%, still more preferably 3% to 35%, and particularly preferably 5 to 30%.
In addition, the alginic acid derivative of the present invention is preferably released more slowly in a situation where longer-term sustained release is expected under neutral conditions. For example, when the alginic acid derivative of the present invention is prepared in a 0.01% by weight aqueous solution and incubated at 37 ° C. and pH 7.0, the camptothecin derivative is released at a release rate of 0.1% to 30% in 7 days. It is preferable to exhibit a behavior that is released at 1% to 25%, more preferably 3% to 20%.
On the other hand, the alginic acid derivative of the present invention is preferably released quickly in a situation where rapid release is expected under neutral conditions. For example, when an alginic acid derivative of the present invention is prepared in a 0.01% by weight aqueous solution and incubated at 37 ° C. and pH 7.0, the camptothecin derivative is released at a release rate of 20% to 100% in one day. It is preferable to exhibit a behavior of being released at 30% to 100%, more preferably 50% to 100%, particularly preferably 70 to 100%.
　同様に、使用する環境のｐＨに対応した条件下にて遊離率の測定を行なうことで、適する本発明の各アルギン酸誘導体を選択することができる。
　また、本発明のアルギン酸誘導体を架橋剤によってゲル化させることにより、徐放効果をさらに強めることも可能であり、アルギン酸誘導体の遊離率とゲル化の状態（ゲル化の強度、形態等）を調整することで、適した放出速度に調整することも可能である。
　このように、遊離率等を調整することで薬剤の放出期間を調整することが可能であり、例えば、本発明のアルギン酸誘導体を抗癌剤として患部（腫瘍局所、例えば腹腔内）に注射等により投与した際に、７日間以上、好ましくは１５日間以上、より好ましくは３０日間以上、更に好ましくは６０日間以上、カンプトテシン誘導体を徐放し続けることが期待される。また、速やかな放出が望ましい局面に合わせて、薬剤の放出期間を調整することも可能であり、例えば、本発明のアルギン酸誘導体を抗癌剤として患部に注射等により投与した際に、７日間以下、好ましくは３日間以下、より好ましくは２４時間以下で、カンプトテシン誘導体を徐放し続けることが期待される。
　ここで、遊離率とは、アルギン酸誘導体に含まれるカンプトテシン誘導体総量に対する、放出されたカンプトテシン誘導体量の比率を示す。
　本発明のアルギン酸誘導体を用いることにより、薬剤の単独投与よりも、腹腔内等の患部（腫瘍局所）又はその近縁部位に投与した場合において、患部（腫瘍局所）に有効な薬剤量が効率的に保持され、少ない薬剤の量であっても強力な治療効果が期待できる。また、徐放性および持続性の調整により、臨床において投与回数の減少等につなげることもできる。
　以下、本発明のアルギン酸誘導体の構成を説明するために、各構成成分である、アルギン酸、リンカー及びカンプトテシン誘導体について説明した後、アルギン酸誘導体ゲルやこれらの用途等について詳述する。 Similarly, a suitable alginate derivative of the present invention can be selected by measuring the liberation rate under conditions corresponding to the pH of the environment to be used.
Moreover, it is possible to further enhance the sustained release effect by gelling the alginic acid derivative of the present invention with a crosslinking agent, and adjusting the release rate of the alginic acid derivative and the gelation state (gelation strength, form, etc.). By doing so, it is possible to adjust to a suitable release rate.
In this way, it is possible to adjust the release period of the drug by adjusting the release rate and the like. For example, the alginic acid derivative of the present invention is administered as an anticancer agent by injection or the like to the affected area (tumor local area, for example, intraperitoneally) In this case, it is expected that the camptothecin derivative is continuously released for 7 days or more, preferably 15 days or more, more preferably 30 days or more, and further preferably 60 days or more. It is also possible to adjust the drug release period according to the situation where rapid release is desired. For example, when the alginic acid derivative of the present invention is administered as an anticancer agent to an affected area by injection or the like, preferably 7 days or less, preferably Is expected to continue the sustained release of the camptothecin derivative in 3 days or less, more preferably 24 hours or less.
Here, the release rate indicates the ratio of the amount of released camptothecin derivative to the total amount of camptothecin derivative contained in the alginic acid derivative.
By using the alginic acid derivative of the present invention, the amount of drug effective at the affected area (tumor local area) is more effective when administered to the affected area (tumor local area) such as the abdominal cavity or a site close thereto than the single administration of the drug. Even if the amount of the drug is small, a strong therapeutic effect can be expected. In addition, adjustment of sustained release and sustainability can lead to a reduction in the number of administrations in the clinic.
Hereinafter, in order to explain the configuration of the alginic acid derivative of the present invention, the alginic acid, the linker, and the camptothecin derivative, which are the respective constituent components, will be described, and then the alginic acid derivative gel and their uses will be described in detail.
　≪アルギン酸又はその塩≫
　本発明において、アルギン酸又はその塩としては、「アルギン酸の１価金属塩」であるアルギン酸のＤ－マンヌロン酸またはＬ－グルロン酸のカルボン酸の水素原子を、Ｎａ+やＫ+などの１価金属イオンとイオン交換することでつくられる水溶性の塩が好ましい。アルギン酸の１価金属塩としては、具体的には、アルギン酸ナトリウム、アルギン酸カリウムなどを挙げることができるが、特には、アルギン酸ナトリウムが好ましい。後述するようにアルギン酸の１価金属塩の溶液は、架橋剤と混合したときにゲルを形成する性質を利用して、本発明のアルギン酸誘導体の形態を調整することもできる。
　アルギン酸は、褐藻類の海藻から抽出し、精製して製造される天然多糖類の一種である。又、Ｄ－マンヌロン酸（Ｍ）とＬ－グルロン酸（Ｇ）が重合したポリマーである。アルギン酸のＤ－マンヌロン酸とＬ－グルロン酸の構成比（Ｍ／Ｇ比）は、主に海藻等の由来となる生物の種類によって異なり、また、その生物の生育場所や季節による影響を受け、Ｍ／Ｇ比が約０．４の高Ｇ型からＭ／Ｇ比が約５の高Ｍ型まで高範囲にわたる。アルギン酸のＭ／Ｇ比、ＭとＧの配列の仕方等によってアルギン酸の物理化学的性質が異なり、また好ましい用途が異なる場合がある。本発明で使用するアルギン酸又はその塩としては、その最終使用用途に応じて、適切なＭ／Ｇ比のものを用いるのがよい。
　アルギン酸の工業的な製造方法には、酸法とカルシウム法などがあるが、本発明ではいずれの製法で製造されたものも使用することができる。精製により、ＨＰＬＣ法による定量値が８０～１２０質量％の範囲に含まれるものが好ましく、９０～１１０質量％の範囲に含まれるものがより好ましく、９５～１０５質量％の範囲に含まれるものがさらに好ましい。本発明においては、ＨＰＬＣ法による定量値が前記の範囲に含まれるものを高純度アルギン酸と称する。本発明で使用するアルギン酸又はその塩は、高純度アルギン酸であることが好ましい。市販品としては、例えば、キミカアルギンシリーズとして、（株）キミカより販売されているもの、好ましくは、高純度食品・医薬品用グレードのものを購入して使用することができる。市販品を、さらに適宜精製して使用することも可能である。例えば、低エンドトキシン処理することが好ましい。精製法や低エンドトキシン処理方法は、例えば特開２００７－７５４２５（特許文献１）に記載されている方法を採用することができる。 ≪Alginic acid or its salt≫
In the present invention, alginic acid or a salt thereof is a monovalent metal such as Na + or K + , which is a “monovalent metal salt of alginic acid” and a hydrogen atom of carboxylic acid of D-mannuronic acid or L-guluronic acid of alginic acid. Water-soluble salts produced by ion exchange with ions are preferred. Specific examples of the monovalent metal salt of alginic acid include sodium alginate and potassium alginate, and sodium alginate is particularly preferable. As will be described later, a solution of a monovalent metal salt of alginic acid can adjust the form of the alginic acid derivative of the present invention by utilizing the property of forming a gel when mixed with a crosslinking agent.
Alginic acid is a kind of natural polysaccharide that is produced by extracting and purifying from brown seaweed seaweed. Further, it is a polymer obtained by polymerizing D-mannuronic acid (M) and L-guluronic acid (G). The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid of alginic acid varies depending on the type of organism mainly derived from seaweed, etc. It ranges from a high G type with an M / G ratio of about 0.4 to a high M type with an M / G ratio of about 5. Depending on the M / G ratio of alginic acid, the arrangement of M and G, etc., the physicochemical properties of alginic acid may differ, and the preferred application may differ. As the alginic acid or a salt thereof used in the present invention, one having an appropriate M / G ratio may be used depending on the end use application.
Industrial methods for producing alginic acid include an acid method and a calcium method. In the present invention, those produced by any method can be used. What is contained in the range of 80 to 120% by mass of the quantitative value by HPLC method by purification is preferred, more preferably in the range of 90 to 110% by mass, and in the range of 95 to 105% by mass. Further preferred. In the present invention, a substance whose quantitative value by HPLC method is included in the above range is referred to as high purity alginic acid. The alginic acid or a salt thereof used in the present invention is preferably high-purity alginic acid. As a commercially available product, for example, a product sold by Kimika Co., Ltd., preferably a high-purity food / pharmaceutical grade product can be purchased and used as the Kimika Argin series. Commercially available products can be used after further appropriate purification. For example, a low endotoxin treatment is preferable. As the purification method and the low endotoxin treatment method, for example, the method described in JP-A-2007-75425 (Patent Document 1) can be employed.
　本発明で使用するアルギン酸又はその塩としては、その最終使用用途に応じて、適切な重量平均分子量のものを用いるのがよい。例えば、腹腔内投与用の抗癌剤として用いる場合には、重量平均分子量が１万～１，０００万のものを用いるのが好ましく、より好ましくは１０万以上５００万以下、さらに好ましくは２０万以上３００万以下である。より具体的には、例えば重量平均分子量が１６９万Ｄａ（±２０％）又は１３４万Ｄａ（±２０％）であるような物性を有するアルギン酸又はその塩を用いることが好ましい。 As the alginic acid or a salt thereof used in the present invention, one having an appropriate weight average molecular weight may be used according to the end use application. For example, when used as an anticancer agent for intraperitoneal administration, those having a weight average molecular weight of 10,000 to 10,000,000 are preferably used, more preferably 100,000 to 5,000,000, still more preferably 200,000 to 300,000. 10,000 or less. More specifically, it is preferable to use alginic acid having a physical property such as a weight average molecular weight of 1.69 million Da (± 20%) or 1.34 million Da (± 20%) or a salt thereof.
　ここで、一般に天然物由来の高分子物質は、単一の分子量を持つのではなく、種々の分子量を持つ分子の集合体であるため、ある一定の幅を持った分子量分布として測定される。代表的な測定手法はゲルろ過クロマトグラフィーである。ゲルろ過クロマトグラフィーにより得られる分子量分布の代表的な情報としては、重量平均分子量（Ｍｗ）、数平均分子量（Ｍｎ）、分散比（Ｍｗ／Ｍｎ）があげられる。
　分子量の大きい高分子の平均分子量への寄与を重視したのが重量平均分子量であり、下記式で表される。
　　Ｍｗ＝Σ（ＷｉＭｉ）／Ｗ＝Σ（ＨｉＭｉ）／Σ（Ｈｉ）
　数平均分子量は、高分子の総重量を高分子の総数で除して算出される。
　　Ｍｎ＝Ｗ／ΣＮｉ＝Σ（ＭｉＮｉ）／ΣＮｉ＝Σ（Ｈｉ）／Σ（Ｈｉ／Ｍｉ）
　ここで、Ｗは高分子の総重量、Ｗｉはｉ番目の高分子の重量、Ｍｉはｉ番目の溶出時間における分子量、Ｎｉは分子量Ｍｉの個数、Ｈｉはｉ番目の溶出時間における高さである。 Here, generally, a high molecular substance derived from a natural product is not a single molecular weight but an aggregate of molecules having various molecular weights, and thus is measured as a molecular weight distribution having a certain width. A typical measurement technique is gel filtration chromatography. Representative information on the molecular weight distribution obtained by gel filtration chromatography includes weight average molecular weight (Mw), number average molecular weight (Mn), and dispersion ratio (Mw / Mn).
The weight average molecular weight places importance on the contribution to the average molecular weight of a polymer having a large molecular weight and is represented by the following formula.
Mw = Σ (WiMi) / W = Σ (HiMi) / Σ (Hi)
The number average molecular weight is calculated by dividing the total weight of the polymer by the total number of polymers.
Mn = W / ΣNi = Σ (MiNi) / ΣNi = Σ (Hi) / Σ (Hi / Mi)
Here, W is the total weight of the polymer, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecular weights Mi, and Hi is the height at the i-th elution time. .
　天然物由来の高分子物質の分子量測定では、測定方法により値に違いが生じうることが知られている（ヒアルロン酸の例：Ｃｈｉｋａｋｏ　ＹＯＭＯＴＡ　ｅｔ．ａｌ．　Ｂｕｌｌ．Ｎａｔｌ．Ｈｅａｌｔｈ　Ｓｃｉ．，　Ｖｏｌ．１１７，　ｐｐ１３５－１３９（１９９９）、Ｃｈｉｋａｋｏ　ＹＯＭＯＴＡ　ｅｔ．ａｌ．　Ｂｕｌｌ．Ｎａｔｌ．Ｉｎｓｔ．　Ｈｅａｌｔｈ　Ｓｃｉ．，　Ｖｏｌ．１２１，　ｐｐ３０－３３（２００３））。アルギン酸の分子量測定については、固有粘度（Ｉｎｔｒｉｎｓｉｃ　ｖｉｓｃｏｓｉｔｙ）から算出する方法、ＳＥＣ－ＭＡＬＬＳ（Ｓｉｚｅ　Ｅｘｃｌｕｓｉｏｎ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ　ｗｉｔｈ　Ｍｕｌｔｉｐｌｅ　Ａｎｇｌｅ　Ｌａｓｅｒ　Ｌｉｇｈｔ　Ｓｃａｔｔｅｒｉｎｇ　Ｄｅｔｅｃｔｉｏｎ）により算出する方法が記載された文献がある（ＡＳＴＭ　Ｆ２０６４－００（２００６），ＡＳＴＭ　Ｉｎｔｅｒｎａｔｉｏｎａｌ発行）。なお、当該文献では、サイズ排除クロマトグラフィー（＝ゲルろ過クロマトグラフィー）により分子量を測定するにあたっては、プルランを標準物質として用いた較正曲線により算出するだけでは不十分とし、多角度光散乱検出器（ＭＡＬＬＳ）を併用すること（＝ＳＥＣ－ＭＡＬＬＳによる測定）を推奨している。また、ＳＥＣ－ＭＡＬＬＳによる分子量を、アルギン酸のカタログ上の規格値として用いている例もある（ＦＭＣ　Ｂｉｏｐｏｌｙｍｅｒ社、ＰＲＯＮＯＶＡＴＭ　ｓｏｄｉｕｍ　ａｌｇｉｎａｔｅｓ　ｃａｔａｌｏｇｕｅ）。
　なお、通常高分子多糖類の分子量を上記のような手法で算出する場合、１０～２０％の測定誤差を生じうる、例えば、４０万であれば３２～４８万、１００万であれば８０～１２０万程度の範囲で値の変動が生じうる。 It is known that the molecular weight of a high molecular weight substance derived from a natural product may vary depending on the measurement method (example of hyaluronic acid: Chikako YOMOTA et. Al. Bull. Natl. Health Sci., Vol. 117). , Pp135-139 (1999), Chikako YOMOTA et.al.Bull.Natl.Inst.Health Sci., Vol.121, pp30-33 (2003)). Regarding the molecular weight measurement of alginic acid, there is a method of calculating from intrinsic viscosity (Intrinsic viscosity), SEC-MALLS (Size Exclusion Chromatography with Multiple Laser Light Scattering, which is calculated by a method described by Ft. 2006), issued by ASTM International. In this document, when measuring the molecular weight by size exclusion chromatography (= gel filtration chromatography), it is not sufficient to calculate with a calibration curve using pullulan as a standard substance, and a multi-angle light scattering detector ( MALLS) (= measurement by SEC-MALLS) is recommended. In addition, there is an example in which the molecular weight by SEC-MALLS is used as a standard value on a catalog of alginic acid (FMC Biopolymer, PRONOVATM sodium alloys catalog).
When the molecular weight of the high molecular polysaccharide is usually calculated by the method as described above, a measurement error of 10 to 20% can occur. For example, if it is 400,000, it is 32 to 480,000, and if it is 1 million, 80 to Value fluctuations can occur in the range of about 1.2 million.
　本明細書中においてアルギン酸又はその塩の分子量を特定する場合は、特段のことわりがない限り、ゲルろ過クロマトグラフィーにより算出される重量平均分子量である。ゲルろ過クロマトグラフィーの条件としては、例えば、後述する本実施例の条件を採用することができる。 In the present specification, when the molecular weight of alginic acid or a salt thereof is specified, it is a weight average molecular weight calculated by gel filtration chromatography unless otherwise specified. As conditions for the gel filtration chromatography, for example, the conditions of this example described later can be adopted.
　また、本発明で使用するアルギン酸又はその塩としては、その最終使用用途に応じて、適切な粘度のものを用いるのがよい。
　また、本発明で使用するアルギン酸又はその塩としては、その最終使用用途に応じて、適切な物性の組み合わせのものを用いるのがよく、具体的には、適切なＭ／Ｇ比、適切な重量平均分子量、適切な粘度等を有するものを用いるのがよい。
　また、本発明で使用するアルギン酸又はその塩は、エンドトキシンレベルを低下させたものを使用することが好ましい。日局エンドトキシン試験により測定したエンドトキシン値が、１００ＥＵ／ｇ未満のものを用いるのが好ましく、より好ましくは７５ＥＵ／ｇ未満、さらに好ましくは５０ＥＵ／ｇ未満である。本発明において、「実質的にエンドトキシンを含まない」とは、日局エンドトキシン試験により測定したエンドトキシン値が前記の数値範囲にあるものを意味する。 Moreover, as alginic acid or its salt used by this invention, it is good to use the thing of a suitable viscosity according to the end use application.
In addition, as the alginic acid or a salt thereof used in the present invention, it is preferable to use a combination of appropriate physical properties according to the end use application, specifically, an appropriate M / G ratio, an appropriate weight. It is preferable to use one having an average molecular weight, an appropriate viscosity and the like.
In addition, it is preferable to use alginic acid or a salt thereof used in the present invention with a lowered endotoxin level. The endotoxin value measured by the JP endotoxin test is preferably less than 100 EU / g, more preferably less than 75 EU / g, and even more preferably less than 50 EU / g. In the present invention, “substantially free of endotoxin” means that the endotoxin value measured by the JP endotoxin test is in the above numerical range.
　≪リンカー≫
　本発明のアルギン酸誘導体のリンカーは、上述したように、アルギン酸又はその塩由来の１残基とアミド結合で結合しうる官能基を有し、かつ、カンプトテシン誘導体の１残基とエステル結合で結合しうる官能基を有し、アルギン酸誘導体を形成しうる構造を有するものであれば、特に限定はされないが、例えば、以下の式（８）に示される構造を有することが好ましい。
－ＮＨ－（ＣＨ2）n1－［Ｘ1］n2－（ＣＲ1Ｒ2）n3－［Ｙ］n4－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］n7－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－　　　（８）
　式（８）中、－ＮＨは、アルギン酸又はその塩の１残基とアミド結合を形成する末端を示し、Ｃ（＝Ｏ）－は、カンプトテシン誘導体の１残基とエステル結合を形成する末端を示す。
　式（８）中、Ｘ1及びＸ2は、置換基を有しても良いヘテロ原子を示し、好ましくはＯ、Ｓ、ＮＨ及びＮＲ7から選択されるいずれかの原子を示し（Ｒ7はＣ1-6アルキル基、Ｃ2-7アルカノイル基又はＣ1-6アルキルスルホニル基を示し、好ましくは、メチル基、エチル基、アセチル基又はメチルスルホニル基であり、より好ましくはアセチル基であり）、より好ましくはＯ、ＮＨ又はＮＲ7を示し、更に好ましくはＸ1はＯを示し、Ｘ2はＯ、ＮＨ又はＮＲ7を示す。
　式（８）中、Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5及びＲ6はそれぞれ独立して、水素、ハロゲン原子、Ｃ1-10アルキル基、Ｃ1-10アルコキシ基又はＣ1-10アルコキシカルボニル基を示すか、又は、Ｒ1及びＲ2若しくはＲ3及びＲ4が一緒になって＝Ｏを示す。好ましくは、Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5及びＲ6はそれぞれ独立して、水素、フッ素、Ｃ1-6アルキル基、Ｃ1-6アルコキシ基又はＣ1-6アルコキシカルボニル基を示すか、又は、Ｒ1及びＲ2若しくはＲ3及びＲ4が一緒になって＝Ｏを示し、より好ましくはＲ3及びＲ4が一緒になって＝Ｏを示す。
　式（８）中、Ｙは、シクロアルカン環、芳香族環又は複素環（前記シクロアルカン環、芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、好ましくはシクロアルカン環、芳香族環又は複素環、より好ましくはシクロアルカン環、ベンゼン環又は単環式の複素環を示し、更に好ましくはシクロヘキサン環、ベンゼン環又はピリジン環を示し、特に好ましくはベンゼン環又はピリジン環を示す。
　式（８）中、ｎ１は０～１０のいずれかの整数を示し、ｎ２、ｎ４及びｎ７は独立して０または１を示し、ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示す。ただし、ｎ１～ｎ９の全てが０になることはない。好ましくは、ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は合計して１～１２であり、１～１０がより好ましい。また、ｎ１は好ましくは１～３のいずれかの整数であり、より好ましくは１又は２であり、更に好ましくは２である。ｎ３は好ましくは０～２のいずれかの整数であり、より好ましくは０又は１であり、更に好ましくは０である。ｎ５及びｎ６は好ましくは合計して０～２であり、より好ましくは０又は１である。ｎ８及びｎ９は好ましくは合計して０～２であり、より好ましくは０又は１である。 ≪Linker≫
As described above, the linker of the alginic acid derivative of the present invention has a functional group that can be bonded to one residue derived from alginic acid or a salt thereof by an amide bond, and is bonded to one residue of the camptothecin derivative by an ester bond. Although it will not specifically limit if it has a structure which has a functional group which can form an alginic acid derivative, for example, it is preferable to have a structure shown by the following formula | equation (8), for example.
—NH— (CH 2 ) n1 — [X 1 ] n2 — (CR 1 R 2 ) n3 — [Y] n4 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 — (CH 2) n8 - (CR 5 R 6) n9 -C (= O) - (8)
In formula (8), —NH represents a terminal that forms an amide bond with one residue of alginic acid or a salt thereof, and C (═O) — represents a terminal that forms an ester bond with one residue of a camptothecin derivative. Show.
In formula (8), X 1 and X 2 each represents a hetero atom which may have a substituent, preferably any atom selected from O, S, NH and NR 7 (R 7 represents A C 1-6 alkyl group, a C 2-7 alkanoyl group or a C 1-6 alkylsulfonyl group, preferably a methyl group, an ethyl group, an acetyl group or a methylsulfonyl group, more preferably an acetyl group) More preferably O, NH or NR 7 , still more preferably X 1 represents O and X 2 represents O, NH or NR 7 .
In formula (8), R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrogen, halogen atom, C 1-10 alkyl group, C 1-10 alkoxy group or C 1. -10 alkoxycarbonyl group, or R 1 and R 2 or R 3 and R 4 taken together represent = 0. Preferably, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrogen, fluorine, C 1-6 alkyl group, C 1-6 alkoxy group or C 1-6 alkoxycarbonyl. Or R 1 and R 2 or R 3 and R 4 together represent ═O, more preferably R 3 and R 4 together represent ═O.
In formula (8), Y is a cycloalkane ring, aromatic ring or heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group). Preferably a cycloalkane ring, an aromatic ring or a heterocyclic ring, more preferably a cycloalkane ring, a benzene ring or a monocyclic heterocyclic ring, still more preferably a cyclohexane ring, a benzene ring or a pyridine ring, Preferably a benzene ring or a pyridine ring is shown.
In the formula (8), n1 represents any integer of 0 to 10, n2, n4 and n7 independently represent 0 or 1, and n3, n5, n6, n8 and n9 independently represent 0 to 3 Indicates one of the integers. However, all of n1 to n9 do not become zero. Preferably, n3, n5, n6, n8 and n9 are in total 1 to 12, more preferably 1 to 10. N1 is preferably an integer of 1 to 3, more preferably 1 or 2, and still more preferably 2. n3 is preferably an integer of 0 to 2, more preferably 0 or 1, and still more preferably 0. n5 and n6 are preferably 0 to 2 in total, and more preferably 0 or 1. n8 and n9 are preferably 0 to 2 in total, and more preferably 0 or 1.
　本明細書中、特に断りのない限り、「ヘテロ原子」としては、例えば、Ｏ、Ｓ、及びＮまたはＰ等が挙げられる。
　本明細書中、特に断りのない限り、「ハロゲン原子」としては、例えば、フッ素原子、塩素原子、臭素原子及びヨウ素原子等が挙げられる。
　本明細書中、特に断りのない限り、「Ｃ1-6アルキル基」としては、メチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ペンチル、イソペンチル、ネオペンチル、ｔｅｒｔ－ペンチル、１－メチルブチル、２－メチルブチル、１，２－ジメチルプロピル、１－エチルプロピル、ヘキシル、イソヘキシル、１－メチルペンチル、２－メチルペンチル、３－メチルペンチル、１，１－ジメチルブチル、１，２－ジメチルブチル、２，２－ジメチルブチル、１，３－ジメチルブチル、２，３－ジメチルブチル、３，３－ジメチルブチル、１－エチルブチル、２－エチルブチル、１，１，２－トリメチルプロピル、１，２，２－トリメチルプロピル、１－エチル－１－メチルプロピル、１－エチル－２－メチルプロピル、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、シクロプロピルメチル、シクロブチルメチル、シクロペンチルメチル、１－シクロプロピルエチル、２－シクロプロピルエチル、２－シクロブチルエチル、および２－メチルシクロプロピル等が挙げられる。「Ｃ1-10アルキル基」としては、前記「Ｃ1-6アルキル基」として挙げた基に加え、ヘプチル、１－メチルヘキシル、オクチル、２－エチルヘキシル、１，１－ジメチルヘキシル、ノニル、デシル、シクロヘプチル、シクロヘキシルメチル、２－シクロヘキシルエチル、４－メチルシクロヘキシル、４，４－ジメチルシクロヘキシル及び３，３，５，５－テトラメチルシクロヘキシル等が挙げられる。 In the present specification, unless otherwise specified, examples of the “heteroatom” include O, S, and N or P.
Unless otherwise specified, in this specification, examples of the “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
In the present specification, unless otherwise specified, examples of the “C 1-6 alkyl group” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- Pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1-ethylpropyl, hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 1, 2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl- 2-methylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopropylethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-methylcyclopropyl, etc. Is mentioned. Examples of the “C 1-10 alkyl group” include heptyl, 1-methylhexyl, octyl, 2-ethylhexyl, 1,1-dimethylhexyl, nonyl, decyl, in addition to the groups listed as the above “C 1-6 alkyl group”. , Cycloheptyl, cyclohexylmethyl, 2-cyclohexylethyl, 4-methylcyclohexyl, 4,4-dimethylcyclohexyl, 3,3,5,5-tetramethylcyclohexyl and the like.
　本明細書中、特に断りのない限り、「Ｃ1-6アルコキシ基」としては、例えば、メトキシ、エトキシ、プロポキシ、イソプロポキシ、ブトキシ、イソブトキシ、ｓｅｃ－ブトキシ、ｔｅｒｔ－ブトキシ、ペンチルオキシ、イソペンチルオキシ、ネオペンチルオキシ、ｔｅｒｔ－ペンチルオキシ、１－メチルブトキシ、２－メチルブトキシ、１，２－ジメチルプロポキシ、１－エチルプロポキシ、ヘキシルオキシ、イソヘキシルオキシ、１－メチルペンチルオキシ、２－メチルペンチルオキシ、３－メチルペンチルオキシ、１，１－ジメチルブチルオキシ、１，２－ジメチルブチルオキシ、２，２－ジメチルブチルオキシ、１，３－ジメチルブチルオキシ、２，３－ジメチルブチルオキシ、３，３－ジメチルブトキシ、１－エチルブチルオキシ、２－エチルブチルオキシ、１，１，２－トリメチルプロピルオキシ、１，２，２－トリメチルプロピルオキシ、１－エチル－１－メチルプロピルオキシ、１－エチル－２－メチルプロピルオキシ、シクロプロピルオキシ、シクロブチルオキシ、シクロペンチルオキシ、シクロヘキシルオキシ、シクロプロピルメトキシ、シクロブチルメトキシ、シクロペンチルメトキシ、１－シクロプロピルエトキシ、２－シクロプロピルエトキシ、２－シクロブチルエトキシ、２－メチルシクロプロピルオキシ等が挙げられる。「Ｃ1-10アルコキシ基」としては、前記「Ｃ1-6アルコキシ基」として挙げた基に加え、ヘプチルオキシ、オクチルオキシ、２－エチルヘキシルオキシ、ノニルオキシ、デシルオキシ、シクロヘプチルオキシ、シクロヘキシルメトキシ、２－シクロヘキシルエトキシ、４－メチルシクロヘキシルオキシ、４，４－ジメチルシクロヘキシルオキシ、及び３，３，５，５－テトラメチルシクロヘキシルオキシ等が挙げられる。 Unless otherwise specified, in this specification, examples of the “C 1-6 alkoxy group” include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyl. Oxy, neopentyloxy, tert-pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 1,2-dimethylpropoxy, 1-ethylpropoxy, hexyloxy, isohexyloxy, 1-methylpentyloxy, 2-methylpentyl Oxy, 3-methylpentyloxy, 1,1-dimethylbutyloxy, 1,2-dimethylbutyloxy, 2,2-dimethylbutyloxy, 1,3-dimethylbutyloxy, 2,3-dimethylbutyloxy, 3, 3-dimethylbutoxy, 1-ethylbutyloxy, 2-ethylbutyloxy, 1,1,2-trimethylpropyloxy, 1,2,2-trimethylpropyloxy, 1-ethyl-1-methylpropyloxy, 1-ethyl-2-methylpropyloxy, cyclopropyloxy, Examples include cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cyclopropylmethoxy, cyclobutylmethoxy, cyclopentylmethoxy, 1-cyclopropylethoxy, 2-cyclopropylethoxy, 2-cyclobutylethoxy, 2-methylcyclopropyloxy and the like. Examples of the “C 1-10 alkoxy group” include heptyloxy, octyloxy, 2-ethylhexyloxy, nonyloxy, decyloxy, cycloheptyloxy, cyclohexylmethoxy, in addition to the groups listed as the above “C 1-6 alkoxy group”. -Cyclohexylethoxy, 4-methylcyclohexyloxy, 4,4-dimethylcyclohexyloxy, 3,3,5,5-tetramethylcyclohexyloxy and the like.
　本明細書中、特に断りのない限り、「Ｃ1-10アルコキシカルボニル基」とは、－Ｃ（＝Ｏ）－Ｒ（ＲはＣ1-10アルコキシ基）で表される基である。
　本明細書中、特に断りのない限り、「Ｃ2-7アルカノイル基」とは、－Ｃ（＝Ｏ）－Ｒ（ＲはＣ1-6アルキル基）で表される基であり、例えば、アセチル、プロピオニル、ブチリル、イソブチリル等が挙げられる。
　本明細書中、特に断りのない限り、「Ｃ1-6アルキルスルホニル基」とは、－Ｓ（＝Ｏ）2－Ｒ（ＲはＣ1-6アルキル基）で表される基であり、例えば、メチルスルホニル、エチルスルホニル、プロピルスルホニル、イソプロピルスルホニル等が挙げられる。 In the present specification, unless otherwise specified, the “C 1-10 alkoxycarbonyl group” is a group represented by —C (═O) —R (R is a C 1-10 alkoxy group).
In the present specification, unless otherwise specified, the “C 2-7 alkanoyl group” is a group represented by —C (═O) —R (R is a C 1-6 alkyl group). Examples include acetyl, propionyl, butyryl, isobutyryl and the like.
In the present specification, unless otherwise specified, the “C 1-6 alkylsulfonyl group” is a group represented by —S (═O) 2 —R (R is a C 1-6 alkyl group). For example, methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl and the like can be mentioned.
　本明細書中、特に断りのない限り、「シクロアルカン環」としては、シクロプロパン、シクロブタン、シクロペンタン、シクロヘキサン、シクロヘプタン、シクロオクタン、シクロノナン及びシクロデカン等が挙げられる。
　本明細書中、特に断りのない限り、「芳香族環」としては、ベンゼン環、１－ナフタレン環、２－ナフタレン環、２－、３－、４－ビフェニルアントロン環、フェナントレン環及びアセナフテン環等が挙げられる。
　本明細書中、特に断りのない限り、「複素環」とは、窒素原子、硫黄原子または酸素原子のヘテロ原子を１～５個含有する３～１４員環の単環式もしくは縮環式の環を意味する。
　本明細書中、特に断りのない限り、「複素環」には、「部分的に水素化された縮環式複素環」、「非芳香族複素環」が挙げられる。
　本明細書中、特に断りのない限り、「複素環」としては、環員数５～７のものが好ましく、例えば、ピロール、フラン、チオフェン、イミダゾール、ピラゾール、オキサゾール、イソキサゾール、チアゾール、イソチアゾール、１，２，３－トリアゾール、１，２，４－トリアゾール、１，２，３－オキサジアゾール、１，２，４－オキサジアゾール、１，３，４－オキサジアゾール、フラザール、１，２，３－チアジアゾール、１，２，４－チアジアゾール、１，３，４－チアジアゾール、テトラゾール、ピリジン、ピリダジン、ピリミジン、ピラジン、１，２，３－トリアジン、１，２，４－トリアジン、１，３，５－トリアジン、２Ｈ－１，２，３－チアジアジン、４Ｈ－１，２，４－チアジアジン、６Ｈ－１，３，４－チアジアジン、１，４－ジアゼピン、１，４－オキサゼピン等が挙げられる。 In the present specification, unless otherwise specified, examples of the “cycloalkane ring” include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, and cyclodecane.
In this specification, unless otherwise specified, examples of the “aromatic ring” include a benzene ring, 1-naphthalene ring, 2-naphthalene ring, 2-, 3-, 4-biphenylanthrone ring, phenanthrene ring, and acenaphthene ring. Is mentioned.
In this specification, unless otherwise specified, the term “heterocycle” means a 3 to 14-membered monocyclic or condensed ring containing 1 to 5 heteroatoms of nitrogen, sulfur or oxygen. Means a ring.
In the present specification, unless otherwise specified, examples of the “heterocycle” include “partially hydrogenated condensed ring heterocycle” and “non-aromatic heterocycle”.
In the present specification, unless otherwise specified, the “heterocycle” is preferably one having 5 to 7 ring members, such as pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, 1 , 2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, frazal, 1,2 , 3-thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3 , 5-triazine, 2H-1,2,3-thiadiazine, 4H-1,2,4-thiadiazine, 6H-1,3,4-thiadiazine 1,4-diazepine, 1,4-oxazepine and the like.
　本明細書中、特に断りのない限り、前記「縮環式の環」としては、環員数８～１２のものが好ましく、例えば、インドール、イソインドール、ベンゾフラン、イソベンゾフラン、ベンゾチオフェン、イソベンゾチオフェン、ベンゾオキサゾール、１，２－ベンゾイソキサゾール、ベンゾチアゾール、１，２－ベンゾイソチアゾール、１Ｈ－ベンズイミダゾール、１Ｈ－インダゾール、１Ｈ－ベンゾトリアゾール、２，１，３－ベンゾチアジアジン、クロメン、イソクロメン、４Ｈ－１，４－ベンゾオキサジン、４Ｈ－１，４－ベンゾチアジン、キノリン、イソキノリン、シンノリン、キナゾリン、キノキサリン、フタラジン、ベンゾオキサゼピン、ベンゾアゼピン、ベンゾジアゼピン、ナフチリジン、プリン、プテリジン、カルバゾール、カルボリン、アクリジニン、フェノキサジン、フェノチアジン、フェナジン、フェノキサチイン、チアンスレン、チアントレン、フェナンスリジン、フェナンスロリン、インドリジン、チエノ［３，２－ｃ］ピリジン、チアゾロ［５，４－ｃ］ピリジン、ピロロ［１，２－ｂ］ピリダジン、ピラゾロ［１，５－ａ］ピリジン、イミダゾ［１，２－ａ］ピリジン、イミダゾ［１，５－ａ］ピリジン、イミダゾ［１，２－ｂ］ピリダジン、イミダゾ［１，５－ａ］ピリミジン、１，２，４－トリアゾロ［４，３－ａ］ピリジン、１，２，４－トリアゾロ［４，３－ｂ］ピリダジン、１Ｈ－ピラゾロ［３，４－ｂ］ピリジン、１，２，４－トリアゾロ［１，５－ａ］ピリミジン等が挙げられる。 In the present specification, unless otherwise specified, the “condensed ring” preferably has 8 to 12 ring members. For example, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, isobenzothiophene , Benzoxazole, 1,2-benzisoxazole, benzothiazole, 1,2-benzisothiazole, 1H-benzimidazole, 1H-indazole, 1H-benzotriazole, 2,1,3-benzothiadiazine, chromene , Isochromene, 4H-1,4-benzoxazine, 4H-1,4-benzothiazine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, phthalazine, benzoxazepine, benzoazepine, benzodiazepine, naphthyridine, purine, pteridine, carbazole, Luborin, acridinine, phenoxazine, phenothiazine, phenazine, phenoxathiin, thianthrene, thianthrene, phenanthridine, phenanthrolin, indolizine, thieno [3,2-c] pyridine, thiazolo [5,4-c] pyridine, Pyrrolo [1,2-b] pyridazine, pyrazolo [1,5-a] pyridine, imidazo [1,2-a] pyridine, imidazo [1,5-a] pyridine, imidazo [1,2-b] pyridazine, Imidazo [1,5-a] pyrimidine, 1,2,4-triazolo [4,3-a] pyridine, 1,2,4-triazolo [4,3-b] pyridazine, 1H-pyrazolo [3,4 b] Pyridine, 1,2,4-triazolo [1,5-a] pyrimidine and the like.
　本発明の化合物は、リンカーとカンプトテシン誘導体とのエステル結合が加水分解され、カンプトテシン誘導体が遊離される。エステル結合は周辺環境により加水分解の速度が変わる。そのため、エステル結合の形態で、より長期の徐放効果が得られるものもある。例えば、リンカーのＣ（＝Ｏ）末端の近傍に、電子供与基や嵩高い基を導入、若しくは置換基として導入することにより、加水分解の速度が遅くなることがあり、例えば、アルキル基、特に分枝状のアルキル基が挙げられる。またその反対に、リンカーのＣ（＝Ｏ）末端の近傍に、電子吸引基を導入、若しくは置換基として導入することにより、加水分解の速度が速くなることがあり、例えば、ハロゲン原子、ハロアルキル基及びアルコキシカルボニル基等を置換基として導入することが挙げられる。また、Ｃ（＝Ｏ）末端の近傍のリンカー内にヘテロ原子を導入することでも、加水分解の速度が速くなることがあり、例えば、酸素原子や窒素原子等をリンカー骨格に導入することが挙げられる。このようにして、目的とする加水分解速度、すなわち徐放効果に応じて、リンカー内への基の導入若しくはリンカーへの置換基の導入を選択することが可能である。 In the compound of the present invention, the ester bond between the linker and the camptothecin derivative is hydrolyzed to release the camptothecin derivative. The rate of hydrolysis of ester bonds varies depending on the surrounding environment. Therefore, there are some which can obtain a longer-term sustained release effect in the form of an ester bond. For example, by introducing an electron donating group or a bulky group in the vicinity of the C (═O) terminal of the linker, or introducing it as a substituent, the rate of hydrolysis may be reduced. For example, an alkyl group, particularly A branched alkyl group can be mentioned. On the other hand, by introducing an electron withdrawing group in the vicinity of the C (═O) end of the linker or introducing it as a substituent, the hydrolysis rate may be increased. For example, a halogen atom, a haloalkyl group And introducing an alkoxycarbonyl group or the like as a substituent. Also, introduction of a heteroatom into the linker near the C (= O) terminal may increase the rate of hydrolysis, for example, introducing an oxygen atom or a nitrogen atom into the linker skeleton. It is done. In this manner, it is possible to select introduction of a group into the linker or introduction of a substituent into the linker according to the target hydrolysis rate, that is, the sustained release effect.
　より好ましくは、以下に示す一群のリンカーのいずれかでアルギン酸又はその塩と、カンプトテシン誘導体が結ばれていることが好ましい。なお、以下のリンカーにおいて、右側の－ＮＨ－は、アルギン酸又はその塩１残基とアミド結合を形成する末端を示し、左側の－Ｃ（＝Ｏ）－は、カンプトテシン誘導体の水酸基とエステル結合を形成する末端を示す。
Figure JPOXMLDOC01-appb-C000001
 More preferably, it is preferable that alginic acid or a salt thereof and a camptothecin derivative are bonded to any one of a group of linkers shown below. In the following linkers, —NH— on the right side represents a terminal that forms an amide bond with one residue of alginic acid or a salt thereof, and —C (═O) — on the left side represents a hydroxyl group and an ester bond of the camptothecin derivative. The end to be formed is indicated.
Figure JPOXMLDOC01-appb-C000001
　＜カンプトテシン誘導体＞
　カンプトテシン誘導体としては、その化学構造中に下記式のカンプトテシン骨格を有しているものを使用する。
Figure JPOXMLDOC01-appb-C000002
　また、カンプトテシン誘導体は塩の形態であっても構わない。本発明におけるカンプトテシン誘導体としては、特に限定されないが、中でも特に抗癌作用を有するものが望ましく、リンカーとの結合の観点から、水酸基を少なくとも有しているカンプトテシン誘導体が好ましい。水酸基の位置は特に限定されないが、カンプトテシン骨格の２０位の３級アルコール性水酸基又は任意の位置に置換導入されている水酸基が挙げられる。水酸基が置換導入されているカンプトテシン誘導体として、フェノール性水酸基を有しているカンプトテシン誘導体がより好ましく、カンプトテシン骨格の１０位にフェノール性水酸基を有しているものが更に好ましい。すなわち、本発明のアルギン酸誘導体は、カンプトテシン誘導体の２０位の３級アルコール性水酸基がリンカーと結合されてなること、又は、カンプトテシン誘導体の置換導入されたフェノール性水酸基がリンカーと結合されてなることがより好ましく、カンプトテシン骨格の１０位にフェノール性水酸基を有し、当該フェノール性水酸基がリンカーと結合されてなることが更に好ましい。また、より遅い徐放速度が望ましい局面では、本発明のアルギン酸誘導体は、カンプトテシン誘導体の２０位の３級アルコール性水酸基がリンカーと結合されてなることもより好ましい。
　水酸基を有するカンプトテシン誘導体としては、例えばカンプトテシン；７－エチルカンプトテシン；１０－ヒドロキシカンプトテシン；ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）；イリノテカン（ＣＰＴ－１１）；９－（ジメチルアミノ）メチルカンプトテシン；トポテカン（ノギテカン）；エキサテカン；Ｔ－２５１３（１０－（３－アミノプロピロキシ）－７－エチルカンプトテシン）：１０，１１－メチレンジオキシカンプトテシン；７－エチル－１０，１１－メチレンジオキシカンプトテシン；９－アミノ－１０，１１－メチレンジオキシカンプトテシン；９－クロロ－１０，１１－メチレンジオキシカンプトテシン；７－（４－メチル－１－ピペラジニル）メチル－１０，１１－メチレンジオキシカンプトテシン；１０，１１－エチレンジオキシカンプトテシン；ルルトテカン（７－（４－メチル－１－ピペラジニル）メチル－１０，１１－エチレンジオキシカンプトテシン）；ギマテカン（７－（ｔｅｒｔ－ブトキシイミノメチル）カンプトテシン）；９－アミノカンプトテシン；ルビテカン（９－ニトロカンプトテシン）；ベロテカン（７－（２－（Ｎ－イソプロピルアミノ）エチル）－カンプトテシン）；コシテカン（７－（２－（トリメチルシリル）エチル）－カンプトテシン）またはシラテカン（７－（ｔｅｒｔ－ブチルジメチルシリル）－１０－ヒドロキシカンプトテシン）等が挙げられ、ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）、イリノテカン（ＣＰＴ－１１）又はトポテカン（ノギテカン）であることが好ましく、カンプトテシン誘導体がＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）であることがより好ましい。
　フェノール性水酸基を有するカンプトテシン誘導体としては、１０－ヒドロキシカンプトテシン；ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）；トポテカン（ノギテカン）；シラテカン（７－（ｔｅｒｔ－ブチルジメチルシリル）－１０－ヒドロキシカンプトテシン）などが挙げられ、ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）又はトポテカン（ノギテカン）であることが好ましく、ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）であることがより好ましい。 <Camptothecin derivative>
As the camptothecin derivative, one having a camptothecin skeleton of the following formula in its chemical structure is used.
Figure JPOXMLDOC01-appb-C000002
The camptothecin derivative may be in the form of a salt. The camptothecin derivative in the present invention is not particularly limited, but among them, those having an anticancer activity are particularly desirable, and a camptothecin derivative having at least a hydroxyl group is preferable from the viewpoint of binding with a linker. The position of the hydroxyl group is not particularly limited, and examples thereof include a tertiary alcoholic hydroxyl group at the 20th position of the camptothecin skeleton or a hydroxyl group substituted and introduced at an arbitrary position. As the camptothecin derivative having a hydroxyl group substituted, a camptothecin derivative having a phenolic hydroxyl group is more preferable, and a camptothecin derivative having a phenolic hydroxyl group at the 10-position of the camptothecin skeleton is more preferable. That is, in the alginic acid derivative of the present invention, the tertiary alcoholic hydroxyl group at the 20-position of the camptothecin derivative is bonded to the linker, or the phenolic hydroxyl group into which the substitution of the camptothecin derivative is introduced is bonded to the linker. More preferably, it has a phenolic hydroxyl group at the 10-position of the camptothecin skeleton, and the phenolic hydroxyl group is more preferably bonded to a linker. Moreover, in a situation where a slower sustained release rate is desired, it is more preferable that the alginic acid derivative of the present invention has a tertiary alcoholic hydroxyl group at the 20-position of the camptothecin derivative bonded to a linker.
Examples of the camptothecin derivative having a hydroxyl group include camptothecin; 7-ethylcamptothecin; 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); irinotecan (CPT-11); 9- (dimethylamino) methylcamptothecin Exotecan; T-2513 (10- (3-aminopropyloxy) -7-ethylcamptothecin): 10,11-methylenedioxycamptothecin; 7-ethyl-10,11-methylenedioxycamptothecin; 9-amino-10,11-methylenedioxycamptothecin; 9-chloro-10,11-methylenedioxycamptothecin; 7- (4-methyl-1-piperazinyl) methyl-10,11-methylenedioxycamptothecin 10,11-ethylenedioxycamptothecin; luruthecan (7- (4-methyl-1-piperazinyl) methyl-10,11-ethylenedioxycamptothecin); gimatecan (7- (tert-butoxyiminomethyl) camptothecin); 9-aminocamptothecin; rubitecan (9-nitrocamptothecin); belothecan (7- (2- (N-isopropylamino) ethyl) -camptothecin); cositecan (7- (2- (trimethylsilyl) ethyl) -camptothecin) or sirathecan ( 7- (tert-butyldimethylsilyl) -10-hydroxycamptothecin) and the like, and preferably SN-38 (7-ethyl-10-hydroxycamptothecin), irinotecan (CPT-11) or topotecan (Nogitecan). Ku, it is more preferable camptothecin derivative is SN-38 (7- ethyl-10-hydroxycamptothecin).
The camptothecin derivatives having a phenolic hydroxyl group include 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); topotecan (nogitecan); silathecan (7- (tert-butyldimethylsilyl) -10-hydroxycamptothecin SN-38 (7-ethyl-10-hydroxycamptothecin) or topotecan (nogitecan) is preferable, and SN-38 (7-ethyl-10-hydroxycamptothecin) is more preferable.
　＜アルギン酸誘導体の合成方法＞
　アルギン酸誘導体の合成において、リンカーへのカンプトテシン誘導体の結合、及び、リンカーへのアルギン酸又はその塩の結合は、どちらが先でも構わないが、水溶媒中でエステル化を行うことは難しいなどの理由から、リンカーへのカンプトテシン誘導体の結合を先にするほうが好ましい。このような結合を達成する方法としては、ＤＣＣ、ＥＤＣＩ、ＤＭＴ－ＭＭなどの縮合剤を使用する方法、ＨＯＳｕ、ＨＯＢｔなどの縮合補助剤と前記縮合剤とを使用する縮合反応、求核置換反応、活性エステル法、酸無水物法等が挙げられ、縮合反応、及び、求核置換反応を用いて結合させるのが副反応を抑えるなどの理由で好ましい。
　より具体的には、例えば以下のような概念を示すスキームのように、縮合反応（エステル化反応）を利用する方法で合成することができる。下記の反応スキームにおいては、便宜上、リンカーを「ＣＯ－Ｌｉｎｋｅｒ－ＮＨ」として解釈し、ＡＬをアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基として解釈し、Ｂｏｃをブトキシカルボニル基（保護基）として解釈すると、反応の概略を理解し得る。なお、ＣＰＴはカンプトテシン誘導体の略を示すが、これは例示であって、本発明においてカンプトテシン誘導体がカンプトテシンに限定されることを意味するわけではない。 <Synthesis Method of Alginic Acid Derivative>
In the synthesis of the alginic acid derivative, either the binding of the camptothecin derivative to the linker and the binding of alginic acid or a salt thereof to the linker may be performed first, but it is difficult to perform esterification in an aqueous solvent. It is preferable that the camptothecin derivative is first bound to the linker. Examples of a method for achieving such bonding include a method using a condensing agent such as DCC, EDCI, and DMT-MM, a condensation reaction using a condensing agent such as HOSu and HOBt and the condensing agent, and a nucleophilic substitution reaction. And an active ester method, an acid anhydride method, and the like. Bonding using a condensation reaction and a nucleophilic substitution reaction is preferable for the purpose of suppressing side reactions.
More specifically, it can be synthesized by a method utilizing a condensation reaction (esterification reaction), for example, as in a scheme showing the following concept. In the following reaction scheme, for convenience, the linker is interpreted as “CO-Linker-NH”, AL is a residue derived from alginic acid or a salt thereof, and L-guluronic acid and D-mannuronic acid constituting alginic acid are included. Interpreting the C (= O) -group of either monosaccharide and interpreting Boc as a butoxycarbonyl group (protecting group) can give an overview of the reaction. CPT is an abbreviation for a camptothecin derivative, but this is merely an example, and does not mean that the camptothecin derivative is limited to camptothecin in the present invention.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　また、本発明におけるアルギン酸誘導体におけるカンプトテシン誘導体の導入率は、本発明のアルギン酸誘導体の合成工程において、縮合剤、縮合補助剤、リンカー結合カンプトテシン誘導体の投入量を変えることなどにより調整可能である。なお、導入率は、吸光度の測定やＨＰＬＣ、ＮＭＲ等を用いる方法で測定することができる。リンカーの構造、導入率によって、アルギン酸誘導体の水溶性を適宜調整することも可能である。 In addition, the introduction rate of the camptothecin derivative in the alginic acid derivative in the present invention can be adjusted by changing the input amount of the condensing agent, the condensation auxiliary agent, and the linker-bound camptothecin derivative in the synthesis step of the alginic acid derivative of the present invention. The introduction rate can be measured by a method using absorbance measurement, HPLC, NMR, or the like. The water solubility of the alginic acid derivative can be appropriately adjusted depending on the structure and introduction rate of the linker.
　＜アルギン酸又はその塩と結合させる際に用いるアミノ化合物＞
　アルギン酸誘導体の合成において、リンカーとカンプトテシン誘導体を結合させた後、アルギン酸又はその塩と結合させる際に用いるアミノ化合物としては、下記式（９）で表されるアミノ化合物、その塩、又はそれらの溶媒物が挙げられる。
ＮＨ2－（ＣＨ2）n1－［Ｘ1］n2－（ＣＲ1Ｒ2）n3－［Ｙ］n4－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］n7－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（９）
　式（９）中、（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、Ｘ1及びＸ2は、置換基を有しても良いヘテロ原子を示し、Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5及びＲ6はそれぞれ独立して、水素、ハロゲン原子、Ｃ1-10アルキル基、Ｃ1-10アルコキシ基又はＣ1-10アルコキシカルボニル基を示すか、又は、Ｒ1及びＲ2若しくはＲ3及びＲ4が一緒になって＝Ｏを示し、Ｙは、シクロアルカン環、芳香族環又は複素環（前記シクロアルカン環、芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、ｎ１は０～１０のいずれかの整数を示し、ｎ２、ｎ４及びｎ７は独立して０または１を示し、ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ１～ｎ９の全てが０になることはない。
　式（９）において、Ｘ1、Ｘ2、Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5、Ｒ6、Ｙ、ｎ１、ｎ２、ｎ３、ｎ４、ｎ５、ｎ６、ｎ７、ｎ８及びｎ９の好ましい態様は、前記式（１）～（８）にて記載した好ましい態様が挙げられる。 <Amino compound used when combined with alginic acid or a salt thereof>
In the synthesis of an alginic acid derivative, an amino compound used when binding a linker and a camptothecin derivative and then binding to alginic acid or a salt thereof is an amino compound represented by the following formula (9), a salt thereof, or a solvent thereof. Things.
NH 2 - (CH 2) n1 - [X 1] n2 - (CR 1 R 2) n3 - [Y] n4 - (CH 2) n5 - (CR 3 R 4) n6 - [X 2] n7 - (CH 2) n8 - (CR 5 R 6) n9 -C (= O) - (D) (9)
In the formula (9), (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative, X 1 and X 2 represent a hetero atom which may have a substituent, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 each independently represent a hydrogen atom, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group, or R 1 and R 2 or R 3 and R 4 together represent ═O, and Y represents a cycloalkane ring, an aromatic ring or a heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring represents a halogen atom or C 1-10 represents an alkyl which may be substituted with a group), n1 is 0 to indicate an integer of 10, n2, n4 and n7 are independently 0 or 1, n3, n5, n6 , N8 and n9 independently represent any integer of 0 to 3, but all of n1 to n9 0 does not become.
In the formula (9), X 1 , X 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , Y, n1, n2, n3, n4, n5, n6, n7, n8 and n9 Preferred embodiments include the preferred embodiments described in the above formulas (1) to (8).
　＜アルギン酸誘導体ゲル＞
　本発明のアルギン酸誘導体は、一般的にアルギン酸の架橋剤として使用される物質と混合することによってアルギン酸誘導体ゲルを形成することができる。そのような架橋剤としては、アルギン酸の１価金属塩の溶液を架橋することにより、その表面を固定化することができるものであれば、特に限定されないが、Ｃａ2+、Ｍｇ2+、Ｂａ2+、Ｓｒ2+などの２価以上の金属イオン化合物、分子内に２～４個のアミノ基を有する架橋性試薬などが挙げられる。より具体的には、２価以上の金属イオン化合物として、ＣａＣｌ2、ＭｇＣｌ2、ＣａＳＯ4、ＢａＣｌ2等を、分子内に２～４個のアミノ基を有する架橋性試薬として、窒素原子上にリジル（ｌｙｓｙｌ）基（－ＣＯＣＨ（ＮＨ2）－（ＣＨ2）4－ＮＨ2）を有することもあるジアミノアルカン、すなわちジアミノアルカンおよびそのアミノ基がリジル基で置換されてリジルアミノ基を形成している誘導体が包含され、具体的にはジアミノエタン、ジアミノプロパン、Ｎ－（リジル）－ジアミノエタン等を挙げることができるが、入手しやすいこと、ゲルの強度等の理由から、特に、ＣａＣｌ2溶液とするのが好ましい。
　ここで、架橋剤にカルシウムが含まれる場合、カルシウムの濃度が高い方が、ゲル化が早く、また、より硬いゲルを形成することができることが知られている。しかし、カルシウムには細胞毒性があるため、濃度が高すぎると、これを体内に投与した場合に体内（幹部）に悪影響を及ぼすおそれもあり、アルギン酸の量に応じて、適量を使用することがよい。
　本発明のアルギン酸誘導体を架橋してなるアルギン酸誘導体ゲルは、形状を加工し、ビーズやスポンジ状にすることが可能である。例えば、腹腔内投与用あるいは手術時留置用の抗癌剤として、ビーズ状のアルギン酸誘導体ゲルを用いることができる。 <Alginic acid derivative gel>
The alginic acid derivative of the present invention can form an alginic acid derivative gel by mixing with a substance generally used as a crosslinking agent for alginic acid. Such a crosslinking agent is not particularly limited as long as it can fix the surface of the monovalent metal salt of alginic acid by crosslinking, but is not limited to Ca 2+ , Mg 2+ , Ba. Examples thereof include divalent or higher-valent metal ion compounds such as 2+ and Sr 2+, and crosslinkable reagents having 2 to 4 amino groups in the molecule. More specifically, CaCl 2 , MgCl 2 , CaSO 4 , BaCl 2, etc. are used as divalent or higher valent metal ion compounds on the nitrogen atom as crosslinkable reagents having 2 to 4 amino groups in the molecule. Diaminoalkanes that may have a lysyl group (—COCH (NH 2 ) — (CH 2 ) 4 —NH 2 ), ie, diaminoalkanes and their amino groups are substituted with lysyl groups to form lysylamino groups And specific examples include diaminoethane, diaminopropane, N- (lysyl) -diaminoethane, etc., but CaCl 2 solution is particularly preferred because of its availability and gel strength. Is preferable.
Here, it is known that when the crosslinking agent contains calcium, the higher the calcium concentration, the faster the gelation and the formation of a harder gel. However, since calcium is cytotoxic, if the concentration is too high, there is a risk of adverse effects on the body (trunk) when it is administered to the body, and an appropriate amount may be used depending on the amount of alginic acid. Good.
The alginic acid derivative gel obtained by crosslinking the alginic acid derivative of the present invention can be processed into a bead or sponge shape. For example, a beaded alginic acid derivative gel can be used as an anticancer agent for intraperitoneal administration or indwelling at the time of surgery.
　＜徐放性医薬組成物＞
　本発明のアルギン酸誘導体又はアルギン酸誘導体ゲルは、生体内においてカンプトテシン誘導体を徐放する挙動を示すため、徐放性医薬組成物として使用することができる。さらに、本発明の徐放性医薬組成物は、その徐放基材としてアルギン酸又はその塩が用いられている。アルギン酸又はその塩は高分子化合物であるが、一般的に腹腔内投与された高分子化合物やミセル形成分子はリンパ系を介して穏やかに吸収されることが知られている。一方で、腹膜転移の形成過程における癌細胞の腹膜への接着経路の一つとして、乳斑やストマータ等のリンパ系組織への選択的な癌細胞の接着がある。よって、腹膜転移の初期転移巣であるリンパ系において、高分子化合物であるアルギン酸又はその塩が吸収されることから、アルギン酸のターゲッティング効果が期待される。すなわち、本発明の徐放性医薬組成物は、徐放されるカンプトテシン誘導体の抗腫瘍作用と、アルギン酸のリンパ吸収性によるターゲッティング作用が、合わせて期待されるものである。本発明の徐放性医薬組成物の対象疾患、投与ルートは特に限定されるものでは無いが、腹膜転移の治療、予防や緩和などを目的とすることが好ましく、腹腔内へ直接注入する投与ルートで投与されることが好ましい。
　例えば、本発明の徐放性医薬組成物を腹腔内投与用の抗癌剤として使用したときに、患部（腫瘍局所）に注射等により投与した際に、７日間以上、好ましくは１５日間以上、より好ましくは３０日間以上、更に好ましくは６０日間以上、安定してカンプトテシン誘導体を徐放し続けることが期待される。
　また、本発明の徐放性医薬組成物を速やかな放出が望ましい局面で使用する際には、その局面に適した薬剤の放出期間を有するアルギン酸誘導体を選択し、例えば、本発明のアルギン酸誘導体を抗癌剤として患部（腫瘍局所）に注射等により投与した際に、７日間以下、好ましくは３日間以下、より好ましくは２４時間以下で、カンプトテシン誘導体を徐放し続けることが期待される。 <Sustained release pharmaceutical composition>
Since the alginic acid derivative or alginic acid derivative gel of the present invention exhibits a behavior of sustained release of the camptothecin derivative in vivo, it can be used as a sustained-release pharmaceutical composition. Furthermore, the sustained-release pharmaceutical composition of the present invention uses alginic acid or a salt thereof as the sustained-release base. Alginic acid or a salt thereof is a high molecular compound, but it is generally known that a high molecular compound or micelle-forming molecule administered intraperitoneally is gently absorbed through the lymphatic system. On the other hand, there is selective adhesion of cancer cells to lymphoid tissues such as milk spots and stoma as one of the pathways of adhesion of cancer cells to the peritoneum in the formation process of peritoneal metastasis. Therefore, alginic acid or a salt thereof, which is a polymer compound, is absorbed in the lymphatic system, which is an initial metastasis of peritoneal metastasis, and thus a targeting effect of alginic acid is expected. That is, the sustained-release pharmaceutical composition of the present invention is expected to have both the antitumor action of the sustained-release camptothecin derivative and the targeting action due to the lymph absorbability of alginic acid. Although the target disease and administration route of the sustained-release pharmaceutical composition of the present invention are not particularly limited, it is preferably intended for the treatment, prevention or alleviation of peritoneal metastasis, and the administration route for direct injection into the abdominal cavity Are preferably administered.
For example, when the sustained-release pharmaceutical composition of the present invention is used as an anticancer agent for intraperitoneal administration, it is administered for 7 days or more, preferably 15 days or more, more preferably when administered to an affected area (tumor local) by injection or the like. Is expected to stably release the camptothecin derivative stably for 30 days or longer, more preferably 60 days or longer.
In addition, when the sustained-release pharmaceutical composition of the present invention is used in an aspect where rapid release is desired, an alginic acid derivative having a drug release period suitable for the aspect is selected, for example, the alginic acid derivative of the present invention is selected. When administered to the affected area (tumor local area) by injection or the like as an anticancer agent, it is expected that the camptothecin derivative will be sustainedly released in 7 days or less, preferably 3 days or less, more preferably 24 hours or less.
　また、本発明の徐放性医薬組成物の投与量は、含まれるカンプトテシン誘導体の量、投与ルート、投与形態、使用目的、投与対象となる動物の具体的症状、年齢、体重等に応じて、治療効果が最も適切に発揮される様に個別に決定され、特に限定されない。 The dosage of the sustained-release pharmaceutical composition of the present invention depends on the amount of the camptothecin derivative contained, the administration route, the administration form, the purpose of use, the specific symptoms of the animal to be administered, the age, the body weight, etc. It is determined individually so that the therapeutic effect is most appropriately exhibited, and is not particularly limited.
　本発明の徐放性医薬組成物の適用部位は非経口投与により投与可能な部位であれば特に限定されないが、中でも腫瘍局所への投与が好ましく、より好ましくは腹腔内投与である。また、動脈化学塞栓療法、特に肝動脈化学塞栓療法に用いることも好ましく、本発明のアルギン酸誘導体ゲルを含む徐放性医薬組成物を塞栓物質として動脈内投与することも好ましい。 The application site of the sustained-release pharmaceutical composition of the present invention is not particularly limited as long as it can be administered by parenteral administration, but administration to a tumor site is preferable, and intraperitoneal administration is more preferable. It is also preferable to use it for arterial chemoembolization, particularly hepatic arterial chemoembolization, and it is also preferable to administer a sustained-release pharmaceutical composition containing the alginic acid derivative gel of the present invention as an embolic substance into an artery.
　本発明では、例えばアルギン酸誘導体を腹膜転移の患部（腫瘍局所）である腹腔内に適用してもよい。また適用後、適度な粘度が保たれない場合には、適用した誘導体の表面に架橋剤を添加しても良い。誘導体表面をゲル化して、表面を固めることで、腹腔から漏れ出すのを効果的に防ぐことができる。
　先にアルギン酸誘導体を患部（腫瘍局所）に投与し、あとから架橋剤を添加する場合、架橋剤は、適用した組成物の表面から徐々に内部に浸透し、架橋をすすめるのが望ましい。患部（腫瘍局所）との接触部分に、架橋剤の影響を強く及ぼさないためには、架橋剤の適用量を過剰にならないよう調節する。２価以上の金属イオンの適用量としては、アルギン酸の１価金属塩を含有する組成物の表面を固めることができる量であれば、特に限定されない。
　本発明のアルギン酸誘導体を患部（腫瘍局所）に適用する際に、架橋剤により表面をゲル化させ、あるいは全体がゲル化するようあらかじめ架橋剤と混合して適用すると、本発明のアルギン酸誘導体は患部（腫瘍局所）で硬化し、適用した患部（腫瘍局所）に密着した状態で局在させることができる。 In the present invention, for example, an alginic acid derivative may be applied to the abdominal cavity that is an affected area (tumor local area) of peritoneal metastasis. In addition, if an appropriate viscosity cannot be maintained after application, a crosslinking agent may be added to the surface of the applied derivative. Leakage from the abdominal cavity can be effectively prevented by gelling the surface of the derivative and solidifying the surface.
When an alginic acid derivative is first administered to an affected area (tumor local area) and a cross-linking agent is added later, it is desirable that the cross-linking agent gradually penetrates from the surface of the applied composition to promote cross-linking. In order not to exert a strong influence of the cross-linking agent on the contact part with the affected part (tumor local area), the application amount of the cross-linking agent is adjusted so as not to be excessive. The application amount of the divalent or higher metal ion is not particularly limited as long as it can solidify the surface of the composition containing the monovalent metal salt of alginic acid.
When applying the alginic acid derivative of the present invention to the affected area (tumor local area), if the surface is gelled with a cross-linking agent, or mixed with a cross-linking agent in advance so that the whole is gelled, the alginic acid derivative of the present invention is applied to the affected area. It is hardened at (tumor local) and can be localized in close contact with the affected part (tumor local) applied.
　また、アルギン酸誘導体ゲルを徐放性医薬組成物に使用する場合には、時間差、温度差、あるいは生体内のカルシウムイオンとの接触などの環境の変化によりゲル化を進める架橋剤の濃度を調整し、例えば投与前は液体状態を維持し、生体内への投与後に自己ゲル化する組成物とすることもできる。このような架橋剤としては、グルコン酸カルシウム、ＣａＳＯ4、アルギン酸カルシウム塩などを挙げることができる。 In addition, when using alginic acid derivative gels in sustained-release pharmaceutical compositions, the concentration of the cross-linking agent that promotes gelation is adjusted by changes in the environment such as time differences, temperature differences, or contact with calcium ions in vivo. For example, a composition that maintains a liquid state before administration and self-gelates after administration into a living body can be used. Examples of such a crosslinking agent include calcium gluconate, CaSO 4 , calcium alginate, and the like.
　また、アルギン酸誘導体を含む医薬組成物に２価以上の金属イオンを加える方法としては、特に限定されないが、例えば、シリンジ、噴射器（スプレー）などで、２価以上の金属イオンの溶液を組成物表面にかける方法などを挙げることができる。本発明の組成物の表面に架橋剤を適用するタイミングは、患部（腫瘍局所）に本発明の組成物を適用した後でもよいし、同時でもよい。 In addition, the method for adding a divalent or higher metal ion to a pharmaceutical composition containing an alginic acid derivative is not particularly limited. For example, a solution of a divalent or higher metal ion can be prepared with a syringe or a sprayer Examples include a method of applying to the surface. The timing of applying the cross-linking agent to the surface of the composition of the present invention may be after the composition of the present invention is applied to the affected area (tumor local) or simultaneously.
　また、本発明のアルギン酸誘導体ゲルを含む徐放性医薬組成物においては、例えば５００μｍ未満の平均粒径を有するマイクロビーズの形態で含んでいてもよい。 The sustained-release pharmaceutical composition containing the alginic acid derivative gel of the present invention may contain microbeads having an average particle size of less than 500 μm, for example.
　次に、本発明をさらに詳細に説明するために実施例、試験例をあげるが、これらの例は単なる実施であって、本発明を限定するものではなく、また本発明の範囲を逸脱しない範囲で変化させてもよい。
　核磁気共鳴スペクトル（ＮＭＲ）の測定には、ＪＥＯＬ　ＪＮＭ－ＥＣＸ４００　ＦＴ－ＮＭＲ（日本電子）を用いた。液体クロマトグラフィー－質量分析スペクトル（ＬＣ－Ｍａｓｓ）は以下の方法で測定した。［ＵＰＬＣ］Ｗａｔｅｒｓ　ＡＱＵＩＴＹ　ＵＰＬＣシステムおよびＢＥＨ　Ｃ１８カラム（２．１ｍｍ×５０ｍｍ、１．７μｍ）（Ｗａｔｅｒｓ）あるいはＢＥＨ　ｐｈｅｎｙｌカラム（２．１ｍｍ×５０ｍｍ、１．７μｍ）を用い、アセトニトリル：０．０５％トリフルオロ酢酸水溶液＝５：９５（０分）～９５：５（１．０分）～９５：５（１．６分）～５：９５（２．０分）の移動相およびグラジエント条件を用いた。
　1Ｈ－ＮＭＲデータ中、ＮＭＲシグナルのパターンで、ｓはシングレット、ｄはダブレット、ｔはトリプレット、ｑはカルテット、ｍはマルチプレット、ｂｒはブロード、Ｊはカップリング定数、Ｈｚはヘルツ、ＣＤＣｌ3は重クロロホルム、Ｄ2Ｏは重水、ＣＤ3ＯＤは重メタノールを意味する。1Ｈ－ＮＭＲデータ中、水酸基（ＯＨ）、アミノ基（ＮＨ2）、カルボキシル基（ＣＯＯＨ）のプロトン等、ブロードバンドであるため確認ができないシグナルについては、データに記載していない。
　実施例中の「室温」は、通常約０℃から約３５℃の温度を示すものとする。
　実施例中の薬剤（カンプトテシン誘導体）導入率（モル％）は、1Ｈ－ＮＭＲ（Ｄ2Ｏ）から算出されたアルギン酸を構成するＤ－マンヌロン酸又はＬ－グルロン酸の単糖を１単位（モル）とし、アルギン酸を構成する単糖１００単位（モル）に対する導入された薬剤のモル数の割合を示すものとする。 Next, in order to explain the present invention in more detail, examples and test examples will be given. However, these examples are merely implementations, and do not limit the present invention, and do not depart from the scope of the present invention. It may be changed with.
JEOL JNM-ECX400 FT-NMR (JEOL) was used for measurement of nuclear magnetic resonance spectrum (NMR). The liquid chromatography-mass spectrometry spectrum (LC-Mass) was measured by the following method. [UPLC] Using a Waters AQUITY UPLC system and a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) (Waters) or a BEH phenyl column (2.1 mm × 50 mm, 1.7 μm), acetonitrile: 0.05% tri Aqueous fluoroacetic acid = mobile phase and gradient conditions from 5:95 (0 min) to 95: 5 (1.0 min) to 95: 5 (1.6 min) to 5:95 (2.0 min) were used. .
1 H-NMR data, NMR signal pattern, s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, br is broad, J is coupling constant, Hz is Hertz, CDCl 3 Represents deuterated chloroform, D 2 O represents deuterated water, and CD 3 OD represents deuterated methanol. In 1 H-NMR data, signals that cannot be confirmed due to broadband such as hydroxyl (OH), amino group (NH 2 ), and carboxyl group (COOH) protons are not described in the data.
“Room temperature” in the examples generally indicates a temperature of about 0 ° C. to about 35 ° C.
The introduction rate (mol%) of the drug (camptothecin derivative) in the examples is 1 unit of monosaccharide of D-mannuronic acid or L-guluronic acid constituting alginic acid calculated from 1 H-NMR (D 2 O) ( Mol) and the ratio of the number of moles of the introduced drug to 100 units (mol) of monosaccharides constituting alginic acid.
　分子量の測定及び算出は、以下のいずれかの方法にて行なった。
＜実施例１～４：方法Ａ＞
　実施例中で得られた本発明に係るアルギン酸誘導体固体を秤量し、１０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．７）を加え室温で１時間以上撹拌・溶解後、希釈し、０．０５％溶液を調製した。この溶液を孔径０．２２μｍの親水性ＰＶＤＦ製ろ過フィルター（ＭｙｌｅｘＧＶ３３　フィルター、Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ社）に通し不溶物を除いた後、この２００μＬをＳｕｐｅｒｏｓｅ６　Ｉｎｃｒｅａｓｅ　１０／３００　ＧＬカラム（ＧＥヘルスケア社）に供しゲルろ過を実施した。ゲルろ過は、クロマトグラフ装置としてＡＫＴＡ　Ｅｘｐｌｏｒｅｒ１０Ｓを、展開溶媒として１０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．７）を使用し、室温で流速０．８ｍＬ／ｍｉｎの条件で実施した。各試料のクロマトグラムは、波長２２０ｎｍの吸光度をモニターし作製した。得られたクロマトグラムは、Ｕｎｉｃｏｒｎ　５．３１ソフトウエア（ＧＥヘルスケア社）にて解析し、ピークの溶出範囲を決定した。 The molecular weight was measured and calculated by any of the following methods.
<Examples 1 to 4: Method A>
The alginate derivative solid according to the present invention obtained in the examples was weighed, added with 10 mmol / L phosphate buffer (pH 7.7), stirred and dissolved at room temperature for 1 hour or more, diluted, and 0.05% solution Was prepared. This solution was passed through a hydrophilic PVDF filtration filter (Mylex GV33 filter, Merck Millipore) having a pore size of 0.22 μm to remove insoluble materials, and then 200 μL of the solution was applied to a Superose 6 Increase 10/300 GL column (GE Healthcare). Filtration was performed. Gel filtration was carried out using AKTA Explorer 10S as the chromatographic apparatus and 10 mmol / L phosphate buffer (pH 7.7) as the developing solvent at a flow rate of 0.8 mL / min at room temperature. The chromatogram of each sample was prepared by monitoring the absorbance at a wavelength of 220 nm. The obtained chromatogram was analyzed with Unicorn 5.31 software (GE Healthcare) to determine the peak elution range.
　本発明に係るアルギン酸誘導体の分子量は、以下の方法にて求めた。ブルーデキストラン（分子量２００万Ｄａ、ＳＩＧＭＡ社）、チログロブリン（分子量６６．９万Ｄａ、ＧＥヘルスケア社）、フェリチン（分子量４４万Ｄａ、ＧＥヘルスケア社）、コンアルブミン（分子量７．５万Ｄａ、ＧＥヘルスケア社）、及びリボヌクレアーゼＡ（分子量１．３７万Ｄａ、ＧＥヘルスケア社）を標準品として用い、試料と同じ条件でゲルろ過を行い、各成分の溶出液量を決定した。各成分の溶出液量を横軸に、分子量の対数値を縦軸にそれぞれプロットし、２次回帰し、検量線を作成した。この検量線を用いて、先に得られた試料のクロマトグラムの溶出時間ｉにおける分子量（Ｍｉ）を計算した。次いで、溶出時間ｉにおける吸光度を読み取り、Ｈｉとした。これらのデータから重量平均分子量（Ｍｗ）を以下の式から求めた。
Figure JPOXMLDOC01-appb-M000004
 The molecular weight of the alginic acid derivative according to the present invention was determined by the following method. Blue dextran (molecular weight 2 million Da, SIGMA), thyroglobulin (molecular weight 66.9 million Da, GE Healthcare), ferritin (molecular weight 440,000 Da, GE Healthcare), conalbumin (molecular weight 75,000 Da) GE Healthcare) and ribonuclease A (molecular weight: 1.37,000 Da, GE Healthcare) were used as standard products, and gel filtration was performed under the same conditions as the sample to determine the amount of each solution. The amount of eluate of each component was plotted on the horizontal axis, and the logarithmic value of molecular weight was plotted on the vertical axis, followed by quadratic regression to create a calibration curve. Using this calibration curve, the molecular weight (Mi) at the elution time i in the chromatogram of the previously obtained sample was calculated. Next, the absorbance at the elution time i was read and taken as Hi. From these data, the weight average molecular weight (Mw) was determined from the following equation.
Figure JPOXMLDOC01-appb-M000004
　原料のアルギン酸又はその塩の分子量は、以下の方法にて求めた。各アルギン酸を、乾燥減量を考慮して秤量し、超純水を加えて１％水溶液を調製した。次いで、終濃度１０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．７）となるように、１００ｍｍｏｌ／Ｌリン酸緩衝液と超純水により希釈し、０．０５％溶液を調製した。不溶物を孔径０．２２μｍの親水性ＰＶＤＦ製ろ過フィルター（ＭｙｌｅｘＧＶ３３　フィルター、Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ社）により除いた後、２００μＬをゲルろ過に供し、本発明に係るアルギン酸誘導体と同様の条件でゲルろ過を実施した。検出は示差屈折計により実施し、本発明に係るアルギン酸誘導体と同様の方法で重量平均分子量（Ｍｗ）を求めた。 The molecular weight of the raw material alginic acid or a salt thereof was determined by the following method. Each alginic acid was weighed in consideration of loss on drying, and ultrapure water was added to prepare a 1% aqueous solution. Subsequently, it diluted with a 100 mmol / L phosphate buffer and ultrapure water so that it might become a final concentration of 10 mmol / L phosphate buffer (pH 7.7), and prepared 0.05% solution. Insoluble matter was removed by a hydrophilic PVDF filter with a pore size of 0.22 μm (Mylex GV33 filter, Merck Millipore), 200 μL was subjected to gel filtration, and gel filtration was performed under the same conditions as the alginic acid derivative according to the present invention. . Detection was carried out with a differential refractometer, and the weight average molecular weight (Mw) was determined in the same manner as the alginic acid derivative according to the present invention.
＜実施例６～１５：方法Ｂ＞
　アルギン酸誘導体固体を秤量し、１０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７～７．４）を加え室温で１時間以上撹拌・溶解後、希釈し、０．２ｗ／ｖ％溶液を調製した。この溶液を０．４５μｍの再生セルロース製ろ過フィルター（ザルトリウス・ステディム社）に通し不溶物を除いた後、２００μＬを前記方法Ａと同様にゲルろ過に供し、重量平均分子量を求めた。但し、展開溶媒として１０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７～７．４）を使用し、モニターは波長２５６ｎｍの吸光度にて実施した。標準品は、ブルーデキストラン（分子量２００万Ｄａ、ＧＥヘルスケア社）、チログロブリン（分子量６６．９万Ｄａ、ＧＥヘルスケア社）、フェリチン（分子量４４万Ｄａ、ＧＥヘルスケア社）、コンアルブミン（分子量７．５万Ｄａ、ＧＥヘルスケア社）、カーボニックアンハイドロラーゼ（分子量２．３万Ｄａ、ＧＥヘルスケア社）及びアプロチニン（分子量６５００Ｄａ，ＧＥヘルスケア社）を用いた。
　原料のアルギン酸又はその塩は、終濃度１０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７～７．４）となるように、１％水溶液を１００ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７）と超純水により希釈し、０．２％溶液を調製した。この溶液を０．４５μｍの再生セルロース製ろ過フィルター（ザルトリウス・ステディム社）に通し不溶物を除いた後、２００μＬをアルギン酸誘導体（方法Ｂ）と同様の条件でゲルろ過を実施した。検出は示差屈折計により実施し、アルギン酸誘導体（方法Ｂ）と同様の方法で重量平均分子量を求めた。
　なお、本実施例のスキームにおける「ＡＬ」とは、アルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖の－Ｃ（＝Ｏ）基を有する残基を意味する。 <Examples 6 to 15: Method B>
The alginate derivative solid was weighed, 10 mmol / L phosphate buffer (pH 7 to 7.4) was added, stirred and dissolved at room temperature for 1 hour or more, and then diluted to prepare a 0.2 w / v% solution. This solution was passed through a 0.45 μm regenerated cellulose filtration filter (Sartorius Stedim) to remove insoluble matters, and then 200 μL was subjected to gel filtration in the same manner as in Method A, and the weight average molecular weight was determined. However, 10 mmol / L phosphate buffer (pH 7 to 7.4) was used as a developing solvent, and monitoring was performed at an absorbance of a wavelength of 256 nm. Standard products include blue dextran (molecular weight 2 million Da, GE Healthcare), thyroglobulin (molecular weight 66.9 million Da, GE Healthcare), ferritin (molecular weight 440,000 Da, GE Healthcare), conalbumin ( Molecular weight 75,000 Da, GE Healthcare), carbonic anhydrolase (molecular weight 23,000 Da, GE Healthcare) and aprotinin (molecular weight 6500 Da, GE Healthcare) were used.
The raw material alginic acid or salt thereof is diluted with 100 mmol / L phosphate buffer (pH 7) and ultrapure water so that the final concentration is 10 mmol / L phosphate buffer (pH 7 to 7.4). A 0.2% solution was prepared. This solution was passed through a 0.45 μm regenerated cellulose filtration filter (Sartorius Stedim) to remove insoluble matters, and then 200 μL of the solution was subjected to gel filtration under the same conditions as the alginic acid derivative (Method B). Detection was carried out with a differential refractometer, and the weight average molecular weight was determined in the same manner as the alginic acid derivative (Method B).
In the scheme of this example, “AL” is a residue derived from alginic acid or a salt thereof, and is a monosaccharide —C (1) of one of L-guluronic acid and D-mannuronic acid constituting alginic acid. = O) means a residue having a group.
（実施例１）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－アミノプロパン酸基導入アルギン酸（化合物１－４）の合成
スキーム１
Figure JPOXMLDOC01-appb-C000005
 Example 1 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis scheme 1 of [1,2-b] quinolin-9-yl 3-aminopropanoic acid group-introduced alginic acid (compound 1-4)
Figure JPOXMLDOC01-appb-C000005
＜工程１＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－アミノプロパン酸塩酸塩（化合物１－３）の合成
　市販の３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）プロパン酸［ＣＡＳ：３３０３－８４－２］（５３．０ｍｇ）とトリエチルアミン（３９．１μＬ）をテトラヒドロフラン（３．０ｍＬ）に溶解させた。続いて、氷水冷撹拌下、クロロギ酸イソブチル（３６．８μＬ）を加えた。この溶液を室温で３０分撹拌した。この反応液に対し、（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物１－１，１００ｍｇ）とトリエチルアミン（３９．１μＬ）のテトラヒドロフラン（２．０ｍＬ）溶液を、氷水冷撹拌下加え、室温で１８時間撹拌した。反応液を濾過し、生じた濾液を減圧下留去し、化合物１－２の粗生成物（１７２ｍｇ）を得た。
　化合物１－２の粗生成物（１７２ｍｇ）を含む容器に対して、氷冷下撹拌下で、４規定－塩化水素／１，４－ジオキサン（２．０ｍＬ）を加えた。この溶液に対し、更に、１，４－ジオキサン（２．０ｍＬ）を加えた。反応液を室温で１６時間攪拌し、ジイソプロピルエーテル（２０ｍＬ）を加えた。析出した固体をろ取し、ジイソプロピルエーテルで洗浄した後、減圧下乾燥することにより化合物１－１と標記化合物１－３を含む混合物（１３１．９ｍｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．６６　（１Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　７．４９　（１Ｈ，　ｓ），　７．２７　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．１１　（１Ｈ，　ｓ），　５．４１　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．２６　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　４．５２　（２Ｈ，　ｓ），　３．２９　（２Ｈ，　ｔ，　Ｊ　＝　６．６　Ｈｚ），　３．０４　（２Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），　２．８７－２．７５　（２Ｈ，　ｍ），　１．８９－１．８４　（２Ｈ，　ｍ），　１．０９　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　０．８７　（３Ｈ，　ｔ，　Ｊ　＝　７．３　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝４６３、ＲＴ＝０．５８（分），［Ｍ＋Ｈ］+＝４６４ <Step 1> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl 3-aminopropanoic acid hydrochloride (Compound 1-3) Commercially available 3-((tert-butoxycarbonyl) amino) propanoic acid [CAS: 3303-84-2] (53.0 mg) and triethylamine (39.1 μL) were dissolved in tetrahydrofuran (3.0 mL). Subsequently, isobutyl chloroformate (36.8 μL) was added under ice-water cooling and stirring. The solution was stirred at room temperature for 30 minutes. To this reaction solution, (S) -4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] A solution of quinoline-3,14 (4H) -dione [CAS: 86639-52-3] (compound 1-1,100 mg) and triethylamine (39.1 μL) in tetrahydrofuran (2.0 mL) was added with ice-water cooling and stirring. Stir at room temperature for 18 hours. The reaction solution was filtered, and the resulting filtrate was distilled off under reduced pressure to obtain a crude product of compound 1-2 (172 mg).
To a container containing the crude product of compound 1-2 (172 mg), 4N-hydrogen chloride / 1,4-dioxane (2.0 mL) was added with stirring under ice cooling. To this solution was further added 1,4-dioxane (2.0 mL). The reaction was stirred at room temperature for 16 hours and diisopropyl ether (20 mL) was added. The precipitated solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to give a mixture (131.9 mg) containing Compound 1-1 and the title compound 1-3 as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.66 (1H, d, J = 8.8 Hz), 7.49 (1H, s), 7.27 (1H, d, J = 9. 2 Hz), 7.11 (1H, s), 5.41 (1H, d, J = 16.0 Hz), 5.26 (1H, d, J = 16.0 Hz), 4.52 (2H , S), 3.29 (2H, t, J = 6.6 Hz), 3.04 (2H, t, J = 7.2 Hz), 2.87-2.75 (2H, m), 1 .89-1.84 (2H, m), 1.09 (3H, t, J = 7.6 Hz), 0.87 (3H, t, J = 7.3 Hz), LC-MS: M ( free amine) = 463, RT = 0.58 (min), [M + H] + = 464
＜工程２＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－アミノプロパン酸基導入アルギン酸（化合物１－４）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：３２６万Ｄａ～２５．３万Ｄａ（ブロード）、重量平均分子量：１６４万Ｄａ）水溶液（１０．９ｍＬ）に、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（５５．８ｍｇ）、１モル濃度－重曹水（１５１．３μＬ）を加えた。この溶液に対し、氷冷撹拌下、（実施例１）＜工程１＞で得られた化合物１－１と化合物１－３の混合物（５０．４ｍｇ）のエタノール（２ｍＬ）溶液を加え、室温で４時間攪拌した。塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（２１．８ｍＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物１－４（９９ｍｇ）を淡黄色粉体として得た。薬剤導入率は１．２モル％であった。分子量は、２９１万Ｄａから６．６万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３３万Ｄａであった。 <Step 2> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 3-aminopropanoic acid group-introduced alginic acid (compound 1-4) 10,000 Da (broad), weight average molecular weight: 16.64 million Da) in aqueous solution (10.9 mL), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride (55.8 mg), 1 molar concentration-aqueous sodium bicarbonate (151.3 μL) were added. To this solution was added an ethanol (2 mL) solution of a mixture of compound 1-1 and compound 1-3 (50.4 mg) obtained in (Example 1) <Step 1> under ice-cooling and stirring at room temperature. Stir for 4 hours. Sodium chloride (100 mg) was added, ethanol (21.8 mL) was added, and the mixture was stirred at room temperature for 30 min. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 1-4 (99 mg) as a pale yellow powder. The drug introduction rate was 1.2 mol%. The molecular weight showed a broad elution peak from 2.91 million Da to 66,000 Da, and the weight average molecular weight was 1.33 million Da.
（実施例２）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　２－（２－（２－アミノエトキシ）エトキシ）酢酸基導入アルギン酸（化合物２－６）の合成
スキーム２
Figure JPOXMLDOC01-appb-C000006
 Example 2 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis Scheme 2 of [1,2-b] quinolin-9-yl 2- (2- (2-aminoethoxy) ethoxy) acetic Acid Group-Introduced Alginic Acid (Compound 2-6)
Figure JPOXMLDOC01-appb-C000006
＜工程１＞８－ｔｅｒｔ－ブトキシカルボニルアミノ－３，６－ジオキサオクタン酸（化合物２－２）の合成
　２－［２－（２－アミノエトキシ）エトキシ］酢酸［ＣＡＳ：１３４９７８－９７－５］（化合物２－１、３００ｍｇ）の１，４－ジオキサン（３０００μＬ）及び水（１２００μＬ）溶液に対して、氷冷撹拌下、重曹（３０８．９ｍｇ）を加えた。続いて、二炭酸ジ－ｔｅｒｔ－ブチル（６３３．６μＬ）を滴下し、室温で３時間撹拌した。反応液に水（３ｍＬ）を加え、室温で１時間撹拌した。続いて、１規定－塩酸（１０ｍＬ）で反応を停止させ、酢酸エチル（１０ｍＬ、３回）で抽出した。有機層を飽和食塩水（５ｍＬ）で洗浄し、無水硫酸ナトリウムで乾燥させた後、減圧下で溶媒を留去することで、標記化合物２－２（４４６ｍｇ）を無色オイル状物質として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：４．９５　（１Ｈ，　ｂｒ　ｓ），　４．１７　（２Ｈ，　ｓ），　３．７８－３．７６　（２Ｈ，　ｍ），　３．６７－３．６６　（２Ｈ，　ｍ），　３．５９－３．５８　（２Ｈ，　ｍ），　３．３５－３．３４　（２Ｈ，　ｍ），　１．４５　（９Ｈ，　ｓ）． <Step 1> Synthesis of 8-tert-butoxycarbonylamino-3,6-dioxaoctanoic acid (Compound 2-2) 2- [2- (2-aminoethoxy) ethoxy] acetic acid [CAS: 134978-97-5 ] To a 1,4-dioxane (3000 μL) and water (1200 μL) solution of (Compound 2-1, 300 mg), sodium bicarbonate (308.9 mg) was added with stirring under ice cooling. Subsequently, di-tert-butyl dicarbonate (633.6 μL) was added dropwise, and the mixture was stirred at room temperature for 3 hours. Water (3 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. Subsequently, the reaction was quenched with 1N hydrochloric acid (10 mL), and extracted with ethyl acetate (10 mL, 3 times). The organic layer was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give the title compound 2-2 (446 mg) as a colorless oil.
NMR data (CDCl 3 ) (δ: ppm): 4.95 (1H, br s), 4.17 (2H, s), 3.78-3.76 (2H, m), 3.67-3. 66 (2H, m), 3.59-3.58 (2H, m), 3.35-3.34 (2H, m), 1.45 (9H, s).
＜工程２＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　２－（２－（２－アミノエトキシ）エトキシ）酢酸　塩酸塩（化合物２－５）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物２－３，１００ｍｇ）、（実施例２）＜工程１＞で得られた化合物２－２（７０．５ｍｇ）、ベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１１８．４ｍｇ）のジクロロメタン（５０００μＬ）溶液に、氷冷撹拌下トリエチルアミン（７４．６μＬ）を滴下し、室温で１７時間撹拌した。この溶液を濾過し、得られた濾液を減圧下で濃縮した。残留物をシリカゲルカラムクロマトグラフィー（２５％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル、酢酸エチル～１０％メタノール／酢酸エチル）で精製し、化合物２－４を含む画分（１２７．４ｍｇ）を得た。
　化合物２－４を含む画分（１２７．４ｍｇ）を１，４－ジオキサン（０．８６ｍＬ）に溶解させた。この溶液に対し、氷冷撹拌下、４規定－塩化水素／１，４－ジオキサン（０．８６ｍＬ）を加え、室温で３時間撹拌した。反応液を濾過し、回収した固体をジイソプロピルエーテル（１０ｍＬ）でトリチュレートした。得られた固体をろ取し、ジイソプロピルエーテルで洗浄後、減圧乾燥して、標記化合物２－５（１２３ｍｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：７．６８　（１Ｈ，　ｄ，　Ｊ　＝　９．６　Ｈｚ），　７．４６　（１Ｈ，　ｓ），　７．２７　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．１１　（１Ｈ，　ｓ），　５．４０　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．２６　（１Ｈ，　ｄ，　Ｊ　＝　１６．６　Ｈｚ），　４．５５　（２Ｈ，　ｓ），　４．４４　（２Ｈ，　ｓ），　３．７７－３．７６　（２Ｈ，　ｍ），　３．６７－３．６６　（４Ｈ，　ｍ），　３．１０　（２Ｈ，　ｔ，　Ｊ　＝　４．８　Ｈｚ），　２．８７－２．７６　（２Ｈ，　ｍ），　１．８５－１．８３　（２Ｈ，　ｍ），　１．０９　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　０．８６　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５３７，ＲＴ＝０．６２（分），［Ｍ＋Ｈ］+＝５３８ <Step 2> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] quinolin-9-yl 2- (2- (2-aminoethoxy) ethoxy) acetic acid Synthesis of hydrochloride (compound 2-5) (S) -4,11-diethyl-4,9-dihydroxy -1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52-3] (compound (3-3, 100 mg), (Example 2) Compound 2-2 (70.5 mg) obtained in <Step 1>, benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (118.4 mg) in dichloromethane ( 5000 μL) The liquid was added dropwise with stirring under ice cooling triethylamine (74.6μL), and stirred at room temperature for 17 hours. The solution was filtered and the resulting filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (25% ethyl acetate / n-heptane to 100% ethyl acetate, ethyl acetate to 10% methanol / ethyl acetate), and a fraction (127.4 mg) containing compound 2-4 was obtained. Obtained.
The fraction containing compound 2-4 (127.4 mg) was dissolved in 1,4-dioxane (0.86 mL). To this solution was added 4N-hydrogen chloride / 1,4-dioxane (0.86 mL) with stirring under ice cooling, and the mixture was stirred at room temperature for 3 hours. The reaction solution was filtered, and the collected solid was triturated with diisopropyl ether (10 mL). The obtained solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to give the title compound 2-5 (123 mg) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.68 (1H, d, J = 9.6 Hz), 7.46 (1H, s), 7.27 (1H, d, J = 9. 2 Hz), 7.11 (1H, s), 5.40 (1H, d, J = 16.0 Hz), 5.26 (1H, d, J = 16.6 Hz), 4.55 (2H , S), 4.44 (2H, s), 3.77-3.76 (2H, m), 3.67-3.66 (4H, m), 3.10 (2H, t, J = 4 .8 Hz), 2.87-2.76 (2H, m), 1.85-1.83 (2H, m), 1.09 (3H, t, J = 7.6 Hz), 0.86 (3H, t, J = 7.6 Hz), LC-MS: M (free amine) = 537, RT = 0.62 (min), [M + H] + = 538
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　２－（２－（２－アミノエトキシ）エトキシ）酢酸基導入アルギン酸（化合物２－６）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：３２６万Ｄａ～２５．３万Ｄａ（ブロード）、重量平均分子量：１６４万Ｄａ）水溶液（１０．９ｍＬ）と４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（５５．８ｍｇ）の混合物に対し、１モル濃度－重曹水（１００．９μＬ）を加えた。この溶液に対し、氷冷撹拌下、（実施例２）＜工程２＞で得られた化合物２－５（５７．９ｍｇ）のエタノール（２ｍＬ）溶液を加えた。更に、エタノール（２ｍＬ）を加え、室温で１７時間攪拌した。塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（２１．８ｍＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物２－６（１０２ｍｇ）を淡黄色粉体として得た。薬剤導入率は３．０モル％であった。分子量は、３０９万Ｄａから３１．３万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１８３万Ｄａであった。 <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 2- (2- (2-aminoethoxy) ethoxy) acetic acid group-introduced alginic acid (compound 2-6) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd.) Molecular weight: 32.6 million Da to 253,000 Da (broad), weight average molecular weight: 1.64 million Da) aqueous solution (10.9 mL) and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl ) -4-Methylmorpholinium chloride (55.8 mg) was added to a 1 molar concentration-aqueous sodium bicarbonate solution (100.9 μL). To this solution was added an ethanol (2 mL) solution of compound 2-5 (57.9 mg) obtained in (Example 2) <Step 2> under ice-cooling and stirring. Furthermore, ethanol (2 mL) was added, and the mixture was stirred at room temperature for 17 hours. Sodium chloride (100 mg) was added, ethanol (21.8 mL) was added, and the mixture was stirred at room temperature for 30 min. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 2-6 (102 mg) as a pale yellow powder. The drug introduction rate was 3.0 mol%. The molecular weight showed a broad elution peak from 309,000 Da to 313,000 Da, and the weight average molecular weight was 1.83 million Da.
（実施例３）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（アミノメチル）シクロヘキサン－１－カルボン酸基導入アルギン酸（化合物３－４）の合成
スキーム３
Figure JPOXMLDOC01-appb-C000007
 Example 3 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis scheme 3 of [1,2-b] quinolin-9-yl 4- (aminomethyl) cyclohexane-1-carboxylic acid group-introduced alginic acid (compound 3-4)
Figure JPOXMLDOC01-appb-C000007
＜工程１＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（（（ｔｅｒｔ－ブトキシカルボニル）アミノ）メチル）シクロヘキサン－１－カルボン酸（化合物３－２）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物３－１，１５０ｍｇ）及び市販のトランス－４－（ｔｅｒｔ－ブトキシカルボニルアミノメチル）シクロヘキサンカルボン酸［ＣＡＳ：２７６８７－１４－５］（１０３．３ｍｇ）のジクロロメタン（９０００μＬ）溶液に、氷冷撹拌下、ベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１７７．５ｍｇ）及びトリエチルアミン（１１１．９μＬ）を加え、室温で１７時間撹拌した。反応液を減圧下濃縮し、残留物をシリカゲルカラムクロマトグラフィー（２５％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、標記化合物３－２（１８２ｍｇ）を淡黄色固体として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：８．２３　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．８０　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　７．６４　（１Ｈ，　ｓ），　７．５３　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．０　Ｈｚ），　５．７６　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．３１　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．２６　（２Ｈ，　ｓ），　４．６２　（１Ｈ，　ｂｒ　ｓ），　３．７２　（１Ｈ，　ｓ），　３．１６　（２Ｈ，　ｑ，　Ｊ　＝　７．６　Ｈｚ），　３．０５－３．０４　（２Ｈ，　ｍ），　２．６３－２．５６　（１Ｈ，　ｍ），　２．２６　（２Ｈ，　ｄ，　Ｊ　＝　１１．４　Ｈｚ），　１．９５－１．８２　（４Ｈ，　ｍ），　１．６７－１．６１　（２Ｈ，　ｍ），　１．４６－１．３８　（１３Ｈ，　ｍ），　１．１４－１．０２　（５Ｈ，　ｍ），ＬＣ－ＭＳ：Ｍ＝６３１，ＲＴ＝１．０３（分），［Ｍ＋Ｈ］+＝６３２ <Step 1> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl 4-(((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid (compound 3-2) (S) -4,11-diethyl-4 , 9-Dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52- 3] (Compound 3-1 150 mg) and commercially available trans-4- (tert-butoxycarbonylaminomethyl) cyclohexanecarboxylic acid [CAS: 27687-14-5] (103.3 mg) Methane (9000μL) solution, with stirring under ice cooling, benzotriazol-1-yloxy - tris (dimethylamino) phosphonium salt (177.5mg) and triethylamine (111.9μL) was added and stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (25% ethyl acetate / n-heptane to 100% ethyl acetate) to give the title compound 3-2 (182 mg) as a pale yellow solid.
NMR data (CDCl 3 ) (δ: ppm): 8.23 (1H, d, J = 9.2 Hz), 7.80 (1H, d, J = 2.4 Hz), 7.64 (1H, s), 7.53 (1H, dd, J = 9.2, 2.0 Hz), 5.76 (1H, d, J = 16.0 Hz), 5.31 (1H, d, J = 16) .0 Hz), 5.26 (2H, s), 4.62 (1H, br s), 3.72 (1H, s), 3.16 (2H, q, J = 7.6 Hz), 3 .05-3.04 (2H, m), 2.63-2.56 (1H, m), 2.26 (2H, d, J = 11.4 Hz), 1.95-1.82 (4H M), 1.67-1.61 (2H, m), 1.46-1.38 (13H, m), 1.14-1.02 (5H, m), LC -MS: M = 631, RT = 1.03 (min), [M + H] + = 632
＜工程２＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（アミノメチル）シクロヘキサン－１－カルボン酸　塩酸塩（化合物３－３）の合成
　（実施例３）＜工程１＞で得られた化合物３－２（１８２ｍｇ）を１，４－ジオキサン（１．８ｍＬ）に溶解させた。この溶液に対し、氷冷撹拌下、４規定－塩化水素／１，４－ジオキサン（１．８ｍＬ）を加え、反応液を室温で１７時間攪拌し、ジイソプロピルエーテル（３０ｍＬ）を加えた。析出した固体をろ取し、ジイソプロピルエーテルで洗浄した後、減圧下乾燥することにより標記化合物３－３（１７１．５ｍｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：７．５１　（１Ｈ，　ｂｒ　ｓ），　７．３１－７．２５　（１Ｈ，　ｂｒ　ｍ），　７．１０－７．０２　（２Ｈ，　ｂｒ　ｍ），　５．３９　（１Ｈ，　ｄ，　Ｊ　＝　１５．６　Ｈｚ），　５．２３　（１Ｈ，　ｄ，　Ｊ　＝　１５．６　Ｈｚ），　２．８１－２．７１　（４Ｈ，　ｍ），　２．３９　（１Ｈ，　ｂｒ　ｓ），　１．９８　（２Ｈ，　ｂｒ　ｓ），　１．８４－１．８１　（４Ｈ，　ｂｒ　ｍ），　１．６０　（１Ｈ，　ｂｒ　ｓ），　１．３６－１．３３　（２Ｈ，　ｂｒ　ｍ），　１．０７－１．００　（７Ｈ，　ｍ），　０．８６　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５３１，ＲＴ＝０．６５（分），［Ｍ＋Ｈ］+＝５３２ <Step 2> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl 4- (aminomethyl) cyclohexane-1-carboxylic acid Synthesis of hydrochloride (Compound 3-3) (Example 3) Compound 3-2 obtained in <Step 1> (182 mg) was dissolved in 1,4-dioxane (1.8 mL). To this solution was added 4N-hydrogen chloride / 1,4-dioxane (1.8 mL) with stirring under ice cooling, and the reaction mixture was stirred at room temperature for 17 hours, and diisopropyl ether (30 mL) was added. The precipitated solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to give the title compound 3-3 (171.5 mg) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.51 (1H, br s), 7.31-7.25 (1H, br m), 7.10-7.02 (2H, br m) , 5.39 (1H, d, J = 15.6 Hz), 5.23 (1H, d, J = 15.6 Hz), 2.81-2.71 (4H, m), 2.39 ( 1H, br s), 1.98 (2H, br s), 1.84 to 1.81 (4H, br m), 1.60 (1H, br s), 1.36-1.33 (2H, br m), 1.07-1.00 (7H, m), 0.86 (3H, t, J = 7.2 Hz), LC-MS: M (free amine) = 531, RT = 0.65 (Minutes), [M + H] + = 532
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（アミノメチル）シクロヘキサン－１－カルボン酸基導入アルギン酸（化合物３－４）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：３２６万Ｄａ～２５．３万Ｄａ（ブロード）、重量平均分子量：１６４万Ｄａ）水溶液（１０．９ｍＬ）に、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（５５．８ｍｇ）、１モル濃度－重曹水（３０２．６μＬ）を加えた。この溶液に対し、（実施例３）＜工程２＞で得られた化合物３－３（５７．３ｍｇ）のエタノール（１ｍＬ）及び水（１ｍＬ）溶液を加え、室温で１９時間攪拌した。塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（２１．８ｍＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物３－４（１０４ｍｇ）を淡黄色固体として得た。薬剤導入率は１．４モル％であった。分子量は、３２１万Ｄａから６．８万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３２万Ｄａであった。 <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl 4- (aminomethyl) cyclohexane-1-carboxylic acid group-introduced alginic acid (compound 3-4) sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd., molecular weight: 32.6 million Da to 25.3 million Da (broad), weight average molecular weight: 1.64 million Da) in aqueous solution (10.9 mL), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-Methylmorpholinium chloride (55.8 mg), 1 molar concentration-aqueous sodium bicarbonate (302.6 μL) were added. To this solution was added a solution of compound 3-3 (57.3 mg) obtained in Example 3 <Step 2> in ethanol (1 mL) and water (1 mL), and the mixture was stirred at room temperature for 19 hours. Sodium chloride (100 mg) was added, ethanol (21.8 mL) was added, and the mixture was stirred at room temperature for 30 min. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 3-4 (104 mg) as a pale yellow solid. The drug introduction rate was 1.4 mol%. The molecular weight showed a broad elution peak from 321,000 Da to 68,000 Da, and the weight average molecular weight was 1.32 Da.
（実施例４）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　５－アミノペンタン酸基導入アルギン酸（化合物４－５）の合成
スキーム４
Figure JPOXMLDOC01-appb-C000008
 Example 4 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis scheme 4 of [1,2-b] quinolin-4-yl 5-aminopentanoic acid group-introduced alginic acid (compound 4-5)
Figure JPOXMLDOC01-appb-C000008
＜工程１＞（Ｓ）－ｔｅｒｔ－ブチル（４，１１－ジエチル４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　炭酸エステル（化合物４－２）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物４－１，０．５ｇ）をピリジン（４ｍＬ）及びジクロロメタン（１２．５ｍＬ）に懸濁させた。この溶液に対し、氷冷撹拌下、二炭酸ジ－ｔｅｒｔ－ブチル（０．４４ｍＬ）を加え、室温で１時間１０分撹拌した。この反応液に対し、水（１０ｍＬ）を室温で加え、同温で１５時間撹拌した。有機層を無水硫酸ナトリウムで乾燥させた後、減圧下で溶媒を留去した。生じた残渣に対し、メチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）を加え、トリチュレートした。得られた固体を濾過し、減圧下で乾燥させることで、標記化合物４－２（０．５９ｇ）を淡黄色粉体として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：８．２３　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．９０　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），７．６６　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．４　Ｈｚ），　７．６４　（１Ｈ，　ｓ），　５．７６　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），５．３１　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．２６　（２Ｈ，　ｓ），　３．７１　（１Ｈ，　ｓ），　３．１６　（２Ｈ，　ｑ，　Ｊ　＝　７．６　Ｈｚ），１．９５－１．８４　（２Ｈ，　ｍ），　１．６１　（９Ｈ，　ｓ），　１．４０　（３Ｈ，　ｔ，　Ｊ　＝　７．８　Ｈｚ），　１．０４　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ） <Step 1> (S) -tert-butyl (4,11-diethyl 4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7 Synthesis of indolizino [1,2-b] quinolin-4-yl carbonate (compound 4-2) (S) -4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano [ 3 ′, 4 ′: 6,7] Indolizino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52-3] (compound 4-1, 0.5 g) 4 mL) and dichloromethane (12.5 mL) To this solution, di-tert-butyl dicarbonate (0.44 mL) was added with stirring under ice cooling, and the mixture was stirred at room temperature for 1 hour and 10 minutes. Water (10 mL) is added to the reaction solution. The organic layer was dried over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure, and methyl tert-butyl ether (20 mL) was added to the resulting residue and triturated. The obtained solid was filtered and dried under reduced pressure to obtain the title compound 4-2 (0.59 g) as a pale yellow powder.
NMR data (CDCl 3 ) (δ: ppm): 8.23 (1H, d, J = 9.2 Hz), 7.90 (1H, d, J = 2.4 Hz), 7.66 (1H, dd, J = 9.2, 2.4 Hz), 7.64 (1H, s), 5.76 (1H, d, J = 16.4 Hz), 5.31 (1H, d, J = 16 .0 Hz), 5.26 (2H, s), 3.71 (1H, s), 3.16 (2H, q, J = 7.6 Hz), 1.95-1.84 (2H, m ), 1.61 (9H, s), 1.40 (3H, t, J = 7.8 Hz), 1.04 (3H, t, J = 7.2 Hz)
＜工程２＞（Ｓ）－９－（（ｔｅｒｔ－ブトキシカルボニル）オキシ）－４，１１－ジエチル－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　５－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ペンタン酸　塩酸塩（化合物４－４）の合成
　（実施例４）＜工程１＞で得られた化合物４－２（１００ｍｇ）及び市販の５－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ペンタン酸［ＣＡＳ：２７２１９－０７－４］（６１．８ｍｇ）のジクロロメタン（２５００μＬ）溶液に対し、氷冷撹拌下、１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（５８．４ｍｇ）及びＮ，Ｎ－ジメチル－４－アミノピリジン（９．９ｍｇ）を加え、室温で１９時間撹拌した。反応液を減圧下濃縮し、残留物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、化合物４－３（９１ｍｇ）を含む画分を得た。
　化合物４－３を含む画分（９１ｍｇ）を１，４－ジオキサン（１．８ｍＬ）に溶解させた。この溶液に対し、氷冷撹拌下、４規定－塩化水素／１，４－ジオキサン（１．８ｍＬ）を加え、反応液を室温で１５時間攪拌し、ジイソプロピルエーテル（３０ｍＬ）を加えた。析出した固体をろ取し、ジイソプロピルエーテルで洗浄した後、減圧下乾燥することにより標記化合物４－４（６９ｍｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：７．６５　（１Ｈ，　ｄ，　Ｊ　＝　９．１　Ｈｚ），　７．１２　（１Ｈ，　ｄ，　Ｊ　＝　９．６　Ｈｚ），　６．９８　（１Ｈ，　ｓ），　６．４０　（１Ｈ，　ｓ），　５．４９　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．３０　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　３．８０　（１Ｈ，　ｄ，　Ｊ　＝　１９．６　Ｈｚ），　３．６８－３．６４　（１Ｈ，　ｍ），　２．９１　（２Ｈ，　ｔ，　Ｊ　＝　６．８　Ｈｚ），　２．６９－２．６２　（２Ｈ，　ｍ），　２．３５　（２Ｈ，　ｄ，　Ｊ　＝　６．９　Ｈｚ），　２．１３－２．１１　（２Ｈ，　ｍ），　１．６８－１．６７　（４Ｈ，　ｍ），　０．９６　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　０．９０　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝４９１，ＲＴ＝０．６５（分），［Ｍ＋Ｈ］+＝４９２ <Step 2> (S) -9-((tert-butoxycarbonyl) oxy) -4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ': 6,7] Synthesis of indolizino [1,2-b] quinolin-4-yl 5-((tert-butoxycarbonyl) amino) pentanoic acid hydrochloride (Compound 4-4) (Example 4) <Step 1 > To a dichloromethane (2500 μL) solution of compound 4-2 (100 mg) obtained in the above and commercially available 5-((tert-butoxycarbonyl) amino) pentanoic acid [CAS: 27219-07-4] (61.8 mg) Under ice-cooling, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (58.4 mg) and N, N-dimethyl-4-aminopyridine (9.9 mg) were added. The mixture was further stirred at room temperature for 19 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 4-3 (91 mg).
The fraction containing Compound 4-3 (91 mg) was dissolved in 1,4-dioxane (1.8 mL). To this solution was added 4N-hydrogen chloride / 1,4-dioxane (1.8 mL) with stirring under ice cooling, the reaction mixture was stirred at room temperature for 15 hours, and diisopropyl ether (30 mL) was added. The precipitated solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to give the title compound 4-4 (69 mg) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.65 (1H, d, J = 9.1 Hz), 7.12 (1H, d, J = 9.6 Hz), 6.98 (1H , S), 6.40 (1H, s), 5.49 (1H, d, J = 16.0 Hz), 5.30 (1H, d, J = 16.0 Hz), 3.80 (1H , D, J = 19.6 Hz), 3.68-3.64 (1H, m), 2.91 (2H, t, J = 6.8 Hz), 2.69-2.62 (2H, m), 2.35 (2H, d, J = 6.9 Hz), 2.13-2.11 (2H, m), 1.68-1.67 (4H, m), 0.96 (3H) , T, J = 7.6 Hz), 0.90 (3H, t, J = 7.6 Hz), LC-MS: M (free amine) = 491, RT = 0 65 (minutes), [M + H] + = 492
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　５－アミノペンタン酸基導入アルギン酸（化合物４－５）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：３２６万Ｄａ～２５．３万Ｄａ（ブロード）、重量平均分子量：１６４万Ｄａ）水溶液（１０．９ｍＬ）に、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（５５．８ｍｇ）、１モル濃度－重曹水（３０２．６μＬ）を加えた。この溶液に対し、（実施例４）＜工程２＞で得られた化合物４－４（５３．３ｍｇ）のエタノール（３ｍＬ）及び水（１ｍＬ）溶液を加え、４０度で３時間攪拌した。塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（２１．８ｍＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物４－５（１０４ｍｇ）を淡黄色粉体として得た。薬剤導入率は３．５モル％であった。分子量は、２８１万Ｄａから４．１万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１１３万Ｄａであった。 <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of 5-aminopentanoic acid group-introduced alginic acid (Compound 4-5) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd., molecular weight: 3.26 million Da to 25.3) 10,000 Da (broad), weight average molecular weight: 16.64 million Da) in aqueous solution (10.9 mL), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride (55.8 mg), 1 molarity-aqueous sodium bicarbonate (302.6 μL) were added. To this solution was added a solution of compound 4-4 (53.3 mg) obtained in (Example 4) <Step 2> in ethanol (3 mL) and water (1 mL), and the mixture was stirred at 40 degrees for 3 hours. Sodium chloride (100 mg) was added, ethanol (21.8 mL) was added, and the mixture was stirred at room temperature for 30 min. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 4-5 (104 mg) as a pale yellow powder. The drug introduction rate was 3.5 mol%. The molecular weight showed a broad elution peak from 2.81 million Da to 41,000 Da, and the weight average molecular weight was 1.13 million Da.
（実施例５）薬剤放出試験１
　実施例１から４で作製した各ＳＮ－３８結合アルギン酸誘導体１ｍｇに、各アルギン酸誘導体の濃度がそれぞれ０．１％ｗ／ｖ濃度となるように２０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．０）または１Ｎ水酸化ナトリウム水溶液を加え、マグネティックスターラー（ＡＳＯＮＥ　ＲＥＭＩＸ　ＲＳ－６Ａ、７５０ｒ．ｐ．ｍ．）を用いて、６時間撹拌した。ゲル状になっていないことを確認し、この溶液を分注した。溶解直後に初期状態（保存０日）として各溶液に存在した遊離ＳＮ－３８量をＬＣ－ＭＳ／ＭＳにて測定した。また、それ以外の分注液は３７℃で３日間インキュベートしたのちに遊離ＳＮ－３８量を測定した。各時点において、１Ｎ水酸化ナトリウム水溶液中の強制分解による遊離ＳＮ－３８量との比を用いて遊離率（％）を算出した。 Example 5 Drug Release Test 1
To 1 mg of each SN-38-conjugated alginate derivative prepared in Examples 1 to 4, 20 mM sodium phosphate buffer (pH 7.0) or 1N so that the concentration of each alginate derivative is 0.1% w / v. Aqueous sodium hydroxide was added, and the mixture was stirred for 6 hours using a magnetic stirrer (ASONE REMIX RS-6A, 750 rpm). After confirming that the gel was not formed, this solution was dispensed. Immediately after dissolution, the amount of free SN-38 present in each solution as an initial state (0 days of storage) was measured by LC-MS / MS. The other dispensed solutions were incubated at 37 ° C. for 3 days, and then the amount of free SN-38 was measured. At each time point, the release rate (%) was calculated using the ratio to the amount of free SN-38 by forced decomposition in 1N aqueous sodium hydroxide solution.
ＬＣ条件は以下の通り
温度：４０℃
流速：０．７ｍＬ／ｍｉｎ
カラム：ＯＤＳ－４：３μｍ（２．１×３０ｍｍ）
溶媒：（Ａ）０．１％　ギ酸水溶液、（Ｂ）１００％　アセトニトリル
グラジエント：

Figure JPOXMLDOC01-appb-I000009
LC conditions are as follows Temperature: 40 ° C
Flow rate: 0.7 mL / min
Column: ODS-4: 3 μm (2.1 × 30 mm)
Solvent: (A) 0.1% formic acid aqueous solution, (B) 100% acetonitrile gradient:

Figure JPOXMLDOC01-appb-I000009
ＭＳ条件は以下の通り
イオン化モード：ＥＳＩ－ｐｏｓｉｔｉｖｅ MS conditions are as follows: Ionization mode: ESI-positive
放出試験における遊離率
Figure JPOXMLDOC01-appb-T000010
 Release rate in release test
Figure JPOXMLDOC01-appb-T000010
　上記リリース試験の結果から、リンカーの構造によって徐放速度を調整でき、かつ、リンカーの種類と薬剤導入率とを合わせて調整することで、長期持続可能な抗癌作用を期待できることが分かった。例えば実施例３で得られた化合物３－４は３日間で５％の遊離率であり、理論上は、充分に３０日間以上にわたる薬剤の放出を期待できる。 From the above release test results, it was found that the sustained release rate can be adjusted depending on the structure of the linker, and a long-term sustainable anticancer effect can be expected by adjusting the linker type and the drug introduction rate together. For example, the compound 3-4 obtained in Example 3 has a release rate of 5% in 3 days, and theoretically, release of the drug over a sufficient period of 30 days or more can be expected.
（実施例６）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－（４－（２－アミノエトキシ）フェニル）プロパン酸基導入アルギン酸（化合物６－７）の合成
Figure JPOXMLDOC01-appb-C000011
 Example 6 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-9-yl 3- (4- (2-aminoethoxy) phenyl) propanoic acid group-introduced alginic acid (compound 6-7)
Figure JPOXMLDOC01-appb-C000011
＜工程１＞３－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）フェニル）プロパン酸メチル（化合物６－３）の合成
　トリフェニルホスフィン（２．１８ｇ）のテトラヒドロフラン（７ｍＬ）溶液に、氷冷撹拌下、アゾジカルボン酸ジエチル（４０％トルエン溶液，３．７８ｍＬ）を加え、室温で３０分間撹拌した。この溶液に対し、室温撹拌下、３－（４－ヒドロキシフェニル）プロパン酸メチル［ＣＡＳ：５５９７－５０－２］（化合物６－１，１ｇ）及びｔｅｒｔ－ブチル（２－ヒドロキシエチル）カルバマート［ＣＡＳ：２６６９０－８０－２］（０．８９ｇ）のテトラヒドロフラン（３ｍＬ）溶液を滴下し、室温で１時間撹拌した。反応液を減圧下濃縮し、残留物をシリカゲルカラムクロマトグラフィー（２５％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、化合物６－２を含む画分を得た。この画分をメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）に溶解させ、１規定－水酸化ナトリウム水溶液（５ｍＬ）で３回、水（５ｍＬ）、飽和食塩水（５ｍＬ）で順次洗浄した。有機層を無水硫酸ナトリウムで乾燥させた後、減圧下で溶媒を留去し、化合物６－２を含む粗生成物（１．１３６ｇ）を得た。
　化合物６－２を含む粗生成物（１．１３６ｇ）及びメタノール（１０ｍＬ）の混合物に対して、水酸化リチウム・一水和物（０．１７ｇ）を室温で加え、６０℃で７時間撹拌した。反応終了後、溶媒を留去し、水（２０ｍＬ）及びメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）を加えた。水層をメチル　ｔｅｒｔ－ブチルエーテル（１０ｍＬ）で３回抽出した。残った水層を１規定－塩酸（２０ｍＬ）で酸性にし、酢酸エチル（１０ｍＬ）で３回抽出した。抽出した有機層を水（１０ｍＬ）及び飽和食塩水（１０ｍＬ）で順次洗浄し、無水硫酸ナトリウムで乾燥させた。乾燥した有機層を濾過し、減圧下で溶媒を留去することで、標記化合物６－３（０．２２６ｇ）をピンク色の固体として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　７．１２　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　６．８１　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　４．９８　（１Ｈ，　ｂｒ　ｓ），　３．９９　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．５２－３．５１　（２Ｈ，　ｍ），　２．９０　（２Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　２．６６－２．６２　（２Ｈ，　ｍ），　１．４５　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝３０９，ＲＴ＝０．８８（分），［Ｍ＋Ｎａ］+＝３３２ <Step 1> Synthesis of methyl 3- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) phenyl) propanoate (Compound 6-3) A solution of triphenylphosphine (2.18 g) in tetrahydrofuran (7 mL) To the mixture, diethyl azodicarboxylate (40% toluene solution, 3.78 mL) was added under ice-cooling, and the mixture was stirred at room temperature for 30 minutes. To this solution, methyl 3- (4-hydroxyphenyl) propanoate [CAS: 5597-50-2] (Compound 6-1, 1 g) and tert-butyl (2-hydroxyethyl) carbamate [CAS] were stirred at room temperature. : 26690-80-2] (0.89 g) in tetrahydrofuran (3 mL) was added dropwise, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (25% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 6-2. This fraction was dissolved in methyl tert-butyl ether (20 mL) and washed successively with 1N aqueous sodium hydroxide solution (5 mL) three times, water (5 mL) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain a crude product (1.136 g) containing compound 6-2.
Lithium hydroxide monohydrate (0.17 g) was added to a mixture of the crude product containing compound 6-2 (1.136 g) and methanol (10 mL) at room temperature, and the mixture was stirred at 60 ° C. for 7 hours. . After completion of the reaction, the solvent was distilled off, and water (20 mL) and methyl tert-butyl ether (20 mL) were added. The aqueous layer was extracted 3 times with methyl tert-butyl ether (10 mL). The remaining aqueous layer was acidified with 1N hydrochloric acid (20 mL) and extracted three times with ethyl acetate (10 mL). The extracted organic layer was washed successively with water (10 mL) and saturated brine (10 mL), and dried over anhydrous sodium sulfate. The dried organic layer was filtered and the solvent was distilled off under reduced pressure to obtain the title compound 6-3 (0.226 g) as a pink solid.
NMR data (CDCl 3 ) (δ: ppm): 7.12 (2H, d, J = 8.8 Hz), 6.81 (2H, d, J = 8.8 Hz), 4.98 (1H, br s), 3.99 (2H, t, J = 5.2 Hz), 3.52-3.51 (2H, m), 2.90 (2H, t, J = 7.6 Hz), 2 .66-2.62 (2H, m), 1.45 (9H, s), LC-MS: M = 309, RT = 0.88 (min), [M + Na] + = 332
＜工程２＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）フェニル）プロパン酸（化合物６－５）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物６－４，１００ｍｇ）及び（実施例６）＜工程１＞で得られた化合物６－３（０．２２６ｇ）のジクロロメタン（５０００μＬ）溶液に、氷冷撹拌下、ベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１１８．３５ｍｇ）及びトリエチルアミン（７４．５９μＬ）を加え、室温で２０時間３０分撹拌した。反応液を濾過し、濾液を減圧下濃縮した。残留物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、標記化合物６－５（１５８ｍｇ）を淡黄色のアモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２１　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．７２　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　７．６４　（１Ｈ，　ｓ），　７．４７　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．８　Ｈｚ），　７．２２　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　６．８７　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　５．７５　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．３１　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．２５　（２Ｈ，　ｓ），　４．９９　（１Ｈ，　ｂｒ　ｓ），　４．０２　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．５６－３．５１　（２Ｈ，　ｍ），　３．１７－３．０５　（４Ｈ，　ｍ），　２．９７－２．９３　（３Ｈ，　ｍ），　１．９６－１．８３　（２Ｈ，　ｍ），　１．４６　（９Ｈ，　ｓ），　１．３９　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　１．０４　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝６８３，ＲＴ＝１．０５（分），［Ｍ＋Ｈ］+＝６８４ <Step 2> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl 3- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) phenyl) propanoic acid (compound 6-5) (S) -4,11- Diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolizino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639 -52-3] (Compound 6-4, 100 mg) and (Example 6) To a solution of Compound 6-3 (0.226 g) obtained in <Step 1> in dichloromethane (5000 μL) under ice-cooling and stirring, Triazol-1-yloxy - tris (dimethylamino) phosphonium salts (118.35mg) and triethylamine (74.59μL) was added and stirred for 20 hours and 30 minutes at room temperature. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound 6-5 (158 mg) as a pale yellow amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.21 (1H, d, J = 9.2 Hz), 7.72 (1H, d, J = 2.4 Hz), 7.64 (1H, s), 7.47 (1H, dd, J = 9.2, 2.8 Hz), 7.22 (2H, d, J = 8.8 Hz), 6.87 (2H, d, J = 8) .8 Hz), 5.75 (1H, d, J = 16.4 Hz), 5.31 (1H, d, J = 16.4 Hz), 5.25 (2H, s), 4.99 ( 1H, br s), 4.02 (2H, t, J = 5.2 Hz), 3.56-3.51 (2H, m), 3.17-3.05 (4H, m), 97-2.93 (3H, m), 1.96-1.83 (2H, m), 1.46 (9H, s), 1.39 (3H, t, J = 7. Hz), 1.04 (3H, t , J = 7.6 Hz), LC-MS: M = 683, RT = 1.05 ( min), [M + H] + = 684
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－（４－（２－アミノエトキシ）フェニル）プロパン酸塩酸塩（化合物６－６）の合成
　＜実施例６＞（工程２）で得られた化合物６－５（０．１５８ｇ）及び１，４－ジオキサン（１．１１ｍＬ）の混合物に対して、水冷撹拌下、４規定－塩化水素／１，４－ジオキサン（１．１１ｍＬ）を加え、反応液を室温で２時間３０分攪拌した。反応終了後、ジイソプロピルエーテル（２０ｍＬ）を加え、析出物を濾過し、標記化合物６－６（０．１４１ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．６２　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．１９－７．１１　（４Ｈ，　ｍ），　６．９９　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．４　Ｈｚ），　６．９４　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　５．３６　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．１５　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　４．７１　（２Ｈ，　ｂｒ　ｓ），　４．２１　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．３８　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　２．８３－２．７５　（６Ｈ，　ｍ），　１．８２　（２Ｈ，　ｄｄ，　Ｊ　＝　１４．８，　７．２　Ｈｚ），　１．１１　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　０．８７　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５８３，ＲＴ＝０．７２（分），［Ｍ＋Ｈ］+＝５８４ <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 3- (4- (2-aminoethoxy) phenyl) propanoic acid hydrochloride (Compound 6-6) <Example 6> Compound obtained in (Step 2) To a mixture of 6-5 (0.158 g) and 1,4-dioxane (1.11 mL), 4N-hydrogen chloride / 1,4-dioxane (1.11 mL) was added with stirring under water cooling, and the reaction solution Was stirred at room temperature for 2 hours 30 minutes. After completion of the reaction, diisopropyl ether (20 mL) was added, and the precipitate was filtered to obtain the title compound 6-6 (0.141 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.62 (1H, d, J = 9.2 Hz), 7.19-7.11 (4H, m), 6.99 (1H, dd, J = 9.2, 2.4 Hz), 6.94 (2H, d, J = 8.8 Hz), 5.36 (1H, d, J = 16.0 Hz), 5.15 (1H, d, J = 16.0 Hz), 4.71 (2H, br s), 4.21 (2H, t, J = 5.2 Hz), 3.38 (2H, t, J = 5.2 Hz). ), 2.83-2.75 (6H, m), 1.82 (2H, dd, J = 14.8, 7.2 Hz), 1.11 (3H, t, J = 7.6 Hz) , 0.87 (3H, t, J = 7.2 Hz), LC-MS: M (free amine) = 583, RT = 0.72 (min), [M + H] + = 584
＜工程４＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　３－（４－（２－アミノエトキシ）フェニル）プロパン酸基導入アルギン酸（化合物６－７）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９．８ｍＬ）に、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（５０．１９ｍｇ）、１モル濃度－重曹水（９０．６９μＬ）を加えた。この溶液に対し、氷冷撹拌下、（実施例６）＜工程３＞で得られた化合物６－６（５６．２４ｍｇ）のエタノール（２ｍＬ）及び水（２ｍＬ）溶液を加え、室温で１８時間攪拌した。塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（１９．６ｍＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物６－７（８４ｍｇ）を白色固体として得た。薬剤導入率は６．５モル％であった。分子量は、２３０万Ｄａから３．２万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１４３万Ｄａであった。 <Step 4> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 3- (4- (2-aminoethoxy) phenyl) propanoic acid group-introduced alginic acid (Compound 6-7) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd.) , Molecular weight: 251,000 Da to 12,000 Da (broad), weight average molecular weight: 1.34 million Da) in aqueous solution (9.8 mL), 4- (4,6-dimethoxy-1,3,5-triazine-2 -Ill) -4-methylmorpholinium chloride (50.19 mg), 1 molar concentration-aqueous sodium bicarbonate (90.69 μL) were added. To this solution was added a solution of compound 6-6 (56.24 mg) obtained in <Example 3><Step3> in ethanol (2 mL) and water (2 mL) under ice-cooling and stirring at room temperature for 18 hours. Stir. Sodium chloride (100 mg) was added, ethanol (19.6 mL) was added, and the mixture was stirred for 30 minutes at room temperature. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 6-7 (84 mg) as a white solid. The drug introduction rate was 6.5 mol%. The molecular weight showed a broad elution peak from 2.3 million Da to 32,000 Da, and the weight average molecular weight was 1.43 million Da.
（実施例７）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　２－（４－（２－アミノエトキシ）フェノキシ）酢酸基導入アルギン酸（化合物７－８）の合成
Figure JPOXMLDOC01-appb-C000012
 Example 7 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-9-yl 2- (4- (2-aminoethoxy) phenoxy) acetic acid group-introduced alginic acid (compound 7-8)
Figure JPOXMLDOC01-appb-C000012
＜工程１＞２－（４－ヒドロキシフェノキシ）酢酸メチル（化合物７－２）の合成
　２－（４－ヒドロキシフェノキシ）酢酸［ＣＡＳ：１８７８－８４－８］（化合物７－１，５００ｍｇ）及びメタノール（５０００μＬ）の混合物に対し、氷冷撹拌下、硫酸（１５．８９μＬ）を加え、反応混合物を６０℃で１時間撹拌した。反応混合物を水（５ｍＬ）及び酢酸エチル（２０ｍＬ）で希釈した。混合溶液を酢酸エチル（１０ｍＬ）で２回抽出し、飽和食塩水（１０ｍＬ）で洗浄した。有機層を無水硫酸ナトリウムで乾燥し、濾過後減圧下で濃縮し、標記化合物７－２（０．５２ｇ）を淡黄色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　６．８３－６．７９　（２Ｈ，　ｍ），　６．７８－６．７４　（２Ｈ，　ｍ），　４．５８　（２Ｈ，　ｓ），　４．５１　（１Ｈ，　ｂｒ　ｓ），　３．８０　（３Ｈ，　ｓ） <Step 1> Synthesis of methyl 2- (4-hydroxyphenoxy) acetate (Compound 7-2) 2- (4-Hydroxyphenoxy) acetic acid [CAS: 1878-84-8] (Compound 7-1,500 mg) and methanol To the mixture (5000 μL), sulfuric acid (15.89 μL) was added with stirring under ice cooling, and the reaction mixture was stirred at 60 ° C. for 1 hour. The reaction mixture was diluted with water (5 mL) and ethyl acetate (20 mL). The mixed solution was extracted twice with ethyl acetate (10 mL) and washed with saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the title compound 7-2 (0.52 g) as a pale yellow amorphous.
NMR data (CDCl 3 ) (δ: ppm): 6.83-6.79 (2H, m), 6.78-6.74 (2H, m), 4.58 (2H, s), 4.51 (1H, br s), 3.80 (3H, s)
＜工程２＞２－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）フェノキシ）酢酸メチル（化合物７－３）の合成
　（実施例７）＜工程１＞で得られた化合物７－２（５２０ｍｇ），ｔｅｒｔ－ブチル　（２－ブロモエチル）カルバマート［ＣＡＳ：３９６８４－８０－５］（７０３．６３ｍｇ）及びアセトニトリル（５２００μＬ）の混合物に対し、水冷撹拌下、炭酸カリウム（７８８．９８ｍｇ）を加え、８０℃で１時間撹拌した。続いて、ヨウ化カリウム（５２１．２２ｍｇ）及びＮ－メチルピロリドン（５２００μＬ）を加え、同温にて４時間撹拌した。反応液を水（１０ｍＬ）及び酢酸エチル（２０ｍＬ）で希釈した。混合液を酢酸エチル（１０ｍＬ）で２回抽出し、飽和食塩水（１０ｍＬ）で洗浄した。有機層を無水硫酸ナトリウムで乾燥し、濾過後減圧下で溶媒を留去した。残留物をシリカゲルカラムクロマトグラフィー（１５％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、化合物７－３及び化合物７－２の混合物を得た。この混合物をメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）に溶解させ、１規定－水酸化ナトリウム水溶液（５ｍＬ）、水（１０ｍＬ）、飽和食塩水（１０ｍＬ）で順次洗浄した。有機層を無水硫酸ナトリウムで乾燥させた後、減圧下で溶媒を留去し、標記化合物７－３（４４８ｍｇ）を無色の油状化合物として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　６．８６－６．８４　（２Ｈ，　ｍ），　６．８３－６．８０　（２Ｈ，　ｍ），　４．９７　（１Ｈ，　ｂｒ　ｓ），　４．５８　（２Ｈ，　ｓ），　３．９８－３．９４　（２Ｈ，　ｍ），　３．８０　（３Ｈ，　ｓ），　３．５３－３．４８　（２Ｈ，　ｍ），　１．４５　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝３２５，ＲＴ＝０．９５（分），［Ｍ＋Ｎａ］+＝３４８ <Step 2> Synthesis of methyl 2- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) phenoxy) acetate (Compound 7-3) (Example 7) Compound 7 obtained in <Step 1> -2 (520 mg), tert-butyl (2-bromoethyl) carbamate [CAS: 39684-80-5] (703.63 mg) and acetonitrile (5200 μL) were mixed with potassium carbonate (788.98 mg) under water-cooling and stirring. And stirred at 80 ° C. for 1 hour. Subsequently, potassium iodide (521.22 mg) and N-methylpyrrolidone (5200 μL) were added, and the mixture was stirred at the same temperature for 4 hours. The reaction was diluted with water (10 mL) and ethyl acetate (20 mL). The mixture was extracted twice with ethyl acetate (10 mL) and washed with saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (15% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a mixture of compound 7-3 and compound 7-2. This mixture was dissolved in methyl tert-butyl ether (20 mL), and washed successively with 1N-aqueous sodium hydroxide solution (5 mL), water (10 mL), and saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to give the title compound 7-3 (448 mg) as a colorless oily compound.
NMR data (CDCl 3 ) (δ: ppm): 6.86-6.84 (2H, m), 6.83-6.80 (2H, m), 4.97 (1H, br s), 4. 58 (2H, s), 3.98-3.94 (2H, m), 3.80 (3H, s), 3.53-3.48 (2H, m), 1.45 (9H, s) , LC-MS: M = 325, RT = 0.95 (min), [M + Na] + = 348
＜工程３＞２－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）フェノキシ）酢酸（化合物７－４）の合成
　（実施例７）＜工程２＞で得られた化合物７－３（０．４４８ｇ）及びメタノール（４．１２ｍＬ）の混合物に対し、室温撹拌下、水酸化ナトリウム（０．１５ｇ）を加え、同温にて３０分撹拌した。反応混合物を水（１０ｍＬ）で希釈し、１規定－塩酸（１０ｍＬ）で酸性にした。混合液を酢酸エチル（１０ｍＬ）で３回抽出し、飽和食塩水（１０ｍＬ）で洗浄した。有機層を無水硫酸ナトリウムで乾燥し、濾過後、減圧下で濃縮することで、標記化合物７－４（０．４５１ｇ）を無色の油状化合物として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　６．８８－６．８１　（４Ｈ，　ｍ），　４．９９　（１Ｈ，　ｂｒ　ｓ），　４．６１　（２Ｈ，　ｓ），　３．９６　（２Ｈ，　ｔ，　Ｊ　＝　４．８　Ｈｚ），　３．５３－３．４９　（２Ｈ，　ｍ），　１．４５　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝３１１，ＲＴ＝０．８３（分），［Ｍ＋Ｎａ］+＝３３４ <Step 3> Synthesis of 2- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) phenoxy) acetic acid (Compound 7-4) (Example 7) Compound 7- obtained in <Step 2> Sodium hydroxide (0.15 g) was added to a mixture of 3 (0.448 g) and methanol (4.12 mL) with stirring at room temperature, and the mixture was stirred at the same temperature for 30 minutes. The reaction mixture was diluted with water (10 mL) and acidified with 1N hydrochloric acid (10 mL). The mixture was extracted 3 times with ethyl acetate (10 mL) and washed with saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound 7-4 (0.451 g) as a colorless oily compound.
NMR data (CDCl 3 ) (δ: ppm): 6.88-6.81 (4H, m), 4.99 (1H, br s), 4.61 (2H, s), 3.96 (2H, t, J = 4.8 Hz), 3.53-3.49 (2H, m), 1.45 (9H, s), LC-MS: M = 311, RT = 0.83 (min), [ M + Na] + = 334
＜工程４＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　２－（４－（２－アミノエトキシ）フェノキシ）酢酸塩酸塩（化合物７－７）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物７－５，１００ｍｇ）、（実施例７）＜工程３＞で得られた化合物７－４（８３．３１ｍｇ）及びベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１１８．３５ｍｇ）のジクロロメタン（１０００μＬ）溶液に、氷冷撹拌下、トリエチルアミン（７４．５９μＬ）を滴下し、室温で１９時間撹拌した。反応液を濾過し、濾液を減圧下濃縮した。残留物をシリカゲルカラムクロマトグラフィー（２５％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル、酢酸エチル～３％メタノール／酢酸エチル）により精製し、化合物７－６（１５０ｍｇ）を含む画分を得た。
　化合物７－６（０．１５ｇ）を含む画分及び１，４－ジオキサン（１．０５ｍＬ）の混合物に対して、水冷撹拌下、４規定－塩化水素／１，４－ジオキサン（１．０５ｍＬ）を加え、反応液を室温で１９時間攪拌した。反応終了後、ジイソプロピルエーテル（２０ｍＬ）を加え、析出物を濾過し、標記化合物７－７（０．１１ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．７１－７．６４　（１Ｈ，　ｂｒ　ｍ），　７．５９－７．４６　（２Ｈ，　ｂｒ　ｍ），　７．２８　（１Ｈ，　ｓ），　７．０３－７．０１　（１Ｈ，　ｂｒ　ｍ），　６．９６　（１Ｈ，　ｓ），　６．８６－６．７８　（７Ｈ，　ｂｒ　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋｓ．），　５．２３　（１Ｈ，　ｄ，　Ｊ　＝　１５．６　Ｈｚ），　５．０３　（１Ｈ，　ｄ，　Ｊ　＝　１５．６　Ｈｚ），　４．４８－４．４２　（１Ｈ，　ｂｒ　ｍ），　４．０８　（３Ｈ，　ｔ，　Ｊ　＝　５．０　Ｈｚ），　３．２９－３．２５　（３Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　２　ｐｅａｋｓ．），　１．７２　（２Ｈ，　ｂｒ　ｓ），　１．１０－１．０３　（４Ｈ，　ｂｒ　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅｋａｓ．），　０．８１－０．７８　（３Ｈ，　ｂｒ　ｍ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５８５，ＲＴ＝０．７０（分），［Ｍ＋Ｈ］+＝５８６ <Step 4> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 2- (4- (2-aminoethoxy) phenoxy) acetic acid hydrochloride (Compound 7-7) (S) -4,11-diethyl-4,9-dihydroxy -1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52-3] (compound 7-5, 100 mg), (Example 7) Compound 7-4 (83.31 mg) obtained in <Step 3> and benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (118.35 mg) in dichloromethane ( 100 The [mu] L) solution with stirring under ice cooling, was added dropwise triethylamine (74.59μL), and stirred at room temperature for 19 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (25% ethyl acetate / n-heptane to 100% ethyl acetate, ethyl acetate to 3% methanol / ethyl acetate) to obtain a fraction containing compound 7-6 (150 mg). .
To a mixture of the fraction containing compound 7-6 (0.15 g) and 1,4-dioxane (1.05 mL), 4N-hydrogen chloride / 1,4-dioxane (1.05 mL) was stirred under water cooling. And the reaction was stirred at room temperature for 19 hours. After completion of the reaction, diisopropyl ether (20 mL) was added, and the precipitate was filtered to obtain the title compound 7-7 (0.11 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.71-7.64 (1H, br m), 7.59-7.46 (2H, br m), 7.28 (1H, s), 7.03-7.01 (1H, br m), 6.96 (1H, s), 6.86-6.78 (7H, br m, Overlapped with other peaks.), 5.23 (1H, d , J = 15.6 Hz), 5.03 (1H, d, J = 15.6 Hz), 4.48-4.42 (1H, br m), 4.08 (3H, t, J = 5 .0 Hz), 3.29-3.25 (3H, m, Overlapped with 2 peaks.), 1.72 (2H, brs), 1.10-1.03 (4H, br m, Overwrapped with other) pekas.) 0.81-0.78 (3H, br m), LC-MS: M (free amine) = 585, RT = 0.70 ( min), [M + H] + = 586
＜工程５＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　２－（４－（２－アミノエトキシ）フェノキシ）酢酸基導入アルギン酸（化合物７－８）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）に、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（６８．６４ｍｇ）、１モル濃度－重曹水（６８．８３μＬ）を加えた。この溶液に対し、水冷撹拌下、（実施例７）＜工程４＞で得られた化合物７－７（４２．６９ｍｇ）のエタノール（２ｍＬ）及び水（１ｍＬ）溶液を加え、４０℃で４時間攪拌した。室温に冷却後、塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（１９７７６μＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物７－８（１１１．５ｍｇ）を黄色固体として得た。薬剤導入率は１．８０モル％であった。分子量は、２２９万Ｄａから６．１万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３６万Ｄａであった。 <Step 5> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl 2- (4- (2-aminoethoxy) phenoxy) acetic acid group-introduced alginic acid (compound 7-8) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd.) (Molecular weight: 251,000 Da to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) in aqueous solution (9888 μL), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-Methylmorpholinium chloride (68.64 mg), 1 molar concentration-aqueous sodium bicarbonate (68.83 μL) were added. To this solution was added a solution of compound 7-7 (42.69 mg) obtained in <Example 4><Step4> in ethanol (2 mL) and water (1 mL) under water-cooling and stirring at 40 ° C. for 4 hours. Stir. After cooling to room temperature, sodium chloride (100 mg) was added, ethanol (19776 μL) was added, and the mixture was stirred for 30 minutes at room temperature. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 7-8 (111.5 mg) as a yellow solid. The drug introduction rate was 1.80 mol%. The molecular weight showed a broad elution peak from 2.29 million Da to 61,000 Da, and the weight average molecular weight was 1.36 million Da.
（実施例８）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（２－アミノエトキシ）安息香酸基導入アルギン酸（化合物８－７）の合成
Figure JPOXMLDOC01-appb-C000013
 Example 8 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-9-yl 4- (2-aminoethoxy) benzoic acid group-introduced alginic acid (compound 8-7)
Figure JPOXMLDOC01-appb-C000013
＜工程１＞４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）安息香酸メチル（化合物８－２）の合成
　トリフェニルホスフィン（２．５９ｇ）のテトラヒドロフラン（７ｍＬ）溶液に、水冷撹拌下、アゾジカルボン酸ジエチル（４０％トルエン溶液，４．４８ｍＬ）を加え、室温で３０分間撹拌した。この溶液に対し、室温撹拌下、４－ヒドロキシ安息香酸メチル［ＣＡＳ：９９－７６－３］（化合物８－１，１ｇ）及びｔｅｒｔ－ブチル（２－ヒドロキシエチル）カルバマート［ＣＡＳ：２６６９０－８０－２］（１．０６ｇ）のテトラヒドロフラン（３ｍＬ）溶液を滴下し、室温で１７時間撹拌した。反応液を減圧下濃縮し、残留物をシリカゲルカラムクロマトグラフィー（５％酢酸エチル／ｎ－ヘプタン～４０％酢酸エチル）により精製し、化合物８－２を含む画分を得た。この画分をメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）に溶解させ、１規定－水酸化ナトリウム水溶液（５ｍＬ）で３回、水（５ｍＬ）、飽和食塩水（５ｍＬ）で順次洗浄した。有機層を無水硫酸ナトリウムで乾燥させた後、減圧下で溶媒を留去し、標記化合物８－２（１．１３６ｇ）を、ピンク色の油状化合物として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　７．９８　（２Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　６．９０　（２Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　４．９７　（１Ｈ，　ｂｒ　ｓ），　４．０７　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．８９　（３Ｈ，　ｓ），　３．５６　（２Ｈ，　ｑ，　Ｊ　＝　５．２　Ｈｚ），　１．４５　（９Ｈ，　ｓ）． <Step 1> Synthesis of methyl 4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzoate (Compound 8-2) A solution of triphenylphosphine (2.59 g) in tetrahydrofuran (7 mL) was stirred with water cooling , Diethyl azodicarboxylate (40% toluene solution, 4.48 mL) was added, and the mixture was stirred at room temperature for 30 min. To this solution, methyl 4-hydroxybenzoate [CAS: 99-76-3] (Compound 8-1, 1 g) and tert-butyl (2-hydroxyethyl) carbamate [CAS: 26690-80-] were stirred at room temperature. 2] (1.06 g) in tetrahydrofuran (3 mL) was added dropwise and stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (5% ethyl acetate / n-heptane to 40% ethyl acetate) to obtain a fraction containing compound 8-2. This fraction was dissolved in methyl tert-butyl ether (20 mL) and washed successively with 1N aqueous sodium hydroxide solution (5 mL) three times, water (5 mL) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and then the solvent was distilled off under reduced pressure to obtain the title compound 8-2 (1.136 g) as a pink oily compound.
NMR data (CDCl 3 ) (δ: ppm): 7.98 (2H, d, J = 8.4 Hz), 6.90 (2H, d, J = 8.4 Hz), 4.97 (1H, br s), 4.07 (2H, t, J = 5.2 Hz), 3.89 (3H, s), 3.56 (2H, q, J = 5.2 Hz), 1.45 (9H , S).
＜工程２＞４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）安息香酸（化合物８－３）の合成
　（実施例８）＜工程１＞で得られた化合物８－２（０．６８３ｇ）及びメタノール（６．８３ｍＬ）の混合物に対し、水酸化リチウム・一水和物（０．２９ｇ）を室温で加えた。この反応混合物を、６５℃で２時間撹拌し、更に水酸化リチウム・一水和物（０．２９ｇ）を加え、１５時間撹拌した。反応終了後、減圧下で溶媒を濃縮し、残留物を水（１０ｍＬ）に溶解させた。この水溶液を１規定－塩酸（２０ｍＬ）で酸性にし、酢酸エチル（２０ｍＬ）に溶解させた。この溶液を酢酸エチル（１０ｍＬ）で２回抽出し、水（１０ｍＬ）及び飽和食塩水（１０ｍＬ）で順次洗浄した。有機層を無水硫酸ナトリウムで乾燥させ、濾過後減圧下で濃縮することで、標記化合物８－３（０．５５ｇ）を白色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．０４　（２Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　６．９３　（２Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　４．９７　（１Ｈ，　ｂｒ　ｓ），　４．０９　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．５９－３．５４　（２Ｈ，　ｂｒ　ｍ），　１．４６　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝２８１，ＲＴ＝０．８４（分），［Ｍ＋Ｎａ］+＝３０４ <Step 2> Synthesis of 4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzoic acid (Compound 8-3) (Example 8) Compound 8-2 obtained in <Step 1> (0. 683 g) and methanol (6.83 mL), lithium hydroxide monohydrate (0.29 g) was added at room temperature. The reaction mixture was stirred at 65 ° C. for 2 hours, further added with lithium hydroxide monohydrate (0.29 g), and stirred for 15 hours. After completion of the reaction, the solvent was concentrated under reduced pressure, and the residue was dissolved in water (10 mL). The aqueous solution was acidified with 1N hydrochloric acid (20 mL) and dissolved in ethyl acetate (20 mL). This solution was extracted twice with ethyl acetate (10 mL) and washed successively with water (10 mL) and saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 8-3 (0.55 g) as a white amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.04 (2H, d, J = 8.4 Hz), 6.93 (2H, d, J = 8.4 Hz), 4.97 (1H, br s), 4.09 (2H, t, J = 5.2 Hz), 3.59-3.54 (2H, br m), 1.46 (9H, s), LC-MS: M = 281 , RT = 0.84 (min), [M + Na] + = 304
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ベンゾエート（化合物８－５）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物８－４，１００ｍｇ）、（実施例８）＜工程２＞で得られた化合物８－３（７５．２７ｍｇ）及びＮ，Ｎ－ジメチル－４－アミノピリジン（６．２３ｍｇ）のジクロロメタン（４ｍＬ）溶液に、氷冷撹拌下、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（５５．２１ｍｇ）のジクロロメタン（１ｍＬ）溶液を滴下し、室温で１７時間撹拌した。反応混合物を濾過し、濾液を減圧下で濃縮した。残留物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル、酢酸エチル～１０％メタノール／酢酸エチル）により精製し、標記化合物８－５（６２ｍｇ）を白色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２８　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　８．２２　（２Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．９５　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　７．６８　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．８　Ｈｚ），　７．６５　（１Ｈ，　ｓ），　７．０２　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　５．７６　（１Ｈ，　ｄ，　Ｊ　＝　１６．６　Ｈｚ），　５．３２　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．２８　（２Ｈ，　ｓ），　５．００　（１Ｈ，　ｂｒ　ｓ），　４．１４　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．７１　（１Ｈ，　ｓ），　３．６０　（２Ｈ，　ｄｄ，　Ｊ　＝　１０．４，　５．６　Ｈｚ），　３．１８　（２Ｈ，　ｄｄ，　Ｊ　＝　１４．８，　７．２　Ｈｚ），　１．９６－１．８５　（２Ｈ，　ｍ），　１．４７　（９Ｈ，　ｓ），　１．４２　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　１．０５　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝６５５，ＲＴ＝１．０２（分），［Ｍ＋Ｈ］+＝６５６ <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl 4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzoate (Compound 8-5) (S) -4,11-diethyl-4,9- Dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52-3] ( Compound 8-4 (100 mg), (Example 8) Compound 8-3 (75.27 mg) obtained in <Step 2> and N, N-dimethyl-4-aminopyridine (6.23 mg) in dichloromethane (4 mL) ) Stir in ice-cooled solution It was added dropwise N, in dichloromethane (1 mL) solution of N'- dicyclohexylcarbodiimide (55.21mg), and stirred at room temperature for 17 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate, ethyl acetate to 10% methanol / ethyl acetate) to obtain the title compound 8-5 (62 mg) as a white amorphous. .
NMR data (CDCl 3 ) (δ: ppm): 8.28 (1H, d, J = 9.2 Hz), 8.22 (2H, d, J = 9.2 Hz), 7.95 (1H, d, J = 2.4 Hz), 7.68 (1H, dd, J = 9.2, 2.8 Hz), 7.65 (1H, s), 7.02 (2H, d, J = 8) .8 Hz), 5.76 (1H, d, J = 16.6 Hz), 5.32 (1H, d, J = 16.0 Hz), 5.28 (2H, s), 5.00 ( 1H, br s), 4.14 (2H, t, J = 5.2 Hz), 3.71 (1H, s), 3.60 (2H, dd, J = 10.4, 5.6 Hz) , 3.18 (2H, dd, J = 14.8, 7.2 Hz), 1.96-1.85 (2H, m), 1.47 (9H, s), 1.42 (3H, t, J = 7.6 Hz), 1.05 (3H, t, J = 7.6 Hz), LC-MS: M = 655, RT = 1.02 (min), [ M + H] + = 656
＜工程４＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（２－アミノエトキシ）ベンゾエート塩酸塩（化合物８－６）の合成
　（実施例８）＜工程３＞で得られた化合物８－５（０．０６２ｇ）を用い、（実施例６）＜工程３＞と同様の操作を行い、標記化合物８－６（０．０６ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．４０－７．３６　（３Ｈ，　ｂｒ　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｔｗｏ　ｐｅａｋｓ．），　７．０２　（１Ｈ，　ｓ），　６．９３－６．９０　（２Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｔｗｏ　ｐｅａｋｓ．），　６．７３　（２Ｈ，　ｄ，　Ｊ　＝　８．０　Ｈｚ），　４．９２　（１Ｈ，　ｄ，　Ｊ　＝　１５．６　Ｈｚ），　４．７７　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　４．４７　（１Ｈ，　ｄ，　Ｊ　＝　１６．８　Ｈｚ），　４．２６－４．１６　（２Ｈ，　ｍ），　３．３８　（２Ｈ，　ｔ，　Ｊ　＝　４．４　Ｈｚ），　２．５４－２．４６　（２Ｈ，　ｂｒ　ｍ），　１．５１－１．４４　（２Ｈ，　ｂｒ　ｍ），　０．８９　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　０．６８　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝５５５，ＲＴ＝０．６７（分），［Ｍ＋Ｈ］+＝５５６ <Step 4> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 4- (2-aminoethoxy) benzoate hydrochloride (Compound 8-6) (Example 8) Compound 8-5 obtained in <Step 3> (0. The same operation as in Example 6 <Step 3> was carried out using 062 g) to obtain the title compound 8-6 (0.06 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.40-7.36 (3H, br m, Overwrapped with peaks.), 7.02 (1H, s), 6.93-6.90 ( 2H, m, Overlapped with two peaks.), 6.73 (2H, d, J = 8.0 Hz), 4.92 (1H, d, J = 15.6 Hz), 4.77 (1H, d , J = 16.0 Hz), 4.47 (1H, d, J = 16.8 Hz), 4.26-4.16 (2H, m), 3.38 (2H, t, J = 4. 4 Hz), 2.54-2.46 (2H, br m), 1.51-1.44 (2H, br m), 0.89 (3H, t, J = 7.6 Hz), 0. 68 (3H, t, J = 7.2 Hz), LC-MS: M 555, RT = 0.67 (min), [M + H] + = 556
＜工程５＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　４－（２－アミノエトキシ）安息香酸基導入アルギン酸（化合物８－７）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）と（実施例８）＜工程４＞で得られた化合物８－６（６８．６４ｍｇ）を用い、（実施例７）＜工程５＞と同様の操作を行い、標記化合物８－７（１０２ｍｇ）を白色固体として得た。薬剤導入率は１．８０モル％であった。 <Step 5> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl 4- (2-aminoethoxy) benzoic acid group-introduced alginic acid (Compound 8-7) Sodium alginate prepared to 1% by weight (produced by Kimika Co., Ltd., molecular weight: 2510,000) Da to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) Using an aqueous solution (9888 μL) and Compound 8-6 (68.64 mg) obtained in (Example 8) <Step 4>, ( Example 7) The same operation as in <Step 5> was performed to give the title compound 8-7 (102 mg) as a white solid. The drug introduction rate was 1.80 mol%.
（実施例９）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　５－（２－アミノエトキシ）ピコリン酸基導入アルギン酸（化合物９－７）の合成
Figure JPOXMLDOC01-appb-C000014
 ＜工程１＞５－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ピコリン酸メチル（化合物９－２）の合成
　５－ヒドロキシピコリン酸［ＣＡＳ：３０７６６－１２－２］（化合物９－１，１ｇ），ｔｅｒｔ－ブチル（２－ブロモエチル）カルバマート［ＣＡＳ：３９６８４－８０－５］（１．８５ｇ）及びＮ－メチルピロリドン（１０ｍＬ）の混合物に対し、室温で、炭酸カリウム（１．８ｇ）を加え、８０℃で２時間撹拌した。反応混合物を室温まで冷却後、水（２０ｍＬ）を加え、メチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）で３回抽出した。有機層を水（１０ｍＬ）で２回、飽和食塩水（１０ｍＬ）で順次洗浄し、無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で溶媒を留去した。残留物をシリカゲルカラムクロマトグラフィー（１２％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、化合物９－２を含む画分を得た。この画分をメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）に溶解させ、１規定－水酸化ナトリウム水溶液（１０ｍＬ）、水（１０ｍＬ）、飽和食塩水（１０ｍＬ）で順次洗浄した。有機層を無水硫酸ナトリウムで乾燥させた後、減圧下で溶媒を留去し、標記化合物９－２（１．２４ｇ）を、黄色の油状化合物として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．３９　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　８．１１　（１Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　７．２６　（１Ｈ，　ｄｄ，　Ｊ　＝　８．４，　２．８　Ｈｚ，　Ｃｈｌｏｒｏｆｏｒｍ　ｗａｓ　ｏｖｅｒｌａｐｐｅｄ．），　４．９８　（１Ｈ，　ｂｒ　ｓ），　４．１３　（２Ｈ，　ｔ，　Ｊ　＝　４．８　Ｈｚ），　３．９８　（３Ｈ，　ｓ），　３．５８　（２Ｈ，　ｄｄ，　Ｊ　＝　１０．４，　４．８　Ｈｚ），　１．４５　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝２９６，ＲＴ＝０．８０（分），［Ｍ＋Ｈ］+＝２９７ Example 9 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-9-yl 5- (2-aminoethoxy) picolinic acid group-introduced alginic acid (compound 9-7)
Figure JPOXMLDOC01-appb-C000014
 <Step 1> Synthesis of methyl 5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinate (Compound 9-2) 5-hydroxypicolinic acid [CAS: 30766-12-2] (Compound 9-1 , 1 g), tert-butyl (2-bromoethyl) carbamate [CAS: 39684-80-5] (1.85 g) and N-methylpyrrolidone (10 mL) at room temperature with potassium carbonate (1.8 g) And stirred at 80 ° C. for 2 hours. The reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted 3 times with methyl tert-butyl ether (20 mL). The organic layer was washed successively with water (10 mL) twice and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 9-2. This fraction was dissolved in methyl tert-butyl ether (20 mL) and washed successively with 1N aqueous sodium hydroxide solution (10 mL), water (10 mL), and saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to give the title compound 9-2 (1.24 g) as a yellow oily compound.
NMR data (CDCl 3 ) (δ: ppm): 8.39 (1H, d, J = 2.8 Hz), 8.11 (1H, d, J = 8.4 Hz), 7.26 (1H, dd, J = 8.4, 2.8 Hz, Chloroform was overwrapped.), 4.98 (1H, brs), 4.13 (2H, t, J = 4.8 Hz), 3.98 (3H) , S), 3.58 (2H, dd, J = 10.4, 4.8 Hz), 1.45 (9H, s), LC-MS: M = 296, RT = 0.80 (min), [M + H] + = 297
＜工程２＞５－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ピコリン酸（化合物９－３）の合成
　（実施例９）＜工程１＞で得られた化合物９－２（１．２３７ｇ）及びメタノール（１２．３７ｍＬ）の混合物に対し、室温で水酸化ナトリウム（０．５ｇ）を加え、６０℃で３０分撹拌した。室温まで冷却後、溶媒を留去した。続いて、水（２０ｍＬ）を加え、メチル　ｔｅｒｔ－ブチルエーテル（１０ｍＬ）で２回抽出し、水層を１規定－塩酸（１５ｍＬ）で酸性にし、酢酸エチル（１０ｍＬ）で３回抽出した。有機層を無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で溶媒を留去することで、標記化合物９－３（０．９２ｇ）を白色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２６　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　８．１８　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．３６　（１Ｈ，　ｄｄ，　Ｊ　＝　８．８，　２．８　Ｈｚ），　４．９５　（１Ｈ，　ｂｒ　ｓ），　４．１６　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．５９　（２Ｈ，　ｄｄ，　Ｊ　＝　１０．８，　５．６　Ｈｚ），　１．４６　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝２８２，ＲＴ＝０．６７（分），［Ｍ＋Ｎａ］+＝３０５ <Step 2> Synthesis of 5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinic acid (Compound 9-3) (Example 9) Compound 9-2 obtained in <Step 1> (1. 237 g) and methanol (12.37 mL), sodium hydroxide (0.5 g) was added at room temperature, and the mixture was stirred at 60 ° C. for 30 minutes. After cooling to room temperature, the solvent was distilled off. Subsequently, water (20 mL) was added and extracted twice with methyl tert-butyl ether (10 mL). The aqueous layer was acidified with 1N hydrochloric acid (15 mL) and extracted three times with ethyl acetate (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give the title compound 9-3 (0.92 g) as a white amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.26 (1H, d, J = 2.8 Hz), 8.18 (1H, d, J = 9.2 Hz), 7.36 (1H, dd, J = 8.8, 2.8 Hz), 4.95 (1H, br s), 4.16 (2H, t, J = 5.2 Hz), 3.59 (2H, dd, J = 10.8, 5.6 Hz), 1.46 (9H, s), LC-MS: M = 282, RT = 0.67 (min), [M + Na] + = 305
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　５－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ピコリナート（化合物９－５）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物９－４，１００ｍｇ）、（実施例９）＜工程２＞で得られた化合物９－３（７６ｍｇ）、ベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１１８．３５ｍｇ）、トリエチルアミン（７４．５９μＬ）及びジクロロメタン（５０００μＬ）を用い、（実施例６）＜工程２＞と同様の操作を行うことで、標記化合物９－５（１２８ｍｇ）を黄色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．５３　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　８．３０　（２Ｈ，　ｄｄ，　Ｊ　＝　８．８，　８．８　Ｈｚ），　８．００　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　７．７３　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．８　Ｈｚ），　７．６６　（１Ｈ，　ｓ），　７．３７　（１Ｈ，　ｄｄ，　Ｊ　＝　８．８，　２．８　Ｈｚ），　５．７６　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．３２　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．２８　（２Ｈ，　ｓ），　４．２１　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．７１　（１Ｈ，　ｓ），　３．６５－３．６１　（２Ｈ，　ｂｒ　ｍ），　３．１７　（２Ｈ，　ｄｄ，　Ｊ　＝　１５．６，　８．０　Ｈｚ），　１．９６－１．８６　（２Ｈ，　ｍ），　１．４７　（９Ｈ，　ｓ），　１．４０　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　１．０５　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝６５６，ＲＴ＝０．９１（分），［Ｍ＋Ｈ］+＝６５７ <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl 5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinate (compound 9-5) (S) -4,11-diethyl-4,9- Dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52-3] ( Compound 9-4, 100 mg), (Example 9) Compound 9-3 obtained in <Step 2> (76 mg), benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (118.35 mg), triethyl Using Min (74.59MyuL) and dichloromethane (5000μL), (Example 6) <Step 2> and and in the same manner, the title compound 9-5 (128 mg) as yellow amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.53 (1H, d, J = 2.8 Hz), 8.30 (2H, dd, J = 8.8, 8.8 Hz), 8. 00 (1H, d, J = 2.8 Hz), 7.73 (1H, dd, J = 9.2, 2.8 Hz), 7.66 (1H, s), 7.37 (1H, dd , J = 8.8, 2.8 Hz), 5.76 (1H, d, J = 16.4 Hz), 5.32 (1H, d, J = 16.4 Hz), 5.28 (2H) , S), 4.21 (2H, t, J = 5.2 Hz), 3.71 (1H, s), 3.65-3.61 (2H, br m), 3.17 (2H, dd , J = 15.6, 8.0 Hz), 1.96-1.86 (2H, m), 1.47 (9H, s), 1.40 (3H, t, = 7.6 Hz), 1.05 (3H , t, J = 7.6 Hz), LC-MS: M = 656, RT = 0.91 ( min), [M + H] + = 657
＜工程４＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　５－（２－アミノエトキシ）ピコリナート塩酸塩（化合物９－６）の合成
　（実施例９）＜工程３＞で得られた化合物９－５（０．１２８ｇ）を用い、（実施例６）＜工程３＞と同様の操作を行い、標記化合物９－６（０．１７ｇ）を含む粗生成物を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　８．２０　（１Ｈ，　ｄ，　Ｊ　＝　２．０　Ｈｚ），　７．８９　（１Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　７．５９　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．４３　（１Ｈ，　ｄｄ，　Ｊ　＝　８．４，　２．４　Ｈｚ），　７．２５　（２Ｈ，　ｄ，　Ｊ　＝　７．２　Ｈｚ），　７．１３　（１Ｈ，　ｓ），　５．０６　（２Ｈ，　ｓ），　４．４２－４．３６　（２Ｈ，　ｍ），　３．４５　（２Ｈ，　ｔ，　Ｊ　＝　４．８　Ｈｚ），　２．６６　（２Ｈ，　ｂｒ　ｓ），　１．６７－１．５７　（２Ｈ，　ｍ），　１．０１－０．９８　（３Ｈ，　ｂｒ　ｍ），　０．７１　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），　２Ｈ　ｗａｓ　ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｓｏｌｖｅｎｔ　ｐｅａｋ．，ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５５６，ＲＴ＝０．６２（分），［Ｍ＋Ｈ］+＝５５７ <Step 4> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl 5- (2-aminoethoxy) picolinate hydrochloride (Compound 9-6) (Example 9) Compound 9-5 obtained in <Step 3> (0. 128g), and the same operation as in Example 6 <Step 3> was performed to give a crude product containing the title compound 9-6 (0.17 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 8.20 (1H, d, J = 2.0 Hz), 7.89 (1H, d, J = 8.8 Hz), 7.59 (1H , D, J = 9.2 Hz), 7.43 (1H, dd, J = 8.4, 2.4 Hz), 7.25 (2H, d, J = 7.2 Hz), 7.13 (1H, s), 5.06 (2H, s), 4.42-4.36 (2H, m), 3.45 (2H, t, J = 4.8 Hz), 2.66 (2H, br s), 1.67-1.57 (2H, m), 1.01-0.98 (3H, br m), 0.71 (3H, t, J = 7.2 Hz), 2H was overwrapped with solvent peak. , LC-MS: M (free amine) = 556, RT = 0.62 (min), [M + H] + = 557
＜工程５＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　５－（２－アミノエトキシ）ピコリン酸基導入アルギン酸（化合物９－７）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）に、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウムクロリド（６８．６４ｍｇ）、１モル濃度－重曹水（６８．８３μＬ）を加えた。この溶液に対し、水冷撹拌下、（実施例９）＜工程４＞で得られた化合物９－６（４０．７ｍｇ）のジメチルスルホキシド（２ｍＬ）溶液を滴下し、室温で１５時間攪拌した。塩化ナトリウム（１００ｍｇ）を加えた後、エタノール（１９７７６μＬ）を加え、３０分間室温で攪拌した。得られた沈殿をろ取し、エタノールで洗浄後、減圧乾燥して、標記化合物９－７（９９ｍｇ）を白色固体として得た。薬剤導入率は１．２５モル％であった。分子量は、２２５万Ｄａから１．３万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３２万Ｄａであった。 <Step 5> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl 5- (2-aminoethoxy) picolinic acid group-introduced alginic acid (Compound 9-7) Sodium alginate prepared to 1% by weight (produced by Kimika Co., Ltd., molecular weight: 2510,000) Da to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) in aqueous solution (9888 μL), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl Morpholinium chloride (68.64 mg), 1 molar concentration—aqueous sodium bicarbonate (68.83 μL) were added. To this solution, a solution of compound 9-6 (40.7 mg) obtained in (Example 9) <Step 4> in dimethyl sulfoxide (2 mL) was added dropwise with stirring under water cooling, followed by stirring at room temperature for 15 hours. Sodium chloride (100 mg) was added, ethanol (19776 μL) was added, and the mixture was stirred for 30 minutes at room temperature. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound 9-7 (99 mg) as a white solid. The drug introduction rate was 1.25 mol%. The molecular weight showed a broad elution peak from 2.25 million Da to 13,000 Da, and the weight average molecular weight was 1,320,000 Da.
（実施例１０）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　（５－（２－アミノエトキシ）ピコリノイル）グリシン基導入アルギン酸（化合物１０－７）の合成
Figure JPOXMLDOC01-appb-C000015
 Example 10 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-9-yl (5- (2-aminoethoxy) picolinoyl) glycine group-introduced alginic acid (compound 10-7)
Figure JPOXMLDOC01-appb-C000015
＜工程１＞（５－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ピコリノイル）グリシン（化合物１０－３）の合成
　（実施例９）＜工程１＞と同様の操作で合成した化合物１０－１（０．７６８ｇ）及びメタノール（６．９７ｍＬ）の混合物に対し、室温で水酸化ナトリウム（０．２８ｇ）を加え、同温にて３０分撹拌、４０℃にて１時間それぞれ撹拌した。反応終了後、１規定－塩酸（７ｍＬ）を加え、溶液を減圧下で濃縮した。残留物に対し、グリシンメチル塩酸塩［ＣＡＳ：５６８０－７９－５］（０．３２ｇ）、 Ｏ－（７－アザベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（０．９８ｇ）及びアセトニトリル（６．９７ｍＬ）を加えた。続いて、氷冷撹拌下、Ｎ，Ｎ－ジイソプロピルエチルアミン（１．２３ｍＬ）を滴下し、室温にて１時間撹拌した。水（１０ｍＬ）を加え反応を停止させた後、酢酸エチル（１０ｍＬ）で３回抽出した。有機層を水（５ｍＬ）、飽和食塩水（５ｍＬ）で順次洗浄し、無水硫酸ナトリウムで乾燥させた。乾燥させた有機層を濾過後、減圧下で濃縮した。粗生成物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、化合物１０－２（０．６７ｇ）を含む画分を得た。
　化合物１０－２（０．６７ｇ）を含む画分及びメタノール（６．３４ｍＬ）の混合物に対し、室温で、水酸化ナトリウム（０．１４ｇ）を加え、同温にて１７時間撹拌した。反応終了後、溶媒を減圧下で留去し、残留物を水（１０ｍＬ）に溶解させた。メチル　ｔｅｒｔ－ブチルエーテル（１０ｍＬ）で２回抽出した後、水層を１規定－塩酸（３．６ｍＬ）で酸性にし、酢酸エチル（１０ｍＬ）で３回抽出した。有機層を水（１０ｍＬ）及び飽和食塩水（１０ｍＬ）で順次洗浄し、無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で溶媒を留去することで、標記化合物１０－３（０．４４ｇ）を白色のアモルファスとして得た。
ＮＭＲデータ（ＣＤ3ＯＤ）（δ：ｐｐｍ）：　８．３３　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　８．０７　（１Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　７．５５　（１Ｈ，　ｄｄ，　Ｊ　＝　８．４，　２．４　Ｈｚ），　４．１６　（２Ｈ，　ｔ，　Ｊ　＝　５．６　Ｈｚ），　４．１４　（２Ｈ，　ｓ），　３．４７　（２Ｈ，　ｔ，　Ｊ　＝　５．６　Ｈｚ），　１．４４　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ＝３３９，ＲＴ＝０．７３（分），［Ｍ＋Ｈ］+＝３４０ <Step 1> Synthesis of (5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinoyl) glycine (Compound 10-3) (Example 9) Compound 10 synthesized in the same manner as in <Step 1> -1 (0.768 g) and methanol (6.97 mL) were added sodium hydroxide (0.28 g) at room temperature, stirred at the same temperature for 30 minutes, and stirred at 40 ° C. for 1 hour. After completion of the reaction, 1N hydrochloric acid (7 mL) was added, and the solution was concentrated under reduced pressure. To the residue, glycine methyl hydrochloride [CAS: 5680-79-5] (0.32 g), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyl Uronium hexafluorophosphate (0.98 g) and acetonitrile (6.97 mL) were added. Subsequently, N, N-diisopropylethylamine (1.23 mL) was added dropwise with stirring under ice cooling, and the mixture was stirred at room temperature for 1 hour. Water (10 mL) was added to stop the reaction, and the mixture was extracted 3 times with ethyl acetate (10 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. The dried organic layer was filtered and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 10-2 (0.67 g).
To a mixture of the fraction containing compound 10-2 (0.67 g) and methanol (6.34 mL), sodium hydroxide (0.14 g) was added at room temperature, and the mixture was stirred at the same temperature for 17 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved in water (10 mL). After extraction twice with methyl tert-butyl ether (10 mL), the aqueous layer was acidified with 1N hydrochloric acid (3.6 mL) and extracted three times with ethyl acetate (10 mL). The organic layer was washed successively with water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give the title compound 10-3 (0.44 g). Was obtained as a white amorphous.
NMR data (CD 3 OD) (δ: ppm): 8.33 (1H, d, J = 2.4 Hz), 8.07 (1H, d, J = 8.4 Hz), 7.55 (1H , Dd, J = 8.4, 2.4 Hz), 4.16 (2H, t, J = 5.6 Hz), 4.14 (2H, s), 3.47 (2H, t, J = 5.6 Hz), 1.44 (9H, s), LC-MS: M = 339, RT = 0.73 (min), [M + H] + = 340
＜工程２＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　（５－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ピコリノイル）グリシナート（化合物１０－５）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物１０－４，１００ｍｇ）、（実施例１０）＜工程１＞で得られた化合物１０－３（９０．８ｍｇ）、ベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１１８．３５ｍｇ）及びジクロロメタン（５０００μＬ）の混合物に対し、氷冷撹拌下、トリエチルアミン（７４．５９μＬ）を滴下し、室温で２２時間撹拌した。反応混合物を濾過し、濾液を減圧下で濃縮した。残留物をシリカゲルカラムクロマトグラフィー（２５％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル，２％メタノール／酢酸エチル～２０％メタノール／酢酸エチル）により精製し、標記化合物１０－５（７３ｍｇ）を黄色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．４２　（１Ｈ，　ｔ，　Ｊ　＝　６．０　Ｈｚ），　８．２５　（２Ｈ，　ｄｄ，　Ｊ　＝　６．０，　３．２　Ｈｚ），　８．１７　（１Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　７．９２　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　７．６４　（１Ｈ，　ｓ），　７．６０　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．８　Ｈｚ），　７．３１　（１Ｈ，　ｄｄ，　Ｊ　＝　８．８，　２．８　Ｈｚ），　５．７５　（１Ｈ，　ｄ，　Ｊ　＝　１６．８　Ｈｚ），　５．３１　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．２７　（２Ｈ，　ｓ），　４．９６　（１Ｈ，　ｂｒ　ｓ），　４．６１　（２Ｈ，　ｄ，　Ｊ　＝　６．０　Ｈｚ），　４．１３　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．５８　（２Ｈ，　ｄｄ，　Ｊ　＝　９．６，　４．８　Ｈｚ），　３．２８－３．２０　（３Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｔｗｏ　ｐｅａｋｓ．），　１．９５－１．８４　（２Ｈ，　ｍ），　１．４５　（９Ｈ，　ｓ），　１．４０　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋｓ．），　１．０４　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝７１３，ＲＴ＝０．９０（分），［Ｍ＋Ｈ］+＝７１４ <Step 2> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl (5- (2-((tert-butoxycarbonyl) amino) ethoxy) picolinoyl) glycinate (compound 10-5) (S) -4,11-diethyl-4 , 9-Dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [CAS: 86639-52- 3] (Compound 10-4, 100 mg), (Example 10) Compound 10-3 (90.8 mg) obtained in <Step 1>, Benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (1 To a mixture of 8.35Mg) and dichloromethane (5000μL), with stirring under ice cooling, was added dropwise triethylamine (74.59μL), was stirred at room temperature for 22 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (25% ethyl acetate / n-heptane to 100% ethyl acetate, 2% methanol / ethyl acetate to 20% methanol / ethyl acetate) to give the title compound 10-5 (73 mg) in yellow Obtained as amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.42 (1H, t, J = 6.0 Hz), 8.25 (2H, dd, J = 6.0, 3.2 Hz), 8. 17 (1H, d, J = 8.4 Hz), 7.92 (1H, d, J = 2.4 Hz), 7.64 (1H, s), 7.60 (1H, dd, J = 9 .2, 2.8 Hz), 7.31 (1H, dd, J = 8.8, 2.8 Hz), 5.75 (1H, d, J = 16.8 Hz), 5.31 (1H , D, J = 16.0 Hz), 5.27 (2H, s), 4.96 (1H, br s), 4.61 (2H, d, J = 6.0 Hz), 4.13 ( 2H, t, J = 5.2 Hz), 3.58 (2H, dd, J = 9.6, 4.8 Hz), 3.28-3.20 (3H, m, Overlapped with two peaks.), 1.95-1.84 (2H, m), 1.45 (9H, s), 1.40 (3H, t, J = 7.2 Hz, Overwrapped with other peaks.) , 1.04 (3H, t, J = 7.2 Hz), LC-MS: M = 713, RT = 0.90 (min), [M + H] + = 714
＜工程３＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　（５－（２－アミノエトキシ）ピコリノイル）グリシナート塩酸塩（化合物１０－６）の合成
　（実施例１０）＜工程２＞で得られた化合物１０－５（０．０７３ｇ）を用い、（実施例６）＜工程３＞と同様の操作を行い、標記化合物１０－６（０．０５２７ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　８．２６　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　７．９４　（１Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　７．４４　（１Ｈ，　ｄｄ，　Ｊ　＝　８．８，　２．８　Ｈｚ），　７．３０　（１Ｈ，　ｂｒ　ｓ），　７．２１　（１Ｈ，　ｂｒ　ｓ），　６．８６　（１Ｈ，　ｂｒ　ｓ），　６．７９－６．７７　（１Ｈ，　ｂｒ　ｍ），　５．１８　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．０２　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　４．３８－４．２８　（６Ｈ，　ｍ），　３．３８　（２Ｈ，　ｔ，　Ｊ　＝　４．８　Ｈｚ），　３．０７　（１Ｈ，　ｄ，　Ｊ　＝　７．２　Ｈｚ），　３．０３　（１Ｈ，　ｄ，　Ｊ　＝　７．２　Ｈｚ），　２．７２－２．６９　（１Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｔｗｏ　ｐｅａｋｓ．），　２．６１－２．５１　（１Ｈ，　ｂｒ　ｍ），　１．７０－１．５８　（２Ｈ，　ｂｒ　ｍ），　１．０５－１．０１　（３Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋｓ．），　０．７４　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝６１３，ＲＴ＝０．６６（分），［Ｍ＋Ｈ］+＝６１４ <Step 3> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of 5- (2-aminoethoxy) picolinoyl) glycinate hydrochloride (Compound 10-6) (Example 10) Compound 10-5 obtained in <Step 2> (0.073 g) was used, and the same operation as in Example 6 <Step 3> was carried out to obtain the title compound 10-6 (0.0527 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 8.26 (1H, d, J = 2.8 Hz), 7.94 (1H, d, J = 8.8 Hz), 7.44 (1H , Dd, J = 8.8, 2.8 Hz), 7.30 (1H, br s), 7.21 (1H, br s), 6.86 (1H, br s), 6.79-6 .77 (1H, br m), 5.18 (1H, d, J = 16.0 Hz), 5.02 (1H, d, J = 16.0 Hz), 4.38-4.28 (6H M), 3.38 (2H, t, J = 4.8 Hz), 3.07 (1H, d, J = 7.2 Hz), 3.03 (1H, d, J = 7.2 Hz). ), 2.72-2.69 (1H, m, Overlapped with two peaks.), 2.61-1.51 (1H, b rm), 1.70-1.58 (2H, br m), 1.05-1.01 (3H, m, overlapped with other peaks.), 0.74 (3H, t, J = 7.2). Hz), LC-MS: M (free amine) = 613, RT = 0.66 (min), [M + H] + = 614
＜工程４＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　（５－（２－アミノエトキシ）ピコリノイル）グリシナート基導入アルギン酸（化合物１０－７）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）と（実施例１０）＜工程３＞で得られた化合物１０－６（３１．４１ｍｇ）を用い、（実施例７）＜工程５＞と同様の操作を行い、標記化合物１０－７（１１０ｍｇ）を黄色固体として得た。薬剤導入率は１．４６モル％であった。分子量は、２２５万Ｄａから１．６万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３３万Ｄａであった。 <Step 4> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-9-yl (5- (2-aminoethoxy) picolinoyl) glycinate introduced alginic acid (compound 10-7) 251,000 Da to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) aqueous solution (9888 μL) and Example 10 <Compound 10-6 (31.41 mg) obtained in <Step 3> were used. (Example 7) The same operation as in <Step 5> was performed to give the title compound 10-7 (110 mg) as a yellow solid. The drug introduction rate was 1.46 mol%. The molecular weight showed a broad elution peak from 2.25 million Da to 16,000 Da, and the weight average molecular weight was 13.3 million Da.
（実施例１１）（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　Ｎ－アセチル－Ｎ－（４－（２－アミノエトキシ）ベンジル）グリシン基導入アルギン酸（化合物１１－９）の合成
Figure JPOXMLDOC01-appb-C000016
 Example 11 (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-9-yl N-acetyl-N- (4- (2-aminoethoxy) benzyl) glycine group-introduced alginic acid (compound 11-9)
Figure JPOXMLDOC01-appb-C000016
＜工程１＞ｔｅｒｔ－ブチル　（２－（４－フォルミルフェノキシ）エチル）カルバマート（化合物１１－２）の合成
　４－ヒドロキシベンズアルデヒド［ＣＡＳ：１２３－０８－０］（化合物１１－１，１ｇ）、ヨウ化カリウム（１．３６ｇ）、ｔｅｒｔ－ブチル（２－ブロモエチル）カルバマート［ＣＡＳ：３９６８４－８０－５］（２．２ｇ）及びＮ－メチルピロリドン（１０ｍＬ）の混合物に対し、室温で、炭酸カリウム（１．３６ｇ）を加え、８０℃で３時間３０分撹拌した。反応終了後、混合物を室温まで冷却し、水（２０ｍＬ）で薄めた。この混合物を酢酸エチル（１５ｍＬ）で５回抽出し、飽和食塩水（１０ｍＬ）で洗浄した。有機層を無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で濃縮した。粗生成物をメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）に溶解させ、１規定－水酸化ナトリウム水溶液（５ｍＬ）で２回、飽和食塩水（５ｍＬ）で順次洗浄した。有機層を無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で濃縮することで、標記化合物１１－２（１．８８ｇ）を褐色の油状化合物として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　９．８８　（１Ｈ，　ｓ），　７．８５－７．８２　（２Ｈ，　ｍ），　７．０１－６．９８　（２Ｈ，　ｍ），　４．９７　（１Ｈ，　ｂｒ　ｓ），　４．１１　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．５７　（２Ｈ，　ｄｄ，　Ｊ　＝　１０．４，　５．２　Ｈｚ），　１．４５　（９Ｈ，　ｓ）． <Step 1> Synthesis of tert-butyl (2- (4-formylphenoxy) ethyl) carbamate (Compound 11-2) 4-hydroxybenzaldehyde [CAS: 123-08-0] (Compound 11-1, 1 g), Potassium carbonate at room temperature against a mixture of potassium iodide (1.36 g), tert-butyl (2-bromoethyl) carbamate [CAS: 39684-80-5] (2.2 g) and N-methylpyrrolidone (10 mL) (1.36 g) was added, and the mixture was stirred at 80 ° C. for 3 hours and 30 minutes. After completion of the reaction, the mixture was cooled to room temperature and diluted with water (20 mL). The mixture was extracted 5 times with ethyl acetate (15 mL) and washed with saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was dissolved in methyl tert-butyl ether (20 mL) and washed successively with 1N aqueous sodium hydroxide solution (5 mL) twice and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the title compound 11-2 (1.88 g) as a brown oily compound.
NMR data (CDCl 3 ) (δ: ppm): 9.88 (1H, s), 7.85-7.82 (2H, m), 7.01-6.98 (2H, m), 4.97 (1H, br s), 4.11 (2H, t, J = 5.2 Hz), 3.57 (2H, dd, J = 10.4, 5.2 Hz), 1.45 (9H, s ).
＜工程２＞（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ベンジル）グリシン塩酸塩（化合物１１－４）の合成
　（実施例１１）＜工程１＞で得られた化合物１１－２（１．２７ｇ）、グリシンメチル塩酸塩［ＣＡＳ：５６８０－７９－５］（０．６９ｇ）及び塩化メチレン（２５．４ｍＬ）の混合物に対し、氷冷撹拌下、トリエチルアミン（０．７３ｍＬ）を滴下し、同温にて１０分間撹拌した。続いて、氷冷撹拌下、水素化トリアセトキシホウ素ナトリウム（１．５２ｇ）を少量ずつ加え、反応混合物を室温にて２４時間撹拌した。水（１０ｍＬ）を加え、反応を停止させた後、有機層を分離し、減圧下で濃縮した。生じた残留物をメチル　ｔｅｒｔ－ブチルエーテル（２０ｍＬ）に溶解させ、１規定－塩酸（１０ｍＬ）で洗浄した。水層をメチル　ｔｅｒｔ－ブチルエーテル（１０ｍＬ）で２回抽出し、１規定－水酸化ナトリウム水溶液（１０ｍＬ）で塩基性にした。この溶液をメチル　ｔｅｒｔ－ブチルエーテル（１０ｍＬ）で３回抽出し、無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で濃縮することで化合物１１－３（１．２９２ｇ）の粗生成物を無色油状化合物として得た。
　化合物１１－３（１．２９２ｇ）の粗生成物及びメタノール（１２．９２ｍＬ）の混合物に対し、室温で水酸化ナトリウム（０．３１ｇ）を加え、反応混合物を４０℃で３時間撹拌した。反応終了後、室温で４規定－塩化水素／酢酸エチル（１１．４５ｍＬ）を加え、減圧下で濃縮した。生じた残留物をメチル　ｔｅｒｔ－ブチルエーテル（４０ｍＬ）でトリチュレートし、濾過することで、標記化合物１１－４（１．２３ｇ）を白色のアモルファスとして得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．４７　（２Ｈ，　ｄ，　Ｊ　＝　８．０　Ｈｚ），　７．１１　（２Ｈ，　ｄ，　Ｊ　＝　８．４　Ｈｚ），　４．２９　（２Ｈ，　ｓ），　４．１９　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．８３　（２Ｈ，　ｓ），　３．５２　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　１．４５　（９Ｈ，　ｓ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝３２４，ＲＴ＝０．６６（分），［Ｍ＋Ｈ］+＝３２５ <Step 2> Synthesis of (4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzyl) glycine hydrochloride (Compound 11-4) (Example 11) Compound 11- obtained in <Step 1> 2 (1.27 g), glycine methyl hydrochloride [CAS: 5680-79-5] (0.69 g) and methylene chloride (25.4 mL) were mixed with triethylamine (0.73 mL) under ice-cooling and stirring. The solution was added dropwise and stirred at the same temperature for 10 minutes. Subsequently, sodium triacetoxyborohydride (1.52 g) was added little by little under ice-cooling and the reaction mixture was stirred at room temperature for 24 hours. Water (10 mL) was added to stop the reaction, and then the organic layer was separated and concentrated under reduced pressure. The resulting residue was dissolved in methyl tert-butyl ether (20 mL) and washed with 1N hydrochloric acid (10 mL). The aqueous layer was extracted twice with methyl tert-butyl ether (10 mL) and basified with 1N aqueous sodium hydroxide solution (10 mL). This solution was extracted three times with methyl tert-butyl ether (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product of compound 11-3 (1.292 g) as a colorless oily compound. Got as.
To a mixture of the crude product of compound 11-3 (1.292 g) and methanol (12.92 mL), sodium hydroxide (0.31 g) was added at room temperature, and the reaction mixture was stirred at 40 ° C. for 3 hours. After completion of the reaction, 4N-hydrogen chloride / ethyl acetate (11.45 mL) was added at room temperature, and the mixture was concentrated under reduced pressure. The resulting residue was triturated with methyl tert-butyl ether (40 mL) and filtered to give the title compound 11-4 (1.23 g) as a white amorphous.
NMR data (D 2 O) (δ: ppm): 7.47 (2H, d, J = 8.0 Hz), 7.11 (2H, d, J = 8.4 Hz), 4.29 (2H , S), 4.19 (2H, t, J = 5.2 Hz), 3.83 (2H, s), 3.52 (2H, t, J = 5.2 Hz), 1.45 (9H , S), LC-MS: M (free amine) = 324, RT = 0.66 (min), [M + H] + = 325
＜工程３＞Ｎ－アセチル－Ｎ－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ベンジル）グリシン（化合物１１－５）の合成
　（実施例１１）＜工程２＞で得られた化合物１１－４（３００ｍｇ）及び塩化アセチル（８８．６７μＬ）及び塩化メチレン（３０００μＬ）の混合物に対し、氷冷撹拌下、トリエチルアミン（４６３．５４μＬ）を滴下した。反応混合物を室温で２時間撹拌し、１規定－塩酸（５ｍＬ）で反応を停止させた。有機層を分離し、水層を酢酸エチル（１０ｍＬ）で３回抽出し、無水硫酸ナトリウムで乾燥させ、濾過後、減圧下で濃縮した。粗生成物をシリカゲルカラムクロマトグラフィー（１２％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル、酢酸エチル～５０％メタノール／酢酸エチル）により精製し、標記化合物１１－５（１８７ｍｇ）を無色の油状化合物として得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　７．１１　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　６．８９　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　４．９９　（１Ｈ，　ｂｒ　ｓ），　４．５５　（２Ｈ，　ｓ），　４．０５　（２Ｈ，　ｓ），　４．０２　（３Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋ（１Ｈ）），　３．５５－３．５２　（２Ｈ，　ｂｒ　ｍ），　２．２５　（３Ｈ，　ｓ），　１．４５　（１３Ｈ，　ｓ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋ（４Ｈ）），ＬＣ－ＭＳ：Ｍ＝３６６，ＲＴ＝０．７９（分），［Ｍ＋Ｈ］+＝３６７ <Step 3> Synthesis of N-acetyl-N- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzyl) glycine (Compound 11-5) (Example 11) obtained in <Step 2> To a mixture of Compound 11-4 (300 mg), acetyl chloride (88.67 μL) and methylene chloride (3000 μL), triethylamine (463.54 μL) was added dropwise under ice-cooling and stirring. The reaction mixture was stirred at room temperature for 2 hours and quenched with 1N hydrochloric acid (5 mL). The organic layer was separated, and the aqueous layer was extracted three times with ethyl acetate (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate, ethyl acetate to 50% methanol / ethyl acetate) to give the title compound 11-5 (187 mg) as a colorless oily compound. Got as.
NMR data (CDCl 3 ) (δ: ppm): 7.11 (2H, d, J = 8.8 Hz), 6.89 (2H, d, J = 8.8 Hz), 4.99 (1H, br s), 4.55 (2H, s), 4.05 (2H, s), 4.02 (3H, t, J = 5.2 Hz, Overlapped with another peak (1H)), 3.55- 3.52 (2H, br m), 2.25 (3H, s), 1.45 (13H, s, overlapped with other peak (4H)), LC-MS: M = 366, RT = 0.79 ( Min), [M + H] + = 367
＜工程４＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　Ｎ－アセチル－Ｎ－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）ベンジル）グリシナート（化合物１１－７）の合成
　（Ｓ）－４，１１－ジエチル－４，９－ジヒドロキシ－１，１２－ジヒドロ－１４Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－３，１４（４Ｈ）－ジオン［ＣＡＳ：８６６３９－５２－３］（化合物１１－６，１００ｍｇ）、（実施例１１）＜工程３＞で得られた化合物１１－５（１１７．８４ｍｇ）、ベンゾトリアゾール－１－イルオキシ－トリスジメチルアミノホスホニウム塩（１１８．３５ｍｇ）、ジクロロメタン（５０００μＬ）及びトリエチルアミン（７４．５９μＬ）を使用し、（実施例６）＜工程２＞と同様の操作をすることで、標記化合物１１－６（１００ｍｇ）を淡黄色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２２　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．８５　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　７．６４　（１Ｈ，　ｓ），　７．５３　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．４　Ｈｚ），　７．２０　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　６．９３　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ），　５．７５　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　５．３１　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．２６　（２Ｈ，　ｓ），　４．９９　（１Ｈ，　ｂｒ　ｓ），　４．６７　（２Ｈ，　ｓ），　４．３３　（２Ｈ，　ｓ），　４．０４　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　４．００　（１Ｈ，　ｄ，　Ｊ　＝　５．５　Ｈｚ），　３．５５　（２Ｈ，　ｄ，　Ｊ　＝　５．５　Ｈｚ），　３．１５　（２Ｈ，　ｄｄ，　Ｊ　＝　１５．６，　７．６　Ｈｚ），　２．３０　（３Ｈ，　ｓ），　１．９５－１．８２　（３Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋ（１Ｈ）．），　１．４６　（１１Ｈ，　ｓ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋ（２Ｈ）．），　１．３９　（４Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋ（１Ｈ）．），　１．０４　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝７４０，ＲＴ＝０．９２（分），［Ｍ＋Ｈ］+＝７４１ <Step 4> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl N-acetyl-N- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) benzyl) glycinate (Compound 11-7) (S) -4 , 11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-3,14 (4H) -dione [ CAS: 86639-52-3] (Compound 11-6, 100 mg), (Example 11) Compound 11-5 obtained in <Step 3> (117.84 mg), Benzotriazol-1-yloxy-trisdimethylamino The title compound 11-6 (100 mg) was prepared in the same manner as in (Example 6) <Step 2> using a suphonium salt (118.35 mg), dichloromethane (5000 μL) and triethylamine (74.59 μL). Obtained as a pale yellow amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.22 (1H, d, J = 9.2 Hz), 7.85 (1H, d, J = 2.8 Hz), 7.64 (1H, s), 7.53 (1H, dd, J = 9.2, 2.4 Hz), 7.20 (2H, d, J = 8.8 Hz), 6.93 (2H, d, J = 8) .8 Hz), 5.75 (1H, d, J = 16.0 Hz), 5.31 (1H, d, J = 16.4 Hz), 5.26 (2H, s), 4.99 ( 1H, br s), 4.67 (2H, s), 4.33 (2H, s), 4.04 (2H, t, J = 5.2 Hz), 4.00 (1H, d, J = 5.5 Hz), 3.55 (2H, d, J = 5.5 Hz), 3.15 (2H, dd, J = 15.6, 7.6 Hz), 2 30 (3H, s), 1.95-1.82 (3H, m, overwrapped with another peak (1H).), 1.46 (11H, s, overwrapped with another peak (2H).), 1.39 (4H, t, J = 7.6 Hz, Overlapped with other peak (1H).), 1.04 (3H, t, J = 7.6 Hz), LC-MS: M = 740, RT = 0. 92 (min), [M + H] + = 741
＜工程５＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　Ｎ－アセチル－Ｎ－（４－（２－アミノエトキシ）ベンジル）グリシナート塩酸塩（化合物１１－８）の合成
　（実施例１１）＜工程４＞で得られた化合物１１－６（０．１ｇ）を用い、（実施例６）＜工程３＞と同様の操作を行い、標記化合物１１－８（０．１１７４ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．６５　（１Ｈ，　ｄ，　Ｊ　＝　１０．０　Ｈｚ），　７．３６　（１Ｈ，　ｂｒ　ｓ，　Ａ　ｐａｉｒ　ｏｆ　ｓｉｎｇｌｅｔ．），　７．２４　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ，　Ａ　ｐａｉｒ　ｏｆ　ｄｏｕｂｌｅｔ．），　７．１４　（１Ｈ，　ｓ），　７．０６　（１Ｈ，　ｄ，　Ｊ　＝　１０．４　Ｈｚ），　６．９５　（２Ｈ，　ｄ，　Ｊ　＝　８．８　Ｈｚ，　Ａ　ｐａｉｒ　ｏｆ　ｄｏｕｂｌｅｔ．），　５．３５　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　５．１４　（１Ｈ，　ｄ，　Ｊ　＝　１６．８　Ｈｚ），　４．１８－４．１０　（４Ｈ，　ｍ），　３．３３－３．２６　（２Ｈ，　ｍ），　２．２０　（３Ｈ，　ｓ，　Ａ　ｐａｉｒ　ｏｆ　ｓｉｎｇｌｅｔ．），　１．８２－１．７６　（２Ｈ，　ｍ），　１．１３－１．０９　（４Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｏｔｈｅｒ　ｐｅａｋｓ．），　０．８３　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ）．
６Ｈ　ｗａｓ　ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｓｏｌｖｅｎｔ　ｐｅａｋ．，ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝６４０，ＲＴ＝０．６５（分），［Ｍ＋Ｈ］+＝６４１ <Step 5> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl Synthesis of N-acetyl-N- (4- (2-aminoethoxy) benzyl) glycinate hydrochloride (Compound 11-8) (Example 11) Obtained in <Step 4> Using the obtained compound 11-6 (0.1 g), the same operation as in Example 6 <Step 3> was carried out to obtain the title compound 11-8 (0.1174 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.65 (1H, d, J = 10.0 Hz), 7.36 (1H, brs, A pair of singlet.), 7.24 (2H , D, J = 8.8 Hz, A pair of doublet.), 7.14 (1H, s), 7.06 (1H, d, J = 10.4 Hz), 6.95 (2H, d, J = 8.8 Hz, A pair of doublet.), 5.35 (1H, d, J = 17.2 Hz), 5.14 (1H, d, J = 16.8 Hz), 4.18- 4.10 (4H, m), 3.33-3.26 (2H, m), 2.20 (3H, s, A pair of singlet.), 1.82-1.76 (2H, m), 1.13-1.09 (4H, m, Overlap ed with other peaks.), 0.83 (3H, t, J = 7.6 Hz).
6H was overwrapped with solvent peak. , LC-MS: M (free amine) = 640, RT = 0.65 (min), [M + H] + = 641
＜工程６＞（Ｓ）－４，１１－ジエチル－４－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－９－イル　Ｎ－アセチル－Ｎ－（４－（２－アミノエトキシ）ベンジル）グリシン基導入アルギン酸（化合物１１－９）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）と（実施例１１）＜工程５＞で得られた化合物１１－８（４６．４７ｍｇ）を用い、（実施例７）＜工程５＞と同様の操作を行い、標記化合物１１－９（１１８ｍｇ）を淡黄色固体として得た。薬剤導入率は７．２１モル％であった。分子量は、２３１万Ｄａから０．４３万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１２９万Ｄａであった。 <Step 6> (S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-9-yl N-acetyl-N- (4- (2-aminoethoxy) benzyl) glycine group-introduced alginic acid (Compound 11-9) Sodium alginate (stock) Manufactured by Kimika Co., Ltd., molecular weight: 251,000 to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) aqueous solution (9888 μL) and compound 11-8 obtained in (Example 11) <Step 5> 46.47 mg), and the same operation as in Example 7 <Step 5> was performed to give the title compound 11-9 (118 mg) as a pale yellow solid. The drug introduction rate was 7.21 mol%. The molecular weight showed a broad elution peak from 23.1 million Da to 0.43 million Da, and the weight average molecular weight was 1,290,000 Da.
（実施例１２）（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２－（２－（２－アミノエトキシ）エトキシ）酢酸基導入アルギン酸（化合物１２－５）の合成
Figure JPOXMLDOC01-appb-C000017
 Example 12 (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-4-yl 2- (2- (2-aminoethoxy) ethoxy) acetic acid group-introduced alginic acid (Compound 12-5)
Figure JPOXMLDOC01-appb-C000017
＜工程１＞（Ｓ）－９－（（ｔｅｒｔ－ブトキシカルボニル）オキシ）－４，１１－ジエチル－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２，２－ジメチル－４－オキソ－３，８，１１－トリオキサ－５－アザトリデカン－１３－オアート（化合物１２－３）の合成
　（実施例４）＜工程１＞で合成した化合物１２－１（１００ｍｇ）、（実施例２）＜工程１＞で合成した化合物１２－２（５９．４ｍｇ）及びＮ，Ｎ－ジメチル－４－アミノピリジン（４．９６ｍｇ）のジクロロメタン（７５０μＬ）溶液に、氷冷撹拌下、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（４１．８９ｍｇ）のジクロロメタン（２５０μＬ）溶液を滴下し、室温で３時間撹拌した。更に、室温で、化合物１２－２（５９．４ｍｇ）のジクロロメタン（５００μＬ）溶液及びＮ，Ｎ’－ジシクロヘキシルカルボジイミド（４１．８９ｍｇ）のジクロロメタン（５００μＬ）溶液を滴下し、同温で１時間撹拌した。混合物を酢酸エチル（３０ｍＬ）で希釈し、濾過後、濾液を減圧下で濃縮した。残留物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル、酢酸エチル～１５％メタノール／酢酸エチル）により精製し、標記化合物１２－３（１４８ｍｇ）を白色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２０　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．９０　（１Ｈ，　ｄ，　Ｊ　＝　２．８　Ｈｚ），　７．６７　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．８　Ｈｚ），　７．１７　（１Ｈ，　ｓ），　５．７０　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　５．４２　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　５．２５　（２Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　５．０３　（１Ｈ，　ｂｒ　ｓ），　４．３８　（２Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　４．３１　（２Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　３．７８－３．６８　（２Ｈ，　ｍ），　３．６２　（２Ｈ，　ｔ，　Ｊ　＝　４．４　Ｈｚ），　３．５２　（２Ｈ，　ｔ，　Ｊ　＝　５．２　Ｈｚ），　３．３０　（２Ｈ，　ｑ，　Ｊ　＝　５．２　Ｈｚ），　３．１６　（２Ｈ，　ｑ，　Ｊ　＝　８．０　Ｈｚ），　２．３２－２．２９　（１Ｈ，　ｍ），　２．１９－２．１７　（１Ｈ，　ｍ），　１．６２　（９Ｈ，　ｓ），　１．４２－１．４０　（１２Ｈ，　ｍ），　０．９８　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝７３７，ＲＴ＝１．１１（分），［Ｍ＋Ｈ］+＝７３８ <Step 1> (S) -9-((tert-butoxycarbonyl) oxy) -4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ': 6,7] Indolizino [1,2-b] quinolin-4-yl 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oate (compound 12-3) (Example 4) Compound 12-1 (100 mg) synthesized in <Step 1>, (Example 2) Compound 12-2 (59.4 mg) synthesized in <Step 1> and N, N-dimethyl- To a solution of 4-aminopyridine (4.96 mg) in dichloromethane (750 μL) was added dropwise a solution of N, N′-dicyclohexylcarbodiimide (41.89 mg) in dichloromethane (250 μL) under ice-cooling and stirring at room temperature. And the mixture was stirred time. Further, at room temperature, a solution of compound 12-2 (59.4 mg) in dichloromethane (500 μL) and N, N′-dicyclohexylcarbodiimide (41.89 mg) in dichloromethane (500 μL) were added dropwise and stirred at the same temperature for 1 hour. . The mixture was diluted with ethyl acetate (30 mL) and after filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate, ethyl acetate to 15% methanol / ethyl acetate) to obtain the title compound 12-3 (148 mg) as a white amorphous. .
NMR data (CDCl 3 ) (δ: ppm): 8.20 (1H, d, J = 9.2 Hz), 7.90 (1H, d, J = 2.8 Hz), 7.67 (1H, dd, J = 9.2, 2.8 Hz), 7.17 (1H, s), 5.70 (1H, d, J = 17.2 Hz), 5.42 (1H, d, J = 17 .2 Hz), 5.25 (2H, d, J = 2.4 Hz), 5.03 (1H, br s), 4.38 (2H, d, J = 17.2 Hz), 4.31 (2H, d, J = 17.2 Hz), 3.78-3.68 (2H, m), 3.62 (2H, t, J = 4.4 Hz), 3.52 (2H, t, J = 5.2 Hz), 3.30 (2H, q, J = 5.2 Hz), 3.16 (2H, q, J = 8.0 Hz), 2.3 -2.29 (1H, m), 2.19-2.17 (1H, m), 1.62 (9H, s), 1.42-1.40 (12H, m), 0.98 (3H , T, J = 7.2 Hz), LC-MS: M = 737, RT = 1.11 (min), [M + H] + = 738
＜工程２＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２－（２－（２－アミノエトキシ）エトキシ）酢酸塩酸塩（化合物１２－４）の合成
　（実施例１２）＜工程１＞で得られた化合物１２－３（０．１４８ｇ）及び１，４－ジオキサン（１．０４ｍＬ）の混合物に対し、水冷撹拌下、４規定－塩化水素／１，４－ジオキサン（１．０４ｍＬ）を加え、室温で２０時間、６０℃で４時間撹拌した。ジイソプロピルエーテル（１０ｍＬ）を加え、析出物を濾過し、減圧下で乾燥させることで、標記化合物１２－４（０．１３ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．６８　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．１６　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．８　Ｈｚ），　７．０２　（１Ｈ，　ｓ），　６．４９－６．４８　（１Ｈ，　ｍ），　５．５５　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．３６　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　４．５８　（１Ｈ，　ｄ，　Ｊ　＝　１８．０　Ｈｚ），　４．５１　（１Ｈ，　ｄ，　Ｊ　＝　１７．６　Ｈｚ），　３．９５－３．９０　（１Ｈ，　ｍ），　３．７８－３．７７　（２Ｈ，　ｍ），　３．６８－３．６６　（４Ｈ，　ｍ），　３．０９　（２Ｈ，　ｔ，　Ｊ　＝　４．８　Ｈｚ），　２．４４－２．４０　（２Ｈ，　ｂｒ　ｍ），　２．１８　（２Ｈ，　ｑ，　Ｊ　＝　７．２　Ｈｚ），　１．０３－０．９４　（７Ｈ，　ｍ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５３７，ＲＴ＝０．６５（分），［Ｍ＋Ｈ］+＝５３８ <Step 2> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of 2- (2- (2-aminoethoxy) ethoxy) acetic acid hydrochloride (Compound 12-4) (Example 12) Compound 12 obtained in <Step 1> -3 (0.148 g) and 1,4-dioxane (1.04 mL) were added with 4N-hydrogen chloride / 1,4-dioxane (1.04 mL) with stirring under water cooling, and at room temperature for 20 hours. , And stirred at 60 ° C. for 4 hours. Diisopropyl ether (10 mL) was added, and the precipitate was filtered and dried under reduced pressure to give the title compound 12-4 (0.13 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.68 (1H, d, J = 9.2 Hz), 7.16 (1H, dd, J = 9.2, 2.8 Hz), 7 .02 (1H, s), 6.49-6.48 (1H, m), 5.55 (1H, d, J = 16.4 Hz), 5.36 (1H, d, J = 16.4) Hz), 4.58 (1H, d, J = 18.0 Hz), 4.51 (1H, d, J = 17.6 Hz), 3.95-3.90 (1H, m), 3. 78-3.77 (2H, m), 3.68-3.66 (4H, m), 3.09 (2H, t, J = 4.8 Hz), 2.44-2.40 (2H, br m), 2.18 (2H, q, J = 7.2 Hz), 1.03-0.94 (7H, m), LC-MS: M (free amine) = 537, RT = 0.65 (min), [M + H] + = 538
＜工程３＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２－（２－（２－アミノエトキシ）エトキシ）酢酸基導入アルギン酸（化合物１２－５）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８００μＬ）と（実施例１２）＜工程２＞で得られた化合物１２－４（４６．４７ｍｇ）を用い、室温で１７時間反応させる以外は（実施例７）＜工程５＞と同様の操作を行い、標記化合物１２－５（１０３．１ｍｇ）を黄色固体として得た。薬剤導入率は１．６５モル％であった。分子量は、２２７万Ｄａから１．３万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３８万Ｄａであった。 <Step 3> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of 2- (2- (2-aminoethoxy) ethoxy) acetic acid group-introduced alginic acid (Compound 12-5) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd.) Molecular weight: 251,000 Da to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) aqueous solution (9800 μL) and compound 12-4 (46.47 mg) obtained in (Example 12) <Step 2> Was used in the same manner as in (Example 7) <Step 5>, except that the mixture was reacted at room temperature for 17 hours to obtain the title compound 12-5 (103.1 mg) as a yellow solid. The drug introduction rate was 1.65 mol%. The molecular weight showed a broad elution peak from 2.27 million Da to 13,000 Da, and the weight average molecular weight was 13.38 million Da.
（実施例１３）（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　３－アミノプロパン酸基導入アルギン酸（化合物１３－４）の合成
Figure JPOXMLDOC01-appb-C000018
 Example 13 (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-4-yl 3-aminopropanoic acid group-introduced alginic acid (compound 13-4)
Figure JPOXMLDOC01-appb-C000018
＜工程１＞（Ｓ）－９－（（ｔｅｒｔ－ブトキシカルボニル）オキシ）－４，１１－ジエチル－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）プロパノエート（化合物１３－２）の合成
　（実施例４）＜工程１＞で合成した化合物１３－１（１００ｍｇ）、３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）プロパン酸（４０．４５ｍｇ）及びＮ，Ｎ－ジメチル－４－アミノピリジン（４．０２ｍｇ）のジクロロメタン（６００μＬ）溶液に、氷冷撹拌下、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（４４．１１ｍｇ）のジクロロメタン（２１０μＬ）溶液を滴下し、室温で２時間撹拌した。反応液を酢酸エチル（２０ｍＬ）で希釈し、濾過後、濾液を減圧下で濃縮した。粗生成物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、標記化合物１３－２（１４２ｍｇ）を淡黄色アモルファスとして得た。
　ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２２　（１Ｈ，　ｄ，　Ｊ　＝　９．６　Ｈｚ），　７．９０　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　７．６７　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．４　Ｈｚ），　７．１６　（１Ｈ，　ｓ），　５．７０　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　５．４１　（１Ｈ，　ｄ，　Ｊ　＝　１７．６　Ｈｚ），　５．２６　（２Ｈ，　ｓ），　５．１７　（１Ｈ，　ｂｒ　ｓ），　３．５１－３．３６　（２Ｈ，　ｍ），　３．１６　（２Ｈ，　ｑ，　Ｊ　＝　７．６　Ｈｚ），　２．８０－２．６０　（２Ｈ，　ｍ），　２．３０－２．０９　（２Ｈ，　ｍ），　１．６１　（９Ｈ，　ｓ），　１．４１－１．３８　（１２Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　２　ｐｅａｋｓ．），　１．００　（３Ｈ，　ｔ，　Ｊ　＝　７．３　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝６６３，ＲＴ＝１．１４（分），［Ｍ＋Ｈ］+＝６６４ <Step 1> (S) -9-((tert-butoxycarbonyl) oxy) -4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ': 6,7] Synthesis of indolizino [1,2-b] quinolin-4-yl 3-((tert-butoxycarbonyl) amino) propanoate (Compound 13-2) (Example 4) Synthesis in <Step 1> Compound 13-1 (100 mg), 3-((tert-butoxycarbonyl) amino) propanoic acid (40.45 mg) and N, N-dimethyl-4-aminopyridine (4.02 mg) in dichloromethane (600 μL) Under ice-cooling, a solution of N, N′-dicyclohexylcarbodiimide (44.11 mg) in dichloromethane (210 μL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate (20 mL), filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound 13-2 (142 mg) as a pale yellow amorphous.
NMR data (CDCl 3 ) (δ: ppm): 8.22 (1H, d, J = 9.6 Hz), 7.90 (1H, d, J = 2.4 Hz), 7.67 (1H, dd, J = 9.2, 2.4 Hz), 7.16 (1H, s), 5.70 (1H, d, J = 17.2 Hz), 5.41 (1H, d, J = 17) .6 Hz), 5.26 (2H, s), 5.17 (1H, br s), 3.51-3.36 (2H, m), 3.16 (2H, q, J = 7.6) Hz), 2.80-2.60 (2H, m), 2.30-2.09 (2H, m), 1.61 (9H, s), 1.41-1.38 (12H, m, Overlapped with 2 peaks.), 1.00 (3H, t, J = 7.3 Hz), LC-MS: M = 663, R = 1.14 (minutes), [M + H] + = 664
＜工程２＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　３－アミノプロパン酸塩酸塩（化合物１３－３）の合成
　（実施例１３）＜工程１＞で得られた化合物１３－２（０．１４ｇ）及び１，４－ジオキサン（０．９８ｍＬ）の混合物に対し、水冷撹拌下、４規定－塩化水素／１，４－ジオキサン（０・９８ｍＬ）を滴下し、６０℃で３時間撹拌した。反応終了後、（実施例１２）＜工程２＞と同様の操作を行い、標記化合物１３－３（０．１ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．７４－７．６７　（１Ｈ，　ｂｒ　ｍ），　７．２１－７．１４　（１Ｈ，　ｂｒ　ｍ），　７．１０－７．０５　（１Ｈ，　ｂｒ　ｍ），　６．６４　（１Ｈ，　ｄ，　Ｊ　＝　４．０　Ｈｚ），　５．５６－５．５１　（１Ｈ，　ｂｒ　ｍ），　５．３９－５．３４　（１Ｈ，　ｂｒ　ｍ），　４．０５　（２Ｈ，　ｂｒ　ｓ），　３．３０－３．２６　（２Ｈ，　ｂｒ　ｍ），　３．０９　（２Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），　２．５５－２．４５　（２Ｈ，　ｂｒ　ｍ），　２．１６　（２Ｈ，　ｑ，　Ｊ　＝　７．２　Ｈｚ），　０．９８　（６Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝４６３，ＲＴ＝０．６３（分），［Ｍ＋Ｈ］+＝４６４ <Step 2> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ Synthesis of 1,2-b] quinolin-4-yl 3-aminopropanoic acid hydrochloride (Compound 13-3) (Example 13) Compounds 13-2 (0.14 g) and 1 obtained in <Step 1> , 4-Dioxane (0.98 mL) was added dropwise 4N-hydrogen chloride / 1,4-dioxane (0.98 mL) with stirring under water cooling, and the mixture was stirred at 60 ° C. for 3 hours. After completion of the reaction, the same operation as in (Example 12) <Step 2> was performed to obtain the title compound 13-3 (0.1 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.74-7.67 (1H, br m), 7.21-7.14 (1H, br m), 7.10-7.05 (1H , Br m), 6.64 (1H, d, J = 4.0 Hz), 5.56-5.51 (1H, br m), 5.39-5.34 (1H, br m), 4 .05 (2H, br s), 3.30-3.26 (2H, br m), 3.09 (2H, t, J = 7.2 Hz), 2.55-2.45 (2H, br m), 2.16 (2H, q, J = 7.2 Hz), 0.98 (6H, t, J = 7.2 Hz), LC-MS: M (free amine) = 463, RT = 0 .63 (min), [M + H] + = 464
＜工程３＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　３－アミノプロパン酸基導入アルギン酸（化合物１３－４）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８００μＬ）と（実施例１３）＜工程２＞で得られた化合物１３－３（４５．３４ｍｇ）を用い、（実施例１２）＜工程３＞と同様の操作を行い、標記化合物１３－４（１０５ｍｇ）を淡黄色固体として得た。薬剤導入率は５．９モル％であった。分子量は、２５２万Ｄａから１．３万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３７万Ｄａであった。 <Step 3> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of 3-aminopropanoic acid group-introduced alginic acid (Compound 13-4) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd., molecular weight: 251,000 Da to 1.2 Ten thousand Da (broad), weight average molecular weight: 1,340,000 Da) using an aqueous solution (9800 μL) and compound 13-3 (45.34 mg) obtained in (Example 13) <Step 2>, (Example 12) < The same operation as in Step 3> was performed to give the title compound 13-4 (105 mg) as a pale yellow solid. The drug introduction rate was 5.9 mol%. The molecular weight showed a broad elution peak from 252,000 Da to 13,000 Da, and the weight average molecular weight was 1.37 million Da.
（実施例１４）（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２-（４-（２-アミノエトキシ）フェノキシ）酢酸基導入アルギン酸（化合物１４－５）の合成
Figure JPOXMLDOC01-appb-C000019
 Example 14 (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-4-yl 2- (4- (2-aminoethoxy) phenoxy) acetic acid group-introduced alginic acid (compound 14-5)
Figure JPOXMLDOC01-appb-C000019
＜工程１＞（Ｓ）－９－（（ｔｅｒｔ－ブトキシカルボニル）オキシ）－４，１１－ジエチル－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２－（４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）エトキシ）フェノキシ）アセテート
　（実施例４）＜工程１＞で合成した化合物１４－１（１００ｍｇ）、（実施例７）＜工程１＞～＜工程３＞に従い合成した化合物１４－２（８２．１７ｍｇ）及びＮ，Ｎ－ジメチル－４－アミノピリジン（４．９６ｍｇ）のジクロロメタン（７５０μＬ）溶液に、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（５４．４６ｍｇ）のジクロロメタン（２５０μＬ）溶液を滴下し、室温で３０分撹拌した。撹拌後、（実施例１３）＜工程１＞と同様の操作を実施することで、標記化合物１４－３（１５２ｍｇ）を白色アモルファスとして得た。
ＮＭＲデータ（ＣＤＣｌ3）（δ：ｐｐｍ）：　８．２５　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．９１　（１Ｈ，　ｄ，　Ｊ　＝　２．４　Ｈｚ），　７．６９　（１Ｈ，　ｄｄ，　Ｊ　＝　９．２，　２．４　Ｈｚ），　７．１８　（１Ｈ，　ｓ），　６．８５－６．８２　（２Ｈ，　ｍ），　６．７８－６．７５　（２Ｈ，　ｍ），　５．６８　（１Ｈ，　ｄ，　Ｊ　＝　１７．４　Ｈｚ），　５．４１　（１Ｈ，　ｄ，　Ｊ　＝　１７．４　Ｈｚ），　５．２７　（１Ｈ，　ｄ，　Ｊ　＝　１８．４　Ｈｚ），　５．２２　（１Ｈ，　ｄ，　Ｊ　＝　１８．８　Ｈｚ），　４．９２　（１Ｈ，　ｂｒ　ｓ），　４．８０　（１Ｈ，　ｄ，　Ｊ　＝　１６．８　Ｈｚ），　４．７５　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　３．８３－３．７２　（２Ｈ，　ｍ），　３．４０　（２Ｈ，　ｄｄ，　Ｊ　＝　１０．４，　５．２　Ｈｚ），　３．１９－３．１４　（２Ｈ，　ｍ），　２．３３－２．１４　（２Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｓｏｌｖｅｎｔ　ｐｅａｋ），　１．６２　（９Ｈ，　ｓ），　１．４３　（９Ｈ，　ｓ），　１．４０　（３Ｈ，　ｔ，　Ｊ　＝　８．０　Ｈｚ），　０．９７　（３Ｈ，　ｔ，　Ｊ　＝　７．２　Ｈｚ），ＬＣ－ＭＳ：Ｍ＝７８５，ＲＴ＝１．２０（分），［Ｍ＋Ｈ］+＝７８６ <Step 1> (S) -9-((tert-butoxycarbonyl) oxy) -4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ': 6,7] Indolizino [1,2-b] quinolin-4-yl 2- (4- (2-((tert-butoxycarbonyl) amino) ethoxy) phenoxy) acetate (Example 4) <Step 1> Compound 14-1 (100 mg) synthesized in Example 1, (Example 7) Compound 14-2 (82.17 mg) synthesized according to <Step 1> to <Step 3> and N, N-dimethyl-4-aminopyridine (4 .96 mg) in dichloromethane (750 μL) was added dropwise a solution of N, N′-dicyclohexylcarbodiimide (54.46 mg) in dichloromethane (250 μL), and the mixture was stirred at room temperature for 30 minutes. After stirring, the same operation as in (Example 13) <Step 1> was carried out to obtain the title compound 14-3 (152 mg) as a white amorphous substance.
NMR data (CDCl 3 ) (δ: ppm): 8.25 (1H, d, J = 9.2 Hz), 7.91 (1H, d, J = 2.4 Hz), 7.69 (1H, dd, J = 9.2, 2.4 Hz), 7.18 (1H, s), 6.85-6.82 (2H, m), 6.78-6.75 (2H, m), 5 .68 (1H, d, J = 17.4 Hz), 5.41 (1H, d, J = 17.4 Hz), 5.27 (1H, d, J = 18.4 Hz), 5.22 (1H, d, J = 18.8 Hz), 4.92 (1H, br s), 4.80 (1H, d, J = 16.8 Hz), 4.75 (1H, d, J = 16 .4 Hz), 3.83-3.72 (2H, m), 3.40 (2H, dd, J = 10.4, 5.2 Hz), 3.19. -3.14 (2H, m), 2.33-2.14 (2H, m, Overlapped with solvent peak), 1.62 (9H, s), 1.43 (9H, s), 1.40 ( 3H, t, J = 8.0 Hz), 0.97 (3H, t, J = 7.2 Hz), LC-MS: M = 785, RT = 1.20 (min), [M + H] + = 786
＜工程２＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２-（４-（２-アミノエトキシ）フェノキシ）酢酸塩酸塩（化合物１４－４）の合成
　（実施例１４）＜工程１＞で得られた化合物１４－３（０．１５ｇ）を用い、（実施例６）＜工程３＞と同様の操作を行い、標記化合物１４－４（０．１３ｇ）を黄色固体として得た。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　７．６５　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　７．１０　（１Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　６．９０　（１Ｈ，　ｓ），　６．８４　（２Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　６．７６　（２Ｈ，　ｄ，　Ｊ　＝　９．２　Ｈｚ），　６．３７　（１Ｈ，　ｓ），　５．４４　（１Ｈ，　ｄ，　Ｊ　＝　１６．４　Ｈｚ），　５．２７　（１Ｈ，　ｄ，　Ｊ　＝　１６．０　Ｈｚ），　４．９７　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　４．９０　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　３．８８－３．７８　（２Ｈ，　ｍ），　３．７３－３．４５　（２Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　ｓｏｌｖｅｎｔ　ｐｅａｋ．），　３．１１－３．０９　（２Ｈ，　ｂｒ　ｍ），　２．３２－２．２７　（２Ｈ，　ｂｒ　ｍ），　２．０６　（２Ｈ，　ｄｄ，　Ｊ　＝　１５．２，　７．２　Ｈｚ），　０．８９　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），　０．８４　（３Ｈ，　ｔ，　Ｊ　＝　７．６　Ｈｚ），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝５８５，ＲＴ＝０．７３（分），［Ｍ＋Ｈ］+＝５８６ <Step 2> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of 2- (4- (2-aminoethoxy) phenoxy) acetic acid hydrochloride (Compound 14-4) (Example 14) Compound 14 obtained in <Step 1> -3 (0.15 g) was used in the same manner as in (Example 6) <Step 3> to obtain the title compound 14-4 (0.13 g) as a yellow solid.
NMR data (D 2 O) (δ: ppm): 7.65 (1H, d, J = 9.2 Hz), 7.10 (1H, d, J = 9.2 Hz), 6.90 (1H , S), 6.84 (2H, d, J = 9.2 Hz), 6.76 (2H, d, J = 9.2 Hz), 6.37 (1H, s), 5.44 (1H) , D, J = 16.4 Hz), 5.27 (1H, d, J = 16.0 Hz), 4.97 (1H, d, J = 17.2 Hz), 4.90 (1H, d , J = 17.2 Hz), 3.88-3.78 (2H, m), 3.73-3.45 (2H, m, Overlapped with solvent peak.), 3.11-3.09 (2H , Br m), 2.32-2.27 (2H, br m), 2.06 (2H, dd, J = 15.2, 7.2 Hz), 0.89 (3H, t, J = 7.6 Hz), 0.84 (3H, t, J = 7.6 Hz), LC-MS: M (free amine) ) = 585, RT = 0.73 (min), [M + H] + = 586
＜工程３＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　２-（４-（２-アミノエトキシ）フェノキシ）酢酸基導入アルギン酸（化合物１４－５）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）と（実施例１４）＜工程２＞で得られた化合物１４－４（２８．４６ｍｇ）を用い、（実施例７）＜工程５＞と同様の操作を行い、標記化合物１４－５（１１２ｍｇ）を淡黄色固体として得た。薬剤導入率は０．８５モル％であった。分子量は、２３０万Ｄａから３．９万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３８万Ｄａであった。 <Step 3> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl 2- (4- (2-aminoethoxy) phenoxy) acetic acid group-introduced alginic acid (compound 14-5) Sodium alginate prepared to 1% by weight (manufactured by Kimika Co., Ltd.) Molecular weight: 251,000 Da to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) aqueous solution (9888 μL) and compound 14-4 (28.46 mg) obtained in Example 14 <Step 2> Was used in the same manner as in (Example 7) <Step 5> to give the title compound 14-5 (112 mg) as a pale yellow solid. The drug introduction rate was 0.85 mol%. The molecular weight showed a broad elution peak from 2.3 million Da to 39,000 Da, and the weight average molecular weight was 1.38 million Da.
（実施例１５）（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　Ｎ－アセチル－Ｎ－（４－（２－アミノエトキシ）ベンジル）グリシン基導入アルギン酸（化合物１５－５）の合成
Figure JPOXMLDOC01-appb-C000020
 Example 15 (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino Synthesis of [1,2-b] quinolin-4-yl N-acetyl-N- (4- (2-aminoethoxy) benzyl) glycine group-introduced alginic acid (Compound 15-5)
Figure JPOXMLDOC01-appb-C000020
＜工程１＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　Ｎ－アセチル－Ｎ－（４－（２－アミノエトキシ）ベンジル）グリシナート塩酸塩（化合物１５－４）の合成
　（実施例４）＜工程１＞で合成した化合物１５－１（１００ｍｇ）、（実施例１１）＜工程１＞～＜工程４＞に従い合成した化合物１５－２（９３．８９ｍｇ）及びＮ，Ｎ－ジメチル－４－アミノピリジン（４．９６ｍｇ）のジクロロメタン（７５０μＬ）溶液に、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（４３．９９ｍｇ）のジクロロメタン（２５０μＬ）溶液を室温で滴下し、同温にて４５時間撹拌した。反応温合物を濾過し、濾液を酢酸エチル（２０ｍＬ）希釈した後、再度濾過した。濾液を減圧下で濃縮し、粗生成物をシリカゲルカラムクロマトグラフィー（１６％酢酸エチル／ｎ－ヘプタン～１００％酢酸エチル）により精製し、化合物１５－３（８７ｍｇ）を含む画分を得た。
　化合物１５－３（０．０８７ｇ）を含む画分を用い、（実施例６）＜工程３＞と同様の操作を行い、標記化合物１５－４（０．０７２ｇ）を黄色固体として得た。本生成物については、ＮＭＲを８０℃で測定した。
ＮＭＲデータ（Ｄ2Ｏ）（δ：ｐｐｍ）：　８．３２　（１Ｈ，　ｄｄ，　Ｊ　＝　１０．４，　９．２　Ｈｚ），　７．８１　（１Ｈ，　ｄ，　Ｊ　＝　９．６　Ｈｚ），　７．７２－７．６４　（２Ｈ，　ｍ），　７．５７　（２Ｈ，　ｄ，　Ｊ　＝　８．０　Ｈｚ），　７．５０　（１Ｈ，　ｄ，　Ｊ　＝　７．２　Ｈｚ），　７．４５　（１Ｈ，　ｄ，　Ｊ　＝　７．６　Ｈｚ，　Ａ　ｐａｉｒ　ｏｆ　ｄｏｕｂｌｅｔ．），　７．２０　（２Ｈ，　ｄ，　Ｊ　＝　７．２　Ｈｚ，　Ａ　ｐａｉｒ　ｏｆ　ｄｏｕｂｌｅｔ　（７．０７　ｐｐｍ）．），　６．０５　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ），　５．８９　（１Ｈ，　ｄ，　Ｊ　＝　１７．２　Ｈｚ，　Ａ　ｐａｉｒ　ｏｆ　ｄｏｕｂｌｅｔ．），　５．３５－５．２７　（１Ｈ，　ｍ），　５．０６－４．７３　（４Ｈ，　ｍ），　４．４６－４．３４　（２Ｈ，　ｍ），　３．７３－３．７０　（１Ｈ，　ｂｒ　ｍ），　３．３９－３．３４　（２Ｈ，　ｍ），　２．６８－２．５１　（５Ｈ，　ｍ，　Ｏｖｅｒｌａｐｐｅｄ　ｗｉｔｈ　Ｍｅ　ｇｒｏｕｐ　ａｎｄ　ｍｅｔｈｙｌｅｎｅ　ｇｒｏｕｐ．），　１．６７－１．６２　（３Ｈ，　ｍ，　Ａ　ｐａｉｒ　ｏｆ　ｔｒｉｐｌｅｔ．），　１．４２－１．３６　（３Ｈ，　ｍ，　Ａ　ｐａｉｒ　ｏｆ　ｔｒｉｐｌｅｔ．），ＬＣ－ＭＳ：Ｍ（ｆｒｅｅ　ａｍｉｎｅ）＝６４０，ＲＴ＝０．６４（分），［Ｍ＋Ｈ］+＝６４１ <Step 1> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of N-acetyl-N- (4- (2-aminoethoxy) benzyl) glycinate hydrochloride (Compound 15-4) Example 4 Synthesis in <Step 1> Compound 15-1 (100 mg), (Example 11) Compound 15-2 (93.89 mg) synthesized according to <Step 1> to <Step 4> and N, N-dimethyl-4-aminopyridine (4.96 mg) ) In dichloromethane (750 μL) was added dropwise a solution of N, N′-dicyclohexylcarbodiimide (43.99 mg) in dichloromethane (250 μL) at room temperature and stirred at the same temperature for 45 hours. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate (20 mL) and filtered again. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 15-3 (87 mg).
Using a fraction containing Compound 15-3 (0.087 g), the same operation as in (Example 6) <Step 3> was carried out to obtain the title compound 15-4 (0.072 g) as a yellow solid. For this product, NMR was measured at 80 ° C.
NMR data (D 2 O) (δ: ppm): 8.32 (1H, dd, J = 10.4, 9.2 Hz), 7.81 (1H, d, J = 9.6 Hz), 7 .72-7.64 (2H, m), 7.57 (2H, d, J = 8.0 Hz), 7.50 (1H, d, J = 7.2 Hz), 7.45 (1H, d, J = 7.6 Hz, A pair of doublet.), 7.20 (2H, d, J = 7.2 Hz, A pair of doublet (7.07 ppm).), 6.05 (1H, d, J = 17.2 Hz), 5.89 (1H, d, J = 17.2 Hz, A pair of doublet.), 5.35-5.27 (1H, m), 5.06-4 .73 (4H, m), 4.46-4.34 (2H, m), 3.73- 70 (1H, br m), 3.39-3.34 (2H, m), 2.68-2. 51 (5H, m, Overlapped with Me group and methylene group.), 1.67-1. 62 (3H, m, A pair of triplet.), 1.42-1.36 (3H, m, A pair of triplet.), LC-MS: M (free amine) = 640, RT = 0.64 ( Min), [M + H] + = 641
＜工程２＞（Ｓ）－４，１１－ジエチル－９－ヒドロキシ－３，１４－ジオキソ－３，４，１２，１４－テトラヒドロ－１Ｈ－ピラノ［３’，４’：６，７］インドリジノ［１，２－ｂ］キノリン－４－イル　Ｎ－アセチル－Ｎ－（４－（２－アミノエトキシ）ベンジル）グリシン基導入アルギン酸（化合物１５－５）の合成
　１重量％に調製したアルギン酸ナトリウム（株式会社キミカ製、分子量：２５１万Ｄａ～１．２万Ｄａ（ブロード）、重量平均分子量：１３４万Ｄａ）水溶液（９８８８μＬ）と（実施例１５）＜工程１＞で得られた化合物１５－４（４６．４７ｍｇ）を用い、（実施例７）＜工程５＞と同様の操作を行い、標記化合物１４－５（１１１ｍｇ）を淡黄色固体として得た。薬剤導入率は２．７９モル％であった。分子量は、２２７万Ｄａから１．５万Ｄａまでブロードな溶出ピークを示し、重量平均分子量は１３８万Ｄａであった。  <Step 2> (S) -4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7] indolidino [ 1,2-b] Quinolin-4-yl Synthesis of N-acetyl-N- (4- (2-aminoethoxy) benzyl) glycine group-introduced alginic acid (Compound 15-5) Sodium alginate (stock) prepared to 1% by weight Manufactured by Kimika Co., Ltd., molecular weight: 251,000 to 12,000 Da (broad), weight average molecular weight: 1,340,000 Da) aqueous solution (9888 μL) and (Example 15) Compound 15-4 obtained in <Step 1> 46.47 mg), and the same operation as in Example 7 <Step 5> was performed to give the title compound 14-5 (111 mg) as a pale yellow solid. The drug introduction rate was 2.79 mol%. The molecular weight showed a broad elution peak from 2.27 million Da to 15,000 Da, and the weight average molecular weight was 1.38 million Da.
（実施例１６）薬剤放出試験２
　本願のＳＮ－３８結合アルギン酸誘導体の放出試験を、実施例５と同様に実施した。具体的には、各種アルギン酸誘導体を２０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．０）または０．１Ｎ水酸化ナトリウム水溶液にて０．０１ｗ／ｖ％濃度に調製し、６時間撹拌溶解した溶液を分注し、３７℃にて放出試験を実施した。１日、３日、７日後、各反応液にメタノールを加え撹拌後、遠心上清に０．０２Ｎ塩酸を加えたものを検体とし、実施例５と同様にＬＣ－ＭＳ／ＭＳ分析による遊離ＳＮ－３８量の測定と遊離率の算出を行った。但し、ＬＣ条件は以下の通りである。
温度：４０℃
流速：０．７ｍＬ／ｍｉｎ
カラム：ＩｎｅｒｔＳｕｓｔａｉｎ　Ｃ１８：５μｍ（２．１×５０ｍｍ）
溶媒：（Ａ）０．０５％　ギ酸水溶液、（Ｂ）１００％　アセトニトリル
グラジエント：

Figure JPOXMLDOC01-appb-I000021
　各時点における遊離率（％）を表２に示す。化合物２－６、化合物７－８、化合物１０－７については、１日の時点で大半のＳＮ－３８が放出されていた。 Example 16 Drug Release Test 2
The release test of the SN-38-linked alginic acid derivative of the present application was carried out in the same manner as in Example 5. Specifically, various alginic acid derivatives were prepared to a concentration of 0.01 w / v% in 20 mM sodium phosphate buffer (pH 7.0) or 0.1 N sodium hydroxide aqueous solution, and the solution obtained by stirring and dissolving for 6 hours was dispensed. The release test was conducted at 37 ° C. One day, three days, and seven days later, methanol was added to each reaction solution, stirred, and 0.02N hydrochloric acid was added to the centrifugal supernatant as a sample, and free SN by LC-MS / MS analysis was performed as in Example 5. The amount of -38 was measured and the release rate was calculated. However, the LC conditions are as follows.
Temperature: 40 ° C
Flow rate: 0.7 mL / min
Column: Inert Sustain C18: 5 μm (2.1 × 50 mm)
Solvent: (A) 0.05% formic acid aqueous solution, (B) 100% acetonitrile gradient:

Figure JPOXMLDOC01-appb-I000021
The release rate (%) at each time point is shown in Table 2. As for Compound 2-6, Compound 7-8, and Compound 10-7, most of SN-38 was released at 1 day.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
　上記放出試験の結果から、リンカーの構造または薬剤におけるリンカーの結合位置によって徐放速度を調整でき、かつ、リンカーの種類、薬剤結合位置と薬剤導入率とを合わせて調整することで、長期持続可能な抗癌作用を期待できることが分かった。具体的には、ＳＮ－３８の３級アルコール性水酸基にリンカーを結合した誘導体は、薬剤放出速度が穏やかである傾向があり、例えば化合物１４－５は、７日間で１５％の遊離率であり、理論上は充分に３０日間以上にわたる薬剤の放出を期待できる。一方、化合物１４－５と同じリンカーを介してＳＮ－３８のフェノール性水酸基に結合した誘導体である化合物７－８は、１日間で大半が放出されており、速やかな薬剤の放出を期待できる。また、化合物９－７は、７日間で４７％の遊離率であり、１４日間程度にわたる薬剤の放出を期待できる。 Based on the results of the above release test, the sustained release rate can be adjusted according to the linker structure or the position of the linker in the drug, and long-term sustainability can be achieved by adjusting the linker type, drug binding position and drug introduction rate. It was found that anticancer activity can be expected. Specifically, a derivative in which a linker is bonded to the tertiary alcoholic hydroxyl group of SN-38 tends to have a slow drug release rate. For example, Compound 14-5 has a release rate of 15% in 7 days. Theoretically, release of the drug over 30 days or more can be expected. On the other hand, most of Compound 7-8, which is a derivative bonded to the phenolic hydroxyl group of SN-38 via the same linker as Compound 14-5, is released in one day, and prompt drug release can be expected. Compound 9-7 has a release rate of 47% in 7 days, and can be expected to release the drug over about 14 days.
（実施例１７）細胞増殖抑制試験
　実施例２で得られた化合物２－６（導入率３．０モル％（５．５ｗ／ｗ％））、実施例６で得られた化合物６－７（導入率６．５モル％（１０．９ｗ／ｗ％））、実施例９で得られた化合物９－７（導入率３．２モル％（５．８ｗ／ｗ％））の細胞増殖抑制作用を、癌細胞を用いて評価した。
　ヒト胃癌由来のＭＫＮ４５細胞（国立研究開発法人医薬基盤・健康・栄養研究所　ＪＣＲＢ細胞バンク）を、９６穴の平底プレート上にウェル当たり１，０００細胞の濃度で播種し、２４時間後、試験化合物のリン酸緩衝生理食塩水（ＰＢＳ）希釈溶液を添加した。さらに７２時間後、CellTiter-Glo（登録商標）Luminescent Cell Viability Assay（プロメガ社）を用い、細胞の生存活性を測定した。各化合物溶液を添加した細胞の生存活性は、比較対照として等量のＰＢＳを添加した細胞を１００％とし算出した。各化合物の希釈溶液を添加した細胞の生存活性を、表３に示す（表記濃度は各化合物の最終濃度（ｗ／ｖ％）を示す）。 Example 17 Cell Growth Inhibition Test Compound 2-6 obtained in Example 2 (introduction rate: 3.0 mol% (5.5 w / w%)), Compound 6-7 obtained in Example 6 ( Introduction rate 6.5 mol% (10.9 w / w%)), cell growth inhibitory action of compound 9-7 obtained in Example 9 (introduction rate 3.2 mol% (5.8 w / w%)) Were evaluated using cancer cells.
MKN45 cells derived from human gastric cancer (National Institute of Biomedical Innovation, Health and Nutrition Research, JCRB Cell Bank) were seeded on a 96-well flat bottom plate at a concentration of 1,000 cells per well, and 24 hours later, test compounds A diluted solution of phosphate buffered saline (PBS) was added. After 72 hours, cell viability was measured using CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay (Promega). The survival activity of the cells to which each compound solution was added was calculated with 100% of the cells to which an equal amount of PBS was added as a comparative control. The survival activity of the cells to which the diluted solution of each compound was added is shown in Table 3 (the notation concentration indicates the final concentration (w / v%) of each compound).
Figure JPOXMLDOC01-appb-T000023
 　上記試験の結果から、ＳＮ－３８結合アルギン酸誘導体はヒト胃癌細胞株の細胞増殖を抑制し、癌細胞に対する生存阻害能を有することが分かった。上記試験に用いた化合物における薬剤導入率は約５～１０ｗ／ｗ％であり、理論上は、化合物の０．０００００１％溶液からＳＮ－３８がすべて遊離した際のＳＮ－３８濃度は０．５～１ｎｇ／ｍＬ程度となる。ＭＫＮ４５細胞におけるＳＮ－３８のＩＣ５０値は０．５４ｎｇ／ｍＬと報告されており（トポテシン点滴静注医薬品インタビューフォーム）、実施例１６の薬剤放出試験におけるＳＮ－３８遊離率も考慮すると、本試験で示された細胞増殖抑制能は遊離したＳＮ－３８によってもたらされた結果だと考えられる。
Figure JPOXMLDOC01-appb-T000023
 From the results of the above test, it was found that the SN-38-conjugated alginic acid derivative suppresses cell growth of human gastric cancer cell lines and has the ability to inhibit survival of cancer cells. The drug introduction rate in the compound used in the above test is about 5 to 10 w / w%. Theoretically, the SN-38 concentration when SN-38 is completely released from a 0.000001% solution of the compound is 0.5. ˜1 ng / mL. The IC50 value of SN-38 in MKN45 cells has been reported to be 0.54 ng / mL (Topotecin Intravenous Drug Interview Form). Considering the SN-38 release rate in the drug release test of Example 16, this study The indicated cytostatic ability is believed to be a result of free SN-38.
（実施例１８）腹腔内投与試験
　マウス腹膜播種モデルを用いて、本願のＳＮ－３８結合アルギン酸誘導体の腹腔内徐放効果を検討した。
　８週齢の雄性Ｃ５７ＢＬ／６Ｎマウス（日本チャールス・リバー）の腹腔内にマウスメラノーマ細胞株であるＢ１６－Ｆ１０細胞（ＡＴＣＣ）１．０×１０5個を播種することによって、マウス腹膜播種モデルを作製した。腫瘍播種７日後のモデルマウスに、５％ぶどう糖液に溶解した各種アルギン酸誘導体（０．１ｗ／ｖ％）を１０ｍＬ／ｋｇで腹腔内投与した。腹腔内投与の２、８、２４、７２、１６８時間後（１時点３－４匹）に採血および腹腔内腫瘍の回収を行い、血漿中ＳＮ－３８濃度および腫瘍組織中ＳＮ－３８濃度を測定した。ＳＮ－３８量の測定は、実施例１６に記載のＬＣ－ＭＳ／ＭＳ測定と同様の方法にて行った。各化合物投与群の各時間経過後の腹膜内腫瘍組織中ＳＮ－３８濃度（ｎｇ／ｇ（組織））及び血漿中濃度（ｎｇ／ｍＬ）を、表４及び表５に示す。 (Example 18) Intraperitoneal administration test Using the mouse peritoneal seeding model, the intraperitoneal sustained release effect of the SN-38-conjugated alginic acid derivative of the present application was examined.
A mouse peritoneal dissemination model was obtained by seeding 1.0 × 10 5 B16-F10 cells (ATCC), a mouse melanoma cell line, into the abdominal cavity of 8-week-old male C57BL / 6N mice (Charles River Japan). Produced. Various alginic acid derivatives (0.1 w / v%) dissolved in 5% glucose solution were intraperitoneally administered to model mice 7 days after tumor seeding at 10 mL / kg. 2, 8, 24, 72, 168 hours after intraperitoneal administration (3-4 animals at one time point), blood was collected and intraperitoneal tumors were collected, and plasma SN-38 concentration and tumor tissue SN-38 concentration were measured did. The amount of SN-38 was measured by the same method as the LC-MS / MS measurement described in Example 16. Tables 4 and 5 show the SN-38 concentration (ng / g (tissue)) and plasma concentration (ng / mL) in the intraperitoneal tumor tissue after each time course of each compound administration group.
表４：腫瘍組織中濃度（ｎｇ／ｇ）
Figure JPOXMLDOC01-appb-T000024
 Table 4: Tumor tissue concentration (ng / g)
Figure JPOXMLDOC01-appb-T000024
表５：血漿中濃度（ｎｇ／ｍＬ）
Figure JPOXMLDOC01-appb-T000025
 Table 5: Plasma concentration (ng / mL)
Figure JPOXMLDOC01-appb-T000025
　上記試験の結果から、ＳＮ－３８結合アルギン酸誘導体を一度投与された動物において、腹腔内腫瘍組織中で７２時間以上にわたりＳＮ－３８が検出されることが分かった。すなわち、本発明のＳＮ－３８結合アルギン酸誘導体は、ＳＮ－３８を腹腔内腫瘍組織に長時間留まらせることが可能であり、腹腔内環境において長期持続可能な抗癌作用を期待できることが分かった。また、すべての時点において、血漿中濃度と比較して腫瘍組織中ＳＮ－３８濃度は高値(４．７～５２倍)であったことから、全身への薬剤曝露が低く、副作用を低減させる条件において、抗腹膜播種効果が期待できる。 From the results of the above test, it was found that SN-38 was detected in the intraperitoneal tumor tissue for 72 hours or longer in the animals once administered with the SN-38-conjugated alginic acid derivative. That is, it was found that the SN-38-binding alginic acid derivative of the present invention can retain SN-38 in the intraperitoneal tumor tissue for a long time and can expect a long-term sustainable anticancer effect in the intraperitoneal environment. In addition, the SN-38 concentration in the tumor tissue was high (4.7 to 52 times) compared with the plasma concentration at all time points. Therefore, the conditions for reducing drug exposure to the whole body and reducing side effects The antiperitoneal seeding effect can be expected.

Claims (11)

  1. 　アルギン酸又はその塩とカンプトテシン誘導体とがリンカーを介して共有結合されてなる構造を有する、アルギン酸誘導体。 An alginic acid derivative having a structure in which alginic acid or a salt thereof and a camptothecin derivative are covalently bonded via a linker.
  2. 　下記式（１）で表される構造を有する、請求項１に記載のアルギン酸誘導体：
    （Ａ）－Ｌ－（Ｄ）　　　（１）
    （式中、
    　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
    　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
    　Ｌは、（Ａ）とアミド結合で結合しうる官能基を有し、かつ（Ｄ）とエステル結合で結合しうる官能基を有するリンカーである）。     The alginic acid derivative according to claim 1, having a structure represented by the following formula (1):
    (A) -L- (D) (1)
    (Where
    (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
    (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
    L is a linker having a functional group that can be bonded to (A) by an amide bond and a functional group that can be bonded to (D) by an ester bond.
  3. 　下記式（２）で表される構造を有する、請求項１に記載のアルギン酸誘導体：
    （Ａ）－ＮＨ－（ＣＨ2）n1－［Ｘ1］n2－（ＣＲ1Ｒ2）n3－［Ｙ］n4－（ＣＨ2）n5－（ＣＲ3Ｒ4）n6－［Ｘ2］n7－（ＣＨ2）n8－（ＣＲ5Ｒ6）n9－Ｃ（＝Ｏ）－（Ｄ）　　　（２）
    （式中、
    　（Ａ）はアルギン酸又はその塩由来の残基であって、アルギン酸を構成するＬ－グルロン酸及びＤ－マンヌロン酸のいずれか一方の単糖のＣ（＝Ｏ）－基を有する１残基を示し、
    　（Ｄ）はカンプトテシン誘導体の水酸基から水素原子を除いた基を示し、
    　Ｘ1及びＸ2は、置換基を有しても良いヘテロ原子を示し、
    　Ｒ1、Ｒ2、Ｒ3、Ｒ4、Ｒ5及びＲ6はそれぞれ独立して、水素、ハロゲン原子、Ｃ1-10アルキル基、Ｃ1-10アルコキシ基又はＣ1-10アルコキシカルボニル基を示すか、又は、Ｒ1及びＲ2若しくはＲ3及びＲ4が一緒になって＝Ｏを示し、
    　Ｙは、シクロアルカン環、芳香族環又は複素環（前記シクロアルカン環、芳香族環又は複素環は、ハロゲン原子又はＣ1-10アルキル基で置換されていてもよい）を示し、
    　ｎ１は０～１０のいずれかの整数を示し、ｎ２、ｎ４及びｎ７は独立して０または１を示し、ｎ３、ｎ５、ｎ６、ｎ８及びｎ９は独立して０～３のいずれかの整数を示すが、ｎ１～ｎ９の全てが０になることはない）。     The alginic acid derivative according to claim 1, having a structure represented by the following formula (2):
    (A) —NH— (CH 2 ) n1 — [X 1 ] n2 — (CR 1 R 2 ) n3 — [Y] n4 — (CH 2 ) n5 — (CR 3 R 4 ) n6 — [X 2 ] n7 - (CH 2) n8 - ( CR 5 R 6) n9 -C (= O) - (D) (2)
    (Where
    (A) is a residue derived from alginic acid or a salt thereof, and one residue having a C (═O) — group of a monosaccharide of either L-guluronic acid or D-mannuronic acid constituting alginic acid. Show
    (D) represents a group obtained by removing a hydrogen atom from a hydroxyl group of a camptothecin derivative;
    X 1 and X 2 represent a hetero atom which may have a substituent,
    R 1 , R 2 , R 3 , R 4 , R 5 and R 6 each independently represents a hydrogen atom, a halogen atom, a C 1-10 alkyl group, a C 1-10 alkoxy group or a C 1-10 alkoxycarbonyl group. Or R 1 and R 2 or R 3 and R 4 together represent ═O,
    Y represents a cycloalkane ring, an aromatic ring or a heterocyclic ring (the cycloalkane ring, aromatic ring or heterocyclic ring may be substituted with a halogen atom or a C 1-10 alkyl group);
    n1 represents any integer of 0 to 10, n2, n4 and n7 each independently represents 0 or 1, n3, n5, n6, n8 and n9 each independently represents any integer of 0 to 3. As shown, all of n1 to n9 are not 0).
  4. 　カンプトテシン誘導体がカンプトテシン；７－エチルカンプトテシン；１０－ヒドロキシカンプトテシン；ＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）；イリノテカン（ＣＰＴ－１１）；９－（ジメチルアミノ）メチルカンプトテシン；トポテカン（ノギテカン）；エキサテカン；Ｔ－２５１３（１０－（３－アミノプロピロキシ）－７－エチルカンプトテシン）；１０，１１－メチレンジオキシカンプトテシン；７－エチル－１０，１１－メチレンジオキシカンプトテシン；９－アミノ－１０，１１－メチレンジオキシカンプトテシン；９－クロロ－１０，１１－メチレンジオキシカンプトテシン；７－（４－メチル－１－ピペラジニル）メチル－１０，１１－メチレンジオキシカンプトテシン；１０，１１－エチレンジオキシカンプトテシン；ルルトテカン（７－（４－メチル－１－ピペラジニル）メチル－１０，１１－エチレンジオキシカンプトテシン）；ギマテカン（７－（ｔｅｒｔ－ブトキシイミノメチル）カンプトテシン）；９－アミノカンプトテシン；ルビテカン（９－ニトロカンプトテシン）；ベロテカン（７－（２－（Ｎ－イソプロピルアミノ）エチル）－カンプトテシン）；コシテカン（７－（２－（トリメチルシリル）エチル）－カンプトテシン）及びシラテカン（７－（ｔｅｒｔ－ブチルジメチルシリル）－１０－ヒドロキシカンプトテシン）からなる群から選ばれる、請求項１～３のいずれか１項に記載のアルギン酸誘導体。 Camptothecin derivatives are camptothecin; 7-ethylcamptothecin; 10-hydroxycamptothecin; SN-38 (7-ethyl-10-hydroxycamptothecin); irinotecan (CPT-11); 9- (dimethylamino) methylcamptothecin; topotecan (nogitecan); Exatecan; T-2513 (10- (3-aminopropyloxy) -7-ethylcamptothecin); 10,11-methylenedioxycamptothecin; 7-ethyl-10,11-methylenedioxycamptothecin; 9-amino-10, 11-methylenedioxycamptothecin; 9-chloro-10,11-methylenedioxycamptothecin; 7- (4-methyl-1-piperazinyl) methyl-10,11-methylenedioxycamptothecin; 10,11-ethylene Oxycamptothecin; luruthecan (7- (4-methyl-1-piperazinyl) methyl-10,11-ethylenedioxycamptothecin); gimatecan (7- (tert-butoxyiminomethyl) camptothecin); 9-aminocamptothecin; rubitecan (9 -Nitrocamptothecin); belothecan (7- (2- (N-isopropylamino) ethyl) -camptothecin); cositecan (7- (2- (trimethylsilyl) ethyl) -camptothecin) and cilatecan (7- (tert-butyldimethylsilyl) The alginic acid derivative according to any one of claims 1 to 3, which is selected from the group consisting of) -10-hydroxycamptothecin).
  5. 　カンプトテシン誘導体がＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）、イリノテカン（ＣＰＴ－１１）又はトポテカン（ノギテカン）である、請求項４に記載のアルギン酸誘導体。 The alginic acid derivative according to claim 4, wherein the camptothecin derivative is SN-38 (7-ethyl-10-hydroxycamptothecin), irinotecan (CPT-11) or topotecan (Nogitecan).
  6. 　カンプトテシン誘導体がＳＮ－３８（７－エチル－１０－ヒドロキシカンプトテシン）である、請求項５に記載のアルギン酸誘導体。 The alginic acid derivative according to claim 5, wherein the camptothecin derivative is SN-38 (7-ethyl-10-hydroxycamptothecin).
  7. 　カンプトテシン誘導体の２０位の３級アルコール性水酸基がリンカーと結合されてなる、又は、カンプトテシン誘導体がフェノール性水酸基を有し、前記フェノール性水酸基がリンカーと結合されてなる、請求項１～６のいずれか１項に記載のアルギン酸誘導体。 The tertiary alcoholic hydroxyl group at position 20 of the camptothecin derivative is bonded to a linker, or the camptothecin derivative has a phenolic hydroxyl group, and the phenolic hydroxyl group is bonded to a linker. The alginic acid derivative according to claim 1.
  8. 　請求項１～７のいずれか１項に記載のアルギン酸誘導体を架橋してなる、アルギン酸誘導体ゲル。 An alginic acid derivative gel obtained by crosslinking the alginic acid derivative according to any one of claims 1 to 7.
  9. 　請求項１～７のいずれか１項に記載のアルギン酸誘導体、又は、請求項８に記載のアルギン酸誘導体ゲルを含む、徐放性医薬組成物。 A sustained-release pharmaceutical composition comprising the alginic acid derivative according to any one of claims 1 to 7 or the alginic acid derivative gel according to claim 8.
  10. 　抗癌剤としての、請求項９に記載の徐放性医薬組成物。 The sustained-release pharmaceutical composition according to claim 9 as an anticancer agent.
  11. 　カンプトテシン誘導体を徐放するための、請求項１～７のいずれか１項に記載のアルギン酸誘導体、又は、請求項８に記載のアルギン酸誘導体ゲルの使用。 Use of the alginic acid derivative according to any one of claims 1 to 7 or the alginic acid derivative gel according to claim 8 for the sustained release of a camptothecin derivative.
PCT/JP2019/013286 2018-03-28 2019-03-27 Anti-cancer agent-bonded alginic acid derivative WO2019189419A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-062049 2018-03-28
JP2018062049A JP2021095424A (en) 2018-03-28 2018-03-28 Anti-cancer agent-bonded alginic acid derivative

Publications (1)

Publication Number Publication Date
WO2019189419A1 true WO2019189419A1 (en) 2019-10-03

Family

ID=68062148

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/013286 WO2019189419A1 (en) 2018-03-28 2019-03-27 Anti-cancer agent-bonded alginic acid derivative

Country Status (2)

Country Link
JP (1) JP2021095424A (en)
WO (1) WO2019189419A1 (en)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0824325A (en) * 1994-05-13 1996-01-30 Kuraray Co Ltd Polymer gel for medical treatment
JPH1072467A (en) * 1995-08-02 1998-03-17 Tanabe Seiyaku Co Ltd Camptothecin derivative
JPH1095802A (en) * 1995-12-28 1998-04-14 Tanabe Seiyaku Co Ltd Camptothecine derivative
JPH1171280A (en) * 1997-06-26 1999-03-16 Tanabe Seiyaku Co Ltd Medicine composition
JP2001504453A (en) * 1996-09-30 2001-04-03 バイエル・アクチエンゲゼルシヤフト Glycoconjugates from modified camptothecin derivatives (20-O-linked)
JP2002539132A (en) * 1999-03-06 2002-11-19 バイエル アクチェンゲゼルシャフト Method for producing 20- (S) -camptothecin sugar-conjugate
JP2003527443A (en) * 2000-03-17 2003-09-16 セル・セラピューティックス・インコーポレーテッド Polyglutamic acid-camptothecin conjugate and method for preparing the same
CN101745119A (en) * 2010-01-25 2010-06-23 中国药科大学 Polysaccharide conjugate of carboxylic acid drug, preparation method thereof and application thereof
CN103705940A (en) * 2013-12-30 2014-04-09 中国药科大学 Preparation and anti-tumor application of natural active drug-polysaccharide targeted compound
JP2014532733A (en) * 2011-11-11 2014-12-08 チア タイ ティアンチン ファーマシューティカル グループ カンパニー リミテッド Trolox derivative-modified lipophilic anticancer compound, and preparation, production method and use thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0824325A (en) * 1994-05-13 1996-01-30 Kuraray Co Ltd Polymer gel for medical treatment
JPH1072467A (en) * 1995-08-02 1998-03-17 Tanabe Seiyaku Co Ltd Camptothecin derivative
JPH1095802A (en) * 1995-12-28 1998-04-14 Tanabe Seiyaku Co Ltd Camptothecine derivative
JP2001504453A (en) * 1996-09-30 2001-04-03 バイエル・アクチエンゲゼルシヤフト Glycoconjugates from modified camptothecin derivatives (20-O-linked)
JPH1171280A (en) * 1997-06-26 1999-03-16 Tanabe Seiyaku Co Ltd Medicine composition
JP2002539132A (en) * 1999-03-06 2002-11-19 バイエル アクチェンゲゼルシャフト Method for producing 20- (S) -camptothecin sugar-conjugate
JP2003527443A (en) * 2000-03-17 2003-09-16 セル・セラピューティックス・インコーポレーテッド Polyglutamic acid-camptothecin conjugate and method for preparing the same
CN101745119A (en) * 2010-01-25 2010-06-23 中国药科大学 Polysaccharide conjugate of carboxylic acid drug, preparation method thereof and application thereof
JP2014532733A (en) * 2011-11-11 2014-12-08 チア タイ ティアンチン ファーマシューティカル グループ カンパニー リミテッド Trolox derivative-modified lipophilic anticancer compound, and preparation, production method and use thereof
CN103705940A (en) * 2013-12-30 2014-04-09 中国药科大学 Preparation and anti-tumor application of natural active drug-polysaccharide targeted compound

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"The Practice of Medicinal Chemistry", 25 September 1999, pages: 321 - 338 *

Also Published As

Publication number Publication date
JP2021095424A (en) 2021-06-24

Similar Documents

Publication Publication Date Title
EP2435053B1 (en) Polyal drug conjugates comprising variable rate-releasing linkers
ES2320439T3 (en) UNITED TAXANS COVALENTLY TO HIALURONIC ACID OR DERIVATIVES OF HIALURONIC ACID.
EP2206502B1 (en) Polymer conjugate of steroid
KR101203475B1 (en) Novel Block Copolymer, Micelle Preparation, and Anticancer Agent Containing the Same as Active Ingredient
KR101057102B1 (en) High molecular weight derivatives of camptothecin
CA2831392A1 (en) Conjugated lipomers and uses thereof
TW200810757A (en) Multi-arm polymeric conjugates of 7-ethyl-10-hydroxycamptothecin for treatment of breast, colorectal, pancreatic, ovarian and lung cancers
EP2773379A2 (en) Subcutaneous delivery of polymer conjugates of therapeutic agents
RU2569847C2 (en) Pharmaceutical composition, containing block-copolymer, including boronic acid compound
US20230190943A1 (en) Alginic acid derivative bonded to nonsteroidal anti-inflammatory compound
EP2279006B1 (en) Therapeutic use of pharmaceutical preparations containing antitumoral drugs bound to hyaluronic acid in the treatment of neoplasias
UA75450C2 (en) Use of polysaccharide conjugates for inhibiting metastasis or preventing recurrence of malignant tumor
AU2016318344B2 (en) Polymer conjugate comprising a bioactive agent
US20190142954A1 (en) Polymer-bonded ca4 pharmaceutical compound and preparation method therefor
US11912709B2 (en) Temozolomide compounds, polymers prepared therefrom, and method of treating a disease
WO2019189419A1 (en) Anti-cancer agent-bonded alginic acid derivative
CN101495485B (en) Camptothecin derivatives with antitumor activity
WO2020214436A1 (en) Platinum-containing compounds, and related compositions and uses thereof
CA3015459A1 (en) Pharmaceutical preparation containing camptothecin-based polymeric derivative
US20160362402A1 (en) Novel compounds having anti-allodynic and antihyperalgesic activity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19777541

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19777541

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP