WO2018192550A1 - 多臂靶向抗癌偶联物 - Google Patents

多臂靶向抗癌偶联物 Download PDF

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WO2018192550A1
WO2018192550A1 PCT/CN2018/083746 CN2018083746W WO2018192550A1 WO 2018192550 A1 WO2018192550 A1 WO 2018192550A1 CN 2018083746 W CN2018083746 W CN 2018083746W WO 2018192550 A1 WO2018192550 A1 WO 2018192550A1
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Prior art keywords
compound
cancer
pharmaceutically acceptable
drug conjugate
acceptable salt
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PCT/CN2018/083746
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English (en)
French (fr)
Inventor
袁建栋
黄仰青
宋云松
丁海峰
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博瑞生物医药（苏州）股份有限公司
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Priority claimed from CN201710263113.4A external-priority patent/CN108727582B/zh
Priority claimed from CN201710263126.1A external-priority patent/CN108727584B/zh
Priority claimed from CN201710263114.9A external-priority patent/CN108727583B/zh
Application filed by 博瑞生物医药（苏州）股份有限公司 filed Critical 博瑞生物医药（苏州）股份有限公司
Priority to CA3058029A priority Critical patent/CA3058029A1/en
Priority to KR1020197032879A priority patent/KR102279429B1/ko
Priority to EP18787379.9A priority patent/EP3613792B1/en
Priority to JP2020504758A priority patent/JP6947909B2/ja
Priority to AU2018255458A priority patent/AU2018255458B2/en
Priority to ES18787379T priority patent/ES2859473T3/es
Priority to US16/498,765 priority patent/US11191843B2/en
Publication of WO2018192550A1 publication Critical patent/WO2018192550A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • 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
    • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds

Definitions

  • the present invention relates to multi-arm polymer modified targeted anti-cancer conjugates, and more particularly to the attachment of targeting molecules to anti-cancer drugs by conjugates via a multi-arm polymer.
  • a commonly employed drug delivery method aims to address or at least ameliorate one or more of the following problems, including drug encapsulation, covalent attachment, such as in a liposome, polymer matrix, or monomolecular micelle To a water soluble polymer such as polyethylene glycol, the use of a gene targeting agent, the structure of a salt, and the like.
  • WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a clinical phase III drug nktr 102, which is mainly used for metastatic breast cancer, developed by Nektar Therapeutics.
  • the drug is a water-soluble multi-branched polymer prodrug to increase the drug loading, and the structure is as follows:
  • the compound is linked to irinotecan using a multi-arm PEG to increase water solubility, increase drug loading, and reduce side effects without changing the anticancer effect.
  • the drug still has disadvantages, for example, poor targeting, can not act on specific cancer cells, while killing cancer cells, it also affects the performance of normal cells, and the incidence of adverse reactions is still relatively high.
  • the present invention discloses a novel targeted multi-branched drug conjugate having three or more branches, which can be expressed as:
  • R is an organic center
  • POLY is a polymer
  • L is a multivalent linker
  • T is a targeting molecule
  • D is an active agent
  • q is any integer between 3-8, where L is:
  • the symbol "*” represents the point of attachment of the multivalent linker L to the target molecule T
  • "#” represents the point of attachment of the multivalent linker L to the active agent D
  • "%” represents the point of attachment of the multivalent linker L to the POLY, wherein l is any integer between 2 and 20, and m and n are each an integer between 0 and 10;
  • D is a camptothecin drug as shown in formula (II):
  • R 1 -R 5 are independently selected from the group consisting of hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxy, cyanide Base, nitro, azido, amido, hydrazine, amine, substituted amine, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, alkyl sulfonate Acyloxy; R 6 is H or OR 8 , R 8 is alkyl, alkenyl, cycloalkyl, haloalkyl or hydroxyalkyl; R 7 is hydroxy, amino, or thiol.
  • POLY is a polymer
  • L is a multivalent linker
  • T is a targeting molecule
  • D is an active agent
  • the four together form a "branched" of the multi-branched drug conjugate.
  • Each of the branches and other branches of the multi-branched drug conjugate are independent of each other.
  • Each branch is sent from the organic center "R". However, in general, each branch of the conjugate is the same.
  • R is an organic core group of 1 to 100 atoms. Preferably, R contains from 3 to 50 atoms, more preferably, R contains from about 3 to about 30 atoms. R may be a core of all carbon atoms, or may optionally contain one or more heteroatoms, for example, O, S, N, P, etc., depending on the particular central molecule used. R may be linear, branched or cyclic, emitting at least 3 separate polymer branches. In the formula (I), "q” corresponds to the number of polymer branches emanating from "R".
  • the organic center "R” is derived from a molecule that provides a number of locations where the polymer is attached, approximately equal to the number of polymer branches. More preferably, the main central formula of the multi-chain polymer structure bears at least three or more hydroxyl, sulfur or amino polyhydroxy compounds, polysulfide compounds or polyamine compounds suitable as polymer branches. Residues.
  • a "polyhydroxy compound” is a molecule composed of a plurality (more than two) of available hydroxyl groups.
  • a “polysulfide compound” is a molecule composed of multiple (greater than 2) available sulfur groups.
  • a "polyamine compound” is a molecule composed of a plurality (more than two) of available amine groups.
  • the precursor of the polyhydroxy compound, polyamine compound or polysulfide compound typically comprises from 3 to 25 hydroxyl, thio or amino groups, preferably 3-
  • the 10 hydroxyl, thio or amino groups preferably comprise from 3 to about 8 (e.g., 3, 4, 5, 6, 7, or 8) a hydroxyl, thio or amino group suitable for covalent bonding to POLY.
  • the parent of the polyhydroxy compound or polyamine compound center typically has a structural formula of R-(OH)p or R-(NH 2 )p prior to its action with the polymer.
  • the p value and the q value correspond, since each functional group in the parent organic molecule typically has -OH and -NH 2 , if the position is susceptible or susceptible to reaction They are covalently bonded to the POLY-linked POLY of the polymer.
  • the hydroxyl group of the polyhydroxy compound of the R precursor after linking with the POLY, the hydroxyl group of the polyhydroxy compound of the R precursor has been converted into a polymer branch, and the R described is the residue after the attachment.
  • the organic center molecule is derived from pentaerythritol
  • the parent of the polyol possesses the structural formula C(CH 2 OH) 4 and the organic center group R is expressed as:
  • Preferred illustrative polyols as polymer centers include aliphatic polyhydroxy compounds containing from 1 to 10 carbon atoms and from 1 to 10 hydroxyl groups, for example, ethylene glycol, alkanediol, hydrocarbyl glycol , alkylene hydrocarbyl glycol, hydrocarbyl cycloalkyl glycol, 1,5-decalindiol, 4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylene glycol, dihydroxyalkane, Trihydroxyalkane, tetrahydroxyalkane, and the like.
  • Cycloaliphatic polyhydroxy compounds include linear or closed-loop saccharides and sugar alcohols such as mannitol, sorbitol, hexitol, xylitol, leucovorol, threitol, arabitol, erythritol, Hexahexanol, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagatose, Pyranoside, sucrose, lactose, maltose, and the like.
  • sugar alcohols such as mannitol, sorbitol, hexitol, xylitol, leucovorol, threitol, arabitol, erythritol, Hexahexanol, ribose, arabinose,
  • Aromatic polyhydroxy compounds such as phenylphosphoric diphenol, hydrocarbyl phenylphosphonate, pyrogallol, fluoroglycine phenol, 1,2,4-benzenetriol, resorcinol, hydrocarbyl resorcinol, Dihydrocarbyl resorcinol, orcinol monohydrate, olive phenol, hydroquinone, hydrocarbyl hydroquinone, phenyl hydroquinone, and the like.
  • Other polyhydroxyl centers may include crown ethers, cyclodextrins, dextrin or other carbohydrates.
  • q is the number of polymer branches corresponding to the "R", and the specific number may be from 3 to 20. Typically, the specific numbers for "q" are 3, 4, 5, 6, 7, 8. Specifically, three, four, five, six, seven, and eight polymer branches are emitted centering on "R".
  • R has three polymer branches, and “R” is preferably:
  • R has four polymer branches, and “R” is preferably:
  • R has six polymer branches, and “R” is preferably:
  • R has eight polymer branches, and “R” is preferably:
  • POLY is a polymer, and the POLY in each polymer branch is independently selected.
  • each polymer is the same polymer. More suitably, the polymer branches in each of the structural formula (I) are the same.
  • Preferred polymers are water soluble, and any water soluble polymer can be used to form the conjugates of the present invention, and the polymers referred to herein can be in any geometric form or form.
  • Representative polymers include, but are not limited to, polyethylene glycol, polypropylene glycol, poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylate), poly(hydroxyalkylmethacrylate), poly(sugar) Class), poly( ⁇ -hydroxy acid), poly(acrylic acid), poly(ethylene acetate), polyphosphazine, polyoxazoline, poly(N-acryloylmorpholine), and the like.
  • POLY is polyethylene glycol (PEG), which may be in any geometric form or form, including linear, branched, forked chains, etc., "polyethylene glycol” as used herein, meaning It is covered by any water soluble poly(ethylene oxide).
  • PEG polyethylene glycol
  • the PEG used in the present invention will comprise one of two structures: “(CH 2 CH 2 O) k -” or “(CH 2 CH 2 O) k -CH 2 CH 2 -", depending on Whether one or more of the terminal oxygen has been replaced, for example, during a synthetic transformation.
  • the variable k ranges from 5 to about 500, and the structure of these terminal groups as well as the overall PEG can vary.
  • the polyethylene glycol structure generally also contains a partial terminal moiety residue, similar to the terminal group of POLY, which may be H, NH 2 , OH, CO 2 H, C 1-6 alkyl (eg, methyl, Ethyl, propyl), C 1-6 alkoxy (eg, methoxy, ethoxy), acyl or aryl ends.
  • a partial terminal moiety residue similar to the terminal group of POLY, which may be H, NH 2 , OH, CO 2 H, C 1-6 alkyl (eg, methyl, Ethyl, propyl), C 1-6 alkoxy (eg, methoxy, ethoxy), acyl or aryl ends.
  • POLY a linear polyethylene glycol, and the typical structure is:
  • the oxygen atom labeled "&" is an atom attached to the organic center "R".
  • k ranges from about 5 to 500, preferably from 50 to 200, and r is any integer between 1-10. More preferably, the "POLY" of the present invention is:
  • the POLY of the present invention can also be:
  • the active agent "D" referred to in the present invention is a camptothecin-based anticancer agent, and the camptothecin-based drug is a topoisomerase I inhibitor for clinical use, and has high water solubility and poor water solubility to a normal body.
  • the shortcomings of tissue toxicity and side effects greatly limit the clinical application of camptothecin anticancer agents.
  • R 7 in the D structure is a group covalently bonded to the polyvalent linker L, such as a hydroxyl group, an amino group, or a mercaptan, preferably a hydroxyl group.
  • the active agent D is attached to the multivalent linker L, there should be no significant loss of biological activity.
  • the active agent of the present invention is preferably irinotecan, SN-38, 10-hydroxycamptothecin or lubitotecan. among them:
  • T is a targeting molecule with or without medicinal action, and the role of the targeting molecule is to increase the targeting property, so that the concentration of the conjugate in the target tissue is higher, and the physiological activity or drug is improved. Use the role.
  • “T” can be a single-function targeting molecule or a multifunctional targeting molecule. In some alternatives, “T” can also be a targeting moiety composed of two or more targeting molecules. .
  • the "T” can be an RGD peptide containing an "arginine-glycine-aspartate" sequence, which is a recognition site for the interaction of integrin with its ligand protein. Preferred RGD peptides include iRGD and cRGD and the like. T can also be Lyp-1, Lyp-1, RPARPAR, Angiopep2 or GE11.
  • iRGD The structure of iRGD is as follows:
  • cRGD is a series of compounds, typical compounds include:
  • the preferred cRGD is:
  • tLyp-1 The structure of tLyp-1 is as follows:
  • Lyp-1 The structure of Lyp-1 is as follows:
  • the polypeptide sequence of RPARPAR is arginine-valine-alanine-arginine-valine-alanine-arginine, and its structure is as follows:
  • the polypeptide sequence of Angiopep2 is: TFFYGGSRGKRNNFKTEEY, and its structure is as follows:
  • polypeptide sequence of GE11 is: YHWYGYTPQNVI, and its structure is:
  • the active agent "D" as used in the present invention refers to a part of a non-modified parent active agent or a covalent chain produced by covalent attachment of a drug to a polyvalent linker of the present invention (or its activated or chemically modified). The form of the residue of the previously unmodified parent active agent. When the linker between the active agent moiety and the polyvalent linker is hydrolyzed or digested, the active agent itself is released.
  • the term "residue” is understood to mean a part of a compound which means the remainder after undergoing a substitution reaction with another compound.
  • the active agent D and the multivalent linker L are cleaved, and the active agent D is released without modification, that is, a form in which no covalent bond is formed, and the parent body Separate and exert physiological activity.
  • POLY is a linear polyethylene glycol linkage arm, i.e., the conjugate of the invention comprises the following types of compounds:
  • k ranges from about 5 to 500, preferably from 50 to 200, and r is any integer between 1-10.
  • k is preferably 113. It should be understood by those skilled in the art that in the field of polymers, k represents the degree of polymerization of the polymer, and depends on the molecular weight of the polymer, and is not an absolute value. When k is 113, it means average. The value is 113.
  • the targeting moiety "T" of the conjugate of the invention is selected from one of iRGD, cRGD, tLyp-1, Lyp-1, RPARPAR, Angiopep2 or GE11, and the active agent "D” is selected from One of irinotecan, SN-38, 10-hydroxycamptothecin, and rubiconol.
  • L is selected from the group consisting of:
  • the compounds of the invention are as follows:
  • compound a can also be written in the following form:
  • Compound A is the hydrochloride salt of Compound a:
  • Compound B is the hydrochloride salt of compound b:
  • Compound C is the hydrochloride salt of compound c:
  • Compound D is the hydrochloride salt of compound d:
  • Compound E is the hydrochloride salt of compound e
  • Compound F is the hydrochloride salt of compound f:
  • compound f can also be written in the following form:
  • the branch of the conjugate of the present invention is separately salted with HCl, for example, compound A, compound B, compound C, and compound D, each molecule having 2 molecules of HCl, The entire molecule will carry 8 HCl.
  • Compound E with 6 molecules of HCl per branch, will carry 24 molecules of HCl.
  • Compound F with 3 molecules of HCl per branch, will carry 12 HCl for the entire molecule.
  • 1D is SN-38
  • T is a conjugate of iRGD, cRGD, tLyp-1, Lyp-1, RPARPAR, Angiopep2 or GE11
  • 2D is 10-hydroxycamptothecin
  • T is iRGD, cRGD, tLyp-1, respectively.
  • 3D is Rubiconcan
  • T is a conjugate of iRGD, cRGD, tLyp-1, Lyp-1, RPARPAR, Angiopep2 or GE11, respectively.
  • the conjugate of the present invention is a typical prodrug, and the active agent D is released by hydrolysis or enzymatic action, and is separated from the mother to exert physiological activity.
  • the conjugates of the present invention exhibit high loading capacity so that the total dose can be lowered to treat a particular disease, such as cancer. That is, the conjugate active agent carrier of the present invention is capable of effectively covalently bonding to a plurality of active agent molecules, allowing a greater amount of therapeutic dosage form (i.e., active agent portion) to be administered per certain amount of conjugate.
  • the modification of the conjugate of the present invention by the water-soluble polymer is essentially that the conjugate is also hydrophilic, and in particular, when the active agent is a water-insoluble drug, the bioavailability of the conjugate is improved.
  • the conjugates of the invention are capable of exhibiting a stronger effect than tissues that are not coupled, and are more enriched in the human or other animal tissues.
  • the conjugate drug precursors of the present invention have a number of unique properties, especially where the active agent is an anti-cancer compound.
  • This prodrug inhibits tumor growth with higher efficiency.
  • the small molecule we use is a small molecule known to have anticancer properties. However, by combining with a multi-branched polymer as described above, its efficacy and pharmacokinetics are greatly improved compared to the small molecule (for example, the anticancer compound itself).
  • Suitable solid tumor types include colon cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, glioma, and malignant sarcoma, carcinoma, and lymphoma of the breast, ovary, colon, kidney, bile duct, lung, and brain.
  • the present invention is a multi-arm polymer modified targeted anti-cancer conjugate, wherein the water-soluble polymer modification enhances the water solubility of the conjugate, thereby increasing the drug loading; the targeting molecule increases the target.
  • the directionality makes the concentration of the conjugate higher in the target tissue; L is any ligated linker, which firstly links the targeting molecule with the anticancer drug, and then "targeting molecule, anticancer drug and polymerization" The arms are joined such that the entire conjugate forms an organic whole.
  • the conjugate of the present invention may be a salt formed by a conventional means in the field of medicinal chemistry, and may also be a trifluoroacetate, a sulfate, a phosphate, an acetate or the like.
  • the invention provides a method of making the conjugate.
  • POLY and the organic center R actually constitute a multi-arm polymer.
  • the multi-arm polymer is a multi-arm polyethylene glycol, which is commercially available.
  • various types of four-arm, three-arm, and eight-arm polyethylene glycol derivatives are commercially available from Beijing Keykai Technology Co., Ltd. Commercially available these multi-arm PEGs can participate directly in the reaction.
  • the four-arm polyethylene glycol preferably used is as follows:
  • the preferred four-arm polyethylene glycol is referred to as 4ARM-PEG20K-SCM and has a molecular weight of about 20 kDa.
  • the molecular weight of the three-arm and eight-arm polyethylene glycol used is also preferably about 20 kDa.
  • the coupling reagent was HOBT/DIC
  • DMF was used as the reaction solvent
  • the reaction was monitored by ninhydrin detection, and the following protected amino acids were sequentially attached to the resin:
  • the coupling reagent was HOBT/DIC, DMF was used as the reaction solvent, and the reaction was monitored by the ninhydrin detection method, and the following protected amino acids were sequentially attached to the resin: Fmoc-Cys(Acm)-OH, Fmoc-Asp (Alloc)OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc -Cys(Acm)-OH, DMF after washing, adding trifluoroacetic acid hydrazine (2.0 eq), stirring for 18 hours, washing with DMF, removing Fmoc, condensing Fmoc-Cys(Trt)-OH, washing with DMF, removing Fm
  • Angiopep-2 The sequence of Angiopep-2 is TFFYGGSRGKRNNFKTEEY
  • the coupling reagent was HOBT/DIC, DMF was used as the reaction solvent, and the reaction was monitored by ninhydrin detection method, and the following protected amino acids were sequentially attached to the resin: Fmoc-Tyr(tBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc -Gly-OH, Fmoc-Ser(tBu)-
  • the sequence of GE11 is YHWYGYTPQNVI
  • the molecular weight of the MALDI-TOF test was 25,480.27.
  • the molecular weight of the MALDI-TOF test was 29,013.19.
  • the molecular weight of the MALDI-TOF test was 28,076.21.
  • the molecular weight of the MALDI-TOF test was 27,963.54.
  • the molecular weight of the MALDI-TOF test was 33,812.65.
  • the molecular weight of the MALDI-TOF test was 30,907.82.
  • Irinotecan (raw drug) is purchased.
  • nktr-102 refers to the method disclosed in CN102711837A, as follows:
  • the saline was purchased from Shanghai Huayuan Changfu Pharmaceutical (Group) Co., Ltd.
  • a 1 ml sterile syringe was purchased from Shanghai Kantele Enterprise Development Group Co., Ltd. (Shanghai, China).
  • MDA-MB-231 was cultured in DMEM medium (GIBCO, USA) containing 10% fetal calf serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .
  • Matrigel (BD Matrigel) Matrigel was purchased from BD Corporation of the United States.
  • Biosafety cabinet (model: AC2-6E1), purchased from ESCO; CO 2 water-tight cell incubator (model: 3111), purchased from Thermo Scientific Forma; inverted microscope (model: CKX41SF), purchased from Olympus; electric aspirator ( Model YX930D), purchased from Shanghai Medical Device Industry (Group) Co., Ltd.; balance (METTLER TOLEDO AB135-S), purchased from METTLER TOLEDO; low-speed centrifuge (model LD5-2A), purchased from Beijing Leiboer Centrifuge Co., Ltd.; electronic digital caliper (model: SF2000), purchased from Guilin Guanglu Digital Measurement and Control Co., Ltd.
  • TV initial is the tumor volume measured at the time of group administration
  • TV t is the tumor volume at each measurement during administration.
  • RTV T represents the treatment group RTV
  • RTV C represents the solvent control group RTV.
  • TGI 100% ⁇ [1-(TV t(T) -TV initial(T) ) / (TV t(C) -TV initial(C) )]]
  • TV t(T) represents the tumor volume measured by the treatment group at each time
  • TV initial (T) represents the tumor volume of the treatment group at the time of group administration
  • TV t (C) represents the tumor volume measured by the solvent control group at each time
  • TV Initial (C) indicates the tumor volume of the solvent control group at the time of group administration.
  • animal weight loss rate 100% ⁇ (BW initial - BW t ) / BW initial
  • BW t represents the body weight of the animal measured each time during administration
  • BW initial represents the body weight of the animal at the time of group administration.
  • IR (%) 100% ⁇ (W C - W T ) / W C
  • W C represents the tumor weight of the control group
  • W T represents the tumor weight of the treatment group
  • test data was calculated using Microsoft Office Excel 2007 software and related statistical processing. Data were expressed as mean ⁇ standard error (Mean ⁇ SE) unless otherwise specified. T-test was used for comparison between groups, P ⁇ 0.05 was considered as significant difference.
  • Test articles irinotecan, nktr-102, 12 compounds of the invention.
  • McCoy's 5A medium McCoy's 5A medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, water for injection, physiological saline, lactic acid, sorbitol.
  • FBS fetal bovine serum
  • trypsin trypsin
  • cyan-chain double antibody water for injection, physiological saline, lactic acid, sorbitol.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-7 weeks) were purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., and were raised in SPF animal room at a temperature of 20-25 °C. Relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human colon cancer cell line HT-29, derived from the cell bank of the Institute of Culture Collection of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • HT-29 cell culture HT-29 cells were cultured in 10% fetal bovine serum McCoy's 5A culture medium under 5% CO 2 at 37 ° C; digested with 0.25% trypsin; cell growth In the case, it is passed 2 to 3 times a week, and the passage ratio is 1:4 to 1:6.
  • HT-29 cells in logarithmic growth phase were collected, cell counted and resuspended in serum-free McCoy's 5A medium, adjusted to a cell concentration of 4 ⁇ 10 7 cells/mL; pipetted cells to make them evenly dispersed Then, the tube was placed in a 50 mL centrifuge tube, and the tube was placed in an ice box; the cell suspension was pipetted with a 1 mL syringe, and injected into the right armpit of the nude mouse, and each animal was inoculated with 100 ⁇ L (4 ⁇ 10 6 cells/cell). An HT-29 nude mouse xenograft model was established.
  • Solvent preparation Weigh 0.5g of sorbitol into a 50mL centrifuge tube, add 50mL of water for injection in the centrifuge tube, vortex to completely dissolve the solid substance, and prepare a 1% concentration of sorbitol aqueous solution (w/v) Store in a refrigerator at 4 °C.
  • Formulation of irinotecan preparation Weigh 12.0 mg of irinotecan, add 0.15 mL of 1% lactic acid, vortex to completely dissolve the drug, and then add 2.85 mL of 1% sorbitol aqueous solution separately, vortex and mix. Evenly, the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The effective concentration of irinotecan in the solution was 4.0 mg ⁇ mL -1 .
  • nktr-102 preparation Before each administration, accurately weigh 101.5mg of nktr-102, add 2.5mL of normal saline, vortex to completely dissolve the drug, and the effective concentration of irinotecan in the solution is 4.0mg ⁇ mL -1 .
  • Formulation of the compound for administration of the present invention 120.3 mg of Compound a and Compound A, 137.0 mg of Compound b and Compound B, 132.6 mg of Compound C and Compound C, and 132.0 mg of Compound d, respectively, were weighed before each administration. And compound D, 159.6 mg of compound e and compound E, 145.9 mg of compound f and compound F, add 2.5 mL of physiological saline, vortex, and ultrasonically (if necessary) to completely dissolve the drug, and the effective concentration of irinotecan in the solution It is 4.0 mg ⁇ mL -1 .
  • Animal grouping and dosing schedule The first administration was started on the day of grouping, and the experiment was terminated after about 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the effective dose equivalent to irinotecan is 40mg ⁇ kg -1 .
  • the first group was a solvent control group, and normal saline was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-15 were administered intravenously to the test samples: irinotecan, nktr-102, compound a, compound A, compound b, compound B, compound c, compound C, compound d, compound D, compound e, compound E, compound f and compound F were administered once every 4 days, Q4D x 3.
  • the experimental results show that the compound of the present invention has a good inhibitory effect on the growth of human colon cancer HT-29 nude mouse xenograft model, and is superior to irinotecan and nktr-102.
  • Test articles irinotecan, nktr-102, 12 compounds of the invention.
  • mice Female BALB/c nude mice, 150 inoculated, 90 experimental, age 6-8 weeks, body weight 20-22g ⁇ 20% body weight mean, animal source is Shanghai Xipuer - Bikai Experimental Animal Co., Ltd. ( BK), license number SCXK (Shanghai) 2008-0016. All experimental animals were housed in SPF-level laboratories. The experimenter is responsible for daily care and experimental research. Each rat cage is attached with an identity card containing information such as the experiment number, experimental group, experimenter's name, mouse breed and gender, and the mouse is marked with earrings.
  • Randomization When the tumor volume reached 150-200 mm 3 , the randomized block method was divided into 15 groups, 6 mice in each group, to ensure the tumor volume and mouse body weight were uniform between the groups. The mean value of tumor volume of each group differed from the mean of tumor volume of all experimental animals by no more than ⁇ 10%.
  • Breeding conditions living conditions: IVC system, 6 per cage; temperature: 20 ° C - 26 ° C; humidity: 40% ⁇ 70%; light: 12 hours alternate day and night.
  • Irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat.
  • Drinking water is urban tap water, which is filtered and autoclaved for drinking.
  • the litter is corn cob, Shanghai Maosheng Derivative Technology Co., Ltd., used after autoclaving. Change the litter twice a week. The mice were given the shortest environmental adaptation period before the experiment.
  • Human breast cancer MDA-MB-231 was purchased from the Institute of Cell Biology, Shanghai Academy of Sciences.
  • a subcutaneous xenograft model of human breast cancer MDA-MB-231 nude mice was established, each inoculated with 1 ⁇ 10 6 cells.
  • the administration volume was 10 mL ⁇ kg -1 .
  • the effective dose equivalent to irinotecan is 40mg ⁇ kg -1 .
  • the first group was a solvent control group, and normal saline was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-15 were administered intravenously to the test samples: irinotecan, nktr-102, compound a, compound A, compound b, compound B, compound c, compound C, compound d, compound D, compound e, compound E, compound f and compound F were administered once every 4 days, Q4D x 3.
  • Example 15 For the preparation of the test article, see Example 15. The volume required for a single administration is 3 mL.
  • MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA). The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the subcutaneous transplantation model of tumor nude mice was established by cell inoculation method: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and the cell suspension concentration was adjusted to 1 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 1 x 10 6 / 0.1 ml / mouse.
  • the animals were randomly divided into 15 groups according to the randomized block method, so that the tumor difference of each group was less than 10% of the mean value, 6 groups in each group, and the day of the group was used as Day1.
  • the experimental period was carried out for 3 weeks, and the animal body weight and tumor size were measured twice a week during the experiment. Daily observations record clinical symptoms.
  • the animals were sacrificed, the body weight was weighed, the tumor was removed, weighed and photographed. The results are shown in Table 2.
  • the experimental results show that the compound of the present invention has a good inhibitory effect on human breast cancer MDA-MB-231 xenografts in nude mice, and is superior to irinotecan and nktr-102.
  • Test articles irinotecan, nktr-102, 12 compounds of the invention.
  • mice Female BALB/c nude mice, 150 inoculated, 90 experimental, age 6-8 weeks, body weight 20-22g ⁇ 20% body weight mean, animal source is Shanghai Xipuer - Bikai Experimental Animal Co., Ltd. ( BK), license number SCXK (Shanghai) 2008-0016. All experimental animals were housed in SPF-level laboratories. The experimenter is responsible for daily care and experimental research. Each rat cage is attached with an identity card containing information such as the experiment number, experimental group, experimenter's name, mouse breed and gender, and the mouse is marked with earrings. When the tumor volume reached 150-200 mm 3 , they were divided into 15 groups by random block method, and 6 mice in each group ensured that the tumor volume and mouse body weight were uniform among the groups. The mean value of tumor volume of each group differed from the mean of tumor volume of all experimental animals by no more than ⁇ 10%.
  • breeding conditions living conditions are IVC system, 6 per cage; temperature: 20 ° C - 26 ° C; humidity: 40% ⁇ 70%; light: 12 hours day and night alternate.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat. Drinking water is urban tap water, which is filtered and autoclaved for drinking.
  • the litter is corn cob, Shanghai Maosheng Derivative Technology Co., Ltd., used after autoclaving. Change the litter twice a week. The mice were given the shortest environmental adaptation period before the experiment.
  • a subcutaneous xenograft model of human pancreatic cancer MIA Paca-2 nude mice was established, each inoculated with 3 ⁇ 10 6 cells.
  • the administration volume was 10 mL ⁇ kg -1 .
  • the effective dose equivalent to irinotecan is 40mg ⁇ kg -1 .
  • the first group was a solvent control group, and normal saline was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-15 were administered intravenously to the test samples: irinotecan, nktr-102, compound a, compound A, compound b, compound B, compound c, compound C, compound d, compound D, compound e, compound E, compound f and compound F were administered once every 4 days, Q4D x 3.
  • Example 15 For the preparation of the test article, see Example 15. The volume required for a single administration is 3 mL.
  • MIA Paca-2 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA) and 2.5% HS. The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the subcutaneous transplantation model of tumor nude mice was established by cell inoculation method: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and the cell suspension concentration was adjusted to 3 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 3 x 10 6 /0.1 ml/mouse.
  • the animals were randomly divided into 15 groups according to the randomized block method, so that the tumor difference of each group was less than 10% of the mean value, 6 groups in each group, and the day of the group was used as Day1. medicine.
  • the experimental period was carried out for 3 weeks, and the animal body weight and tumor size were measured twice a week during the experiment. Daily observations record clinical symptoms. On the last day of the experiment, the animals were sacrificed, the body weight was weighed, the tumor was removed, weighed and photographed. The results are shown in Table 3.
  • the experimental results show that the compound of the present invention has a good inhibitory effect on human pancreatic cancer MIA Paca-2 xenografts in nude mice, and is superior to irinotecan and nktr-102.
  • Example 18 Inhibition of in vivo growth of human gastric cancer NCI-N87 cell line in nude mice xenograft model.
  • Test articles irinotecan, nktr-102, 12 compounds of the invention.
  • Reagents RPMI-1640 medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. , temperature 20 ⁇ 25 ° C, relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human gastric cancer cell NCI-N87, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • NCI-N87 cell culture NCI-N87 cells were cultured in 10% fetal bovine serum RPMI-1640 medium in 5% CO 2 at 37 ° C; digested with 0.25% trypsin; The growth situation is 1 to 2 times per week, and the passage ratio is 1:2 to 1:6.
  • NCI-N87 cells were collected in logarithmic growth phase. After cell counting, resuspend in serum-free RPMI-1640 medium, adjust the cell concentration to 5 ⁇ 10 7 cells/mL; pipette the cells with a pipette. Disperse evenly, put into a 50mL centrifuge tube, place the centrifuge tube in an ice box; use a 1mL syringe to aspirate the cell suspension, and inject it into the right armpit of the nude mouse before inoculation, each animal is inoculated with 100 ⁇ L (5 ⁇ 10 6 cells / ), establish a NCI-N87 nude mouse xenograft model.
  • Example 15 For the preparation of the test article, see Example 15. The volume required for a single administration is 3 mL.
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-15 were administered intravenously to the test samples: irinotecan, nktr-102, compound a, compound A, compound b, compound B, compound c, compound C, compound d, compound D, compound e, compound E, compound f and compound F were administered at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan content), Q4D ⁇ 3.
  • the experimental results show that the compound of the present invention has a good inhibitory effect on the tumor growth of the human gastric cancer NCI-N87 cell line xenograft model, and is superior to irinotecan and nktr-102.
  • Example 19 Effect on the survival rate of brain in situ model of U87MG nude mice.
  • Test articles irinotecan, nktr-102, 12 compounds of the invention.
  • Reagents RPMI-1640 medium, trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., raised in SPF animal room, temperature 20 ⁇ 25 ° C, Relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain glioma cell line U87MG, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • NCI-N87 cell culture NCI-N87 cells were cultured in RPMI-1640 medium in 5% CO 2 at 37 ° C; digested with 0.25% trypsin; passaged weekly according to cell growth 1 to 2 times, the passage ratio is 1:2 to 1:6.
  • NCI-N87 cells were collected in logarithmic growth phase. After cell counting, resuspend in serum-free RPMI-1640 medium, adjust the cell concentration to 1 ⁇ 10 8 cells/mL; pipette the cells with a pipette. After dispersing evenly, it was placed in a 50 mL centrifuge tube, and the centrifuge tube was placed in an ice box; the cell suspension was aspirated with a 1 mL syringe, and the human glioma cell line U87MG was cultured in vitro by microinjection using the guidance of an animal stereotactic instrument.
  • Formulation of the administration preparation The preparation of the test sample is shown in Example 15. The volume required for a single administration is 3 mL.
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered to the tail vein every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-15 were given tail vein injections of irinotecan, nktr-102, and test compounds at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan), Q4D ⁇ 3.
  • the experimental results show that the compound of the present invention has a good inhibitory effect on glioma and is superior to irinotecan and nktr-102.

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Abstract

具有如结构式(Ⅰ)的多支链药物偶联物或其药学上可接受的盐，R为有机中心，POLY为聚合物，L为多价接头，T为靶向分子，D为活性剂，q为3-8之间的任一整数，其中L中符号"*"代表多价接头L与靶向分子T的连接点，"#"代表多价接头L与活性剂D的连接点，"％"代表多价接头L与POLY的连接点，其中，l为2-20之间的任一整数，m、n分别为0-10之间的任一整数；T为iRGD、cRGD、tLyp-1、Lyp-1、RPARPAR、Angiopep2或GE11；D为喜树碱类药物。

Description

多臂靶向抗癌偶联物 技术领域
本发明涉及多臂聚合物修饰的靶向抗癌偶联物，更具体地说，本发明涉及将靶向分子通过多臂聚合物与抗癌药物连接成偶联物。
背景技术
多年来，已经提出了用于改进生物活性剂的稳定性和递送的多种方法。与药用试剂的配制以及投递相关联的挑战可以包括：该药用试剂的差的水溶性、毒性、低的生物利用率、不稳定性、以及迅速的体内降解。尽管已经设计了许多办法来改进药用试剂的递送，但是没有一种单独的方法是没有其缺点的。例如，通常采用的药物递送方法的目标在于解决或至少改善一个或多个以下问题，包括如在一种脂质体、聚合物基质、或单分子胶束中的药物胶囊化、共价附接至一种水溶性聚合物如聚乙二醇上、基因靶向剂的使用、盐类的结构、等等。
WO2005028539、WO2010019233、WO2011063156、WO2011063158公开了一种处于临床三期的药物nktr 102，该药物主要用于转移性乳腺癌，由Nektar Therapeutics研发。该药物是一种水溶性的多支链聚合物药物前体，以提高药物的负载，结构如下：
Figure PCTCN2018083746-appb-000001
该化合物是使用多臂PEG与伊立替康连接，以提高水溶性，增加载药量，在抗癌作用不变的情况下，降低副作用。但该药物仍具有缺点，比如，靶向性较差，不能作用于特定的癌细胞，在杀死癌细胞的同时，也会影响正常细胞的性能，而使不良反应发生率仍然比较高。
发明内容
本发明公开了一种全新的具有靶向性的多支链药物偶联物，该偶联物具有三个或更多的支链，该偶联物可表示为下式：
Figure PCTCN2018083746-appb-000002
R为有机中心，POLY为聚合物，L为多价接头，T为靶向分子，D为活性剂，q为3-8之间的任一整数，其中L为：
Figure PCTCN2018083746-appb-000003
符号“*”代表多价接头L与靶向分子T的连接点，“#”代表多价接头L与活性剂D的连接点，“％”代表多价接头L与POLY的连接点，其中，l为2-20之间的任一整数，m、n分别为0-10之间的任一整数；
D为如式(Ⅱ)所示的喜树碱类药物：
Figure PCTCN2018083746-appb-000004
R 1-R 5相互独立地选自于以下基团：氢、卤素、酰基、烷基、取代烷基、烷氧基、取代烷氧基、烯基、炔基、环烷基、羟基、氰基、硝基、叠氮基、酰胺基、肼、胺基、取代胺基、羟基羰基、烷氧羰基、烷氧羰氧基、氨基甲酰氧基、芳基磺酰氧基、烷基磺酰氧基；R 6为H或OR 8，R 8为烷基、烯基、环烷基、卤代烷基或羟烷基；R 7为羟基、氨基、或硫醇。
POLY为聚合物，L为多价接头，T为靶向分子，D为活性剂，四者共同构 成该多支链药物偶联物的“支链”。该多支链药物偶联物的每一个支链和其他的支链是相互独立的。每一个支链从有机中心“R”发出。然而，一般来说，该偶联物的每个支链都是相同的。
现在详细地描述结构式(Ⅰ)中的每一个变量部分。
有机中心，“R”：在结构式(Ⅰ)中，“R”是一个1-100个原子的有机核心基团。比较好地是，R含有3-50个原子，更好地是，R含有大约3-30个原子。R可以为全部为碳原子的核心，也可选择性的含有1个或多个杂原子，例如，O、S、N、P等，依赖于所使用的特殊中心分子。R可以是直链、支链或环状的，发出至少3个独立的聚合物支链。结构式(Ⅰ)中，“q”与从“R”发出的聚合物支链的数量相对应。
有机中心“R”，是从一个分子衍生而来的，该分子提供许多聚合物连接的位置，近似等于聚合物支链的数量。更好地是，多链聚合物结构的主要中心分子式至少带有适合作为聚合物支链的3个及3个以上的羟基、硫基或氨基的多羟基化合物、多硫化合物或多胺化合物的残基。一个“多羟基化合物”是一个由多个(大于2个)可利用的羟基基团组成的分子。一个“多硫化合物”是一个由多个(大于2个)可利用的硫基基团组成的分子。一个“多胺化合物”是一个由多个(大于2个)可利用的胺基基团组成的分子。依据聚合物支链的数量，多羟基化合物、多胺化合物或多硫化合物的母体(在POLY共价键结合前)典型地包括3-25个羟基、硫基或氨基，较好的是3-10个羟基、硫基或氨基，最好是包括从3到大约8个(如3、4、5、6、7或8)适合与POLY共价键结合的羟基、硫基或氨基。
多羟基化合物或多胺化合物中心的母体在和聚合物作用之前典型地有一个结构式R-(OH)p或者R-(NH 2)p。在结构式(Ⅰ)中，p值和q值是相对应的，因为在母体有机分子中的每一个功能性基团，典型地有-OH和-NH 2，如果位置易受影响或易发生反应，它们就和聚合物支链的POLY共价键结合。结构式(Ⅰ)中，与POLY连接后，R母体的多羟基化合物的羟基都已经转换成了一个聚合物支链，所描述的R为连接后的残基。例如，如果有机中心分子是从季戊四醇衍生得来的，多羟基化合物的母体拥有结构式C(CH 2OH) 4，有机中心基R表示为：
Figure PCTCN2018083746-appb-000005
优选的作为聚合物中心的说明性多羟基化合物包括含有1到10个碳原子和1到10个羟基基团的脂肪族多羟基化合物，例如，乙烯乙二醇、烷二醇、烃基乙二醇、亚烷基烃基二醇、烃基环烷基二醇、1,5-萘烷二醇、4,8-二(羟甲基)三环癸烷、环亚烷基二醇、二羟基烷烃、三羟基烷烃、四羟基烷烃等。环脂肪族多羟基化合物包括直链的或者闭环糖类和糖醇，如甘露醇、山梨糖醇、纤维醇、木糖醇、白雀醇、苏糖醇、阿拉伯糖醇、赤藻糖醇、己六醇、核糖、树胶醛糖、木糖、来苏糖、鼠李糖、半乳糖、葡萄糖、果糖、山梨糖、甘露糖、吡喃糖、阿卓糖、塔罗糖、塔格糖、吡喃糖苷、蔗糖、乳糖、麦芽糖等。也可采用芳香族多羟基化合物，如苯磷二酚、烃基苯磷二酚、焦酚、氟代甘氨酸酚、1,2,4-苯三酚、间苯二酚、烃基间苯二酚、二烃基间苯二酚、苔黑酚一水合物、橄榄酚、对苯二酚、烃基对苯二酚、苯基对苯二酚等。其他可能采用的多羟基化合物中心可能包括冠醚、环式糊精、糊精或其它碳水化合物。
在结构式(Ⅰ)中，q为对应“R”上连接的聚合物支链的个数，具体的数字可以为3～20。典型的，“q”的具体数字为3、4、5、6、7、8。具体来讲，以“R”为中心发出三、四、五、六、七、八个聚合物支链。
在一些具体的方案中，“R”具有三个聚合物支链，“R”优选为：
Figure PCTCN2018083746-appb-000006
在一些具体的方案中，“R”具有四个聚合物支链，“R”优选为：
Figure PCTCN2018083746-appb-000007
在一些具体的方案中，“R”具有六个聚合物支链，“R”优选为：
Figure PCTCN2018083746-appb-000008
在一些具体的方案中，“R”具有八个聚合物支链，“R”优选为：
Figure PCTCN2018083746-appb-000009
聚合物，“POLY”：在结构式(Ⅰ)中，“POLY”为聚合物，每一个聚合物支链中的POLY是独立地选择出来的，较好的，每一个聚合物是相同的聚合物，更适宜地，每一个结构式(Ⅰ)中的聚合物支链是相同的。优选的聚合物为水溶性的，任意的水溶性聚合物可以用于形成本发明的偶联物，本发明所指聚合物可以是任意几何形态或形式的。代表性的聚合物包括但并不限于：聚乙二醇、聚丙二醇、聚(乙烯吡咯烷酮)、聚(羟烷基甲基丙烯酸胺)、聚(羟烷基甲基丙烯酸盐)、聚(糖类)、聚(α-羟基酸)、聚(丙烯酸)、聚(乙烯乙酸)、聚膦嗪、聚唑啉、聚(N-丙烯酰吗啉)等。
在典型的化合物中，“POLY”为聚乙二醇(PEG)，可为任意几何形态或形式，包括直链、支链、叉形链等，在此使用的“聚乙二醇”，意思是涵盖任何水溶性聚(氧化乙烯)。典型地，用于本发明中的PEG将包括二个以下结构的一种：“(CH 2CH 2O) k-”或“(CH 2CH 2O) k-CH 2CH 2-”，取决于一个或多个末端氧是否已经例如在一个合成转化期间被置换。变量k范围是从5至约500，并且这些末端基团以及总体PEG的结构可以改变。所述的聚乙二醇结构通常还含有部分末端部分残基，类似于POLY的终端基团，可以为H、NH 2、OH、CO 2H、C 1-6烷基(例如，甲基、乙基、丙基)、C 1-6烷氧基(例如，甲氧基、乙氧基)、酰基或芳基来结束。
本发明优选的“POLY”为直线型的聚乙二醇，典型的结构为：
Figure PCTCN2018083746-appb-000010
代表原子的连接处，标“&”号的氧原子为与有机中心“R”连接的原子。其中k的取值范围为大约5～500，最好为50～200，r为1-10之间的任一整数，更优选的，本发明“POLY”为：
Figure PCTCN2018083746-appb-000011
本发明POLY还可为：
Figure PCTCN2018083746-appb-000012
等。
本发明所指的活性剂“D”为喜树碱类抗癌剂，喜树碱类药物是用于临床的拓扑异构酶Ⅰ抑制剂，在高活性的同时，具有水溶性差、对正常机体组织毒副作用大等缺点，极大的限制了喜树碱类抗癌剂的临床应用。
D结构中的R 7为与多价接头L共价连接的基团，如羟基、氨基、或硫醇，优选为羟基。活性剂D连接到多价接头L时，应没有重大的生物活性损失。
本发明活性剂优选伊立替康、SN-38、10-羟基喜树碱、鲁比替康。其中：
Figure PCTCN2018083746-appb-000013
本发明中，“T”为靶向分子，具有或不具有药用作用，该靶向分子的作用是增加靶向性，使得该偶联物在目标组织的浓度更高，提高生理活性或药用作用。“T”可为单功能的靶向分子，也可为多功能的靶向分子，在一些可选择的方案中，“T”还可为两个或两个以上靶向分子组成的靶向部分。在一些具体的方案中，“T”可为含有“精氨酸-甘氨酸-天冬氨酸”序列的RGD肽，RGD肽是整合素与其配体蛋白相互作用的识别位点。优选的RGD肽包括iRGD和cRGD等。T还可以为Lyp-1、Lyp-1、RPARPAR、Angiopep2或GE11。
iRGD结构如下：
Figure PCTCN2018083746-appb-000014
cRGD是一系列化合物，典型的化合物包括：
Figure PCTCN2018083746-appb-000015
优选的cRGD为：
Figure PCTCN2018083746-appb-000016
tLyp-1的结构如下：
Figure PCTCN2018083746-appb-000017
Lyp-1的结构如下：
Figure PCTCN2018083746-appb-000018
RPARPAR的多肽序列为精氨酸-脯氨酸-丙氨酸-精氨酸-脯氨酸-丙氨酸-精氨酸，其结构如下：
Figure PCTCN2018083746-appb-000019
Angiopep2的多肽序列为：TFFYGGSRGKRNNFKTEEY，其结构如下：
Figure PCTCN2018083746-appb-000020
GE11的多肽序列为：YHWYGYTPQNVI，其结构为：
Figure PCTCN2018083746-appb-000021
本发明所指的活性剂“D”指的是非改性母体活性剂的一部分或由药物与本发明的多价接头共价连接所产生的共价链(或者是它的活化的或化学改性的形式)之前的未改性母体活性剂的残基。活性剂部分与多价接头之间的连接基水解或酶 解时，活性剂本身就得到释放。
根据本发明的目的，术语“残基”应该理解为化合物的一部分，它是指经历了与另一个化合物取代反应后的剩余物。
本发明偶联物进入生物体内，到达靶细胞或靶组织时，活性剂D与多价接头L断裂，活性剂D以它未经改进，即未形成共价键的形式被释放出来，与母体分离，发挥生理活性。
本发明优选的方案中，“POLY”为线状的聚乙二醇连接臂，即本发明偶联物包括以下几种类型的化合物：
四臂：
Figure PCTCN2018083746-appb-000022
三臂：
Figure PCTCN2018083746-appb-000023
八臂：
Figure PCTCN2018083746-appb-000024
其中k的取值范围为大约5～500，最好为50～200，r为1-10之间的任一整数。
本发明优选式(Ⅲ)化合物，在式(Ⅲ)的基础上，k优选113。本领域技术人员应该知晓，在高分子领域，k代表所述的聚合物的聚合度，并取决于所述的聚合物的分子量，并不是绝对的数值，当k取值113时，是指平均值为113。
在更优选的方案中，本发明偶联物的靶向部分“T”选自iRGD、cRGD、tLyp-1、Lyp-1、RPARPAR、Angiopep2或GE11中的一种，活性剂“D”选自伊立替康、SN-38、10-羟基喜树碱、鲁比替康中的一种。
在更优选的方案中，L选自：
Figure PCTCN2018083746-appb-000025
Figure PCTCN2018083746-appb-000026
中的一种。
基于式(Ⅲ)，在一些具体的方案中，本发明化合物如下：
化合物a：D为伊立替康，T为cRGD
Figure PCTCN2018083746-appb-000027
更具体地，化合物a也可写做如下形式：
Figure PCTCN2018083746-appb-000028
化合物A为化合物a的盐酸盐：
Figure PCTCN2018083746-appb-000029
化合物b：D为伊立替康，T为iRGD
Figure PCTCN2018083746-appb-000030
化合物B为化合物b的盐酸盐：
Figure PCTCN2018083746-appb-000031
化合物c：D为伊立替康，T为tLyP-1
Figure PCTCN2018083746-appb-000032
化合物C为化合物c的盐酸盐：
Figure PCTCN2018083746-appb-000033
化合物d：D为伊立替康，T为RPARPAR
Figure PCTCN2018083746-appb-000034
化合物D为化合物d的盐酸盐：
Figure PCTCN2018083746-appb-000035
化合物e：D为伊立替康，T为Angiopep-2
Figure PCTCN2018083746-appb-000036
化合物E为化合物e的盐酸盐
Figure PCTCN2018083746-appb-000037
更具体地，化合物e可写为如下形式：
Figure PCTCN2018083746-appb-000038
化合物f：D为伊立替康，T为GE11：
Figure PCTCN2018083746-appb-000039
化合物F为化合物f的盐酸盐：
Figure PCTCN2018083746-appb-000040
化合物F
更具体地，化合物f也可写做如下形式：
Figure PCTCN2018083746-appb-000041
在这里需要说明的是，成盐时，是本发明偶联物的支链和HCl分别成盐，例如化合物A、化合物B、化合物C、化合物D，每个支链上带有2分子HCl，整个分子将带有8HCl。化合物E，每个支链上带有6分子HCl，整个分子将带有24分子的HCl。化合物F，每个支链上带有3分子HCl，整个分子将带有12个HCl。
根据本发明的发明精神，除以上公开的具体化合物外，本领域技术人员还能根据本发明所述的技术方案和制备方法，制备出更多偶联物，例如：
①D为SN-38，T分别为iRGD、cRGD、tLyp-1、Lyp-1、RPARPAR、Angiopep2或GE11的偶联物；②D为10-羟基喜树碱，T分别为iRGD、cRGD、tLyp-1、Lyp-1、RPARPAR、Angiopep2或GE11的偶联物；③D为鲁比替康，T分别为iRGD、cRGD、tLyp-1、Lyp-1、RPARPAR、Angiopep2或GE11的偶联物。
本发明偶联物是典型的药物前体，通过水解作用或酶解作用，活性剂D被释放出来，与母体分离，发挥生理活性。本发明偶联物呈现出高载荷能力，这样就可以降低总剂量来治疗一种特殊的疾病，例如癌症等。也就是说，本发明偶联物活性剂载体能够有效地和多种活性剂分子以共价键结合，允许每一定的偶联物量可以服用更多量的治疗剂型(也就是活性剂部分)。本发明偶联物通过水溶性聚合物的修饰，本质上是偶联物也是亲水的，特别是活性剂为水难溶药物时，提高偶联物的生物利用度。相比未偶联的药物，本发明偶联物能够表现出更强的作用，在人体或其他动物体内组织更加富集。
本发明中偶联物药物前体有许多独特的性质，尤其是在活性剂为一个抗癌化合物的情况下。这种药物前体能以较高效率来抑制肿瘤的生长。我们使用的这种小分子是一种已知具有抗癌特性的小分子。然而，通过如上所述与多支链聚合物结合，其疗效和药物代谢动力学与该小分子(例如，抗癌化合物本身)相比，有了很大的改进。适合的实体肿瘤类型包括结肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、脑胶质瘤及乳房、卵巢、结肠、肾、胆管、肺和脑的恶性肉瘤、癌和淋巴瘤。
综上，本发明为一个多臂聚合物修饰的靶向抗癌偶联物，其中，水溶性的聚合物修饰可增强该偶联物的水溶性，从而提高载药量；靶向分子增加靶向性，使得该偶联物在目标组织的浓度更高；L为任意的连接接头，其作用是首先将靶向分子与抗癌药物连接起来，再将“靶向分子、抗癌药物与聚合物臂连接起来，使得整个偶联物形成一个有机的整体。
本发明偶联物，药学上可接受的盐优选为盐酸盐，可用药物化学领域的常规手段成盐，还可为三氟乙酸盐、硫酸盐、磷酸盐、醋酸盐等。
另一方面，本发明提供了所述偶联物的制备方法。在本发明偶联物制备过程中，POLY和有机中心R实际上组成了多臂聚合物，在本发明优选的实施例中，该多臂聚合物为多臂聚乙二醇，能够从商业可利用的原材料中获得，例如，可从北京键凯科技有限公司购得多种类型的四臂、三臂、八臂聚乙二醇衍生物。市售的这些多臂PEG可直接参与反应。
制备式(Ⅲ)偶联物时，优选使用的四臂聚乙二醇如下：
Figure PCTCN2018083746-appb-000042
该优选的四臂聚乙二醇被称为4ARM-PEG20K-SCM，其分子量约为20kDa左右。同理，制备式(Ⅳ)和式(Ⅴ)偶联物时，使用的三臂和八臂聚乙二醇的分子量也优选为20kDa左右。
具体实施例
在下面将对本发明进行详细描述。然而，本发明可能具体体现为许多不同的形式，而且它不应该被局限于此处所描述的实施例中，提供这些实施例中的目的是使所披露内容更完整与全面。所用试剂和原料，除了提供制备方法的除外，其余均为市售，其中4ARM-PEG20K-SCM购自北京键凯科技有限公司
名词解释
Figure PCTCN2018083746-appb-000043
Figure PCTCN2018083746-appb-000044
实施例1
Figure PCTCN2018083746-appb-000045
化合物2的制备
向250mL圆底烧瓶中加入3.50g化合物1(1.0eq)，52.5mlDMF，加热至60℃溶解，5-10min后减压蒸去DMF，加入300ml正庚烷减压蒸馏，重复三次，旋干后加入105mlDCM，1.08g Boc-Gly-OH(1.2eq)，63mg DMAP(0.1eq)，滴加1.59gDCC(1.5eq)溶于10mlDCM的溶液，20℃反应4小时，TLC监控反应完毕后，过滤，浓缩至剩余25％体积时加入120ml IPA，蒸去75％的溶剂，加入150ml 正庚烷，室温搅拌1小时，过滤，正庚烷洗涤2次，干燥得淡黄色固体4.02g化合物2。
化合物3的制备
向100mL三口瓶中加入4.02g化合物2，50mlDCM，搅拌溶解后滴加11.6ml TFA，室温反应2h，TLC监控反应完毕后加入150ml乙腈，减压蒸馏120ml溶剂后倒入320mlTBME溶液中，搅拌30min，过滤，滤饼用TBME洗涤得4.00g淡黄色固体化合物3。
实施例2
Figure PCTCN2018083746-appb-000046
化合物5的制备
向250mL三口瓶中加入6.9g化合物4，30ml EA，搅拌溶解后降温至0℃，加40ml 0.3M的HCl/EA,保温反应2h，TLC监控反应完毕浓缩至干，得到化合物5，直接进行下一步反应。
化合物6的制备
将化合物5(1.0eq)用50ml纯化水溶解，加入3.96g碳酸氢钠(2.0eq),用50mlDME将5.30g Fmoc-OSU(1.0eq)溶解，加入到化合物5的反应瓶中，补加 25mlTHF，室温搅拌2小时，TLC监控反应完毕后，蒸去有机溶剂，EA萃取杂质，水相用稀盐酸调节pH至3-4，EA萃取2次，合并有机相，水洗一次，饱和食盐水洗涤后无水硫酸钠干燥，浓缩得8.4g淡黄色油状物化合物6。
化合物7的制备
在100ml反应瓶中加入4.00g化合物6(1.0eq)、2.92g H-Lys(Boc)-OBzl·HCl、40mlDCM溶解，加入2.76gDIEA(3.0eq)，1.74g DEPC(1.5eq)，室温搅拌2小时，TLC监控反应完毕后，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，水洗一次，饱和食盐水洗涤一次后无水硫酸钠干燥，浓缩得7.0g淡黄色油状物化合物7，不纯化直接进行下一步反应。(利用同样的方法制备化合物16)
化合物8的制备
用140ml 25％的DEA/DCM将7.0g化合物7溶解，室温搅拌6小时，TLC监控反应完毕后，浓缩至干，加入100ml，50mlEA，用稀盐酸将pH调节至3-4，分液，水相用EA萃取2次后浓缩至干，得3.5g淡黄色固体化合物8。(利用同样的方法制备化合物17)
实施例3 连有保护基的靶向分子cRGD(化合物11)的制备
Figure PCTCN2018083746-appb-000047
化合物9的制备
利用2Cl-Trt Resin，Fmoc保护法，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测，依次将下列保护氨基酸连接到树脂上：
Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OBzl)-OH、Fmoc-D-Phe-OH、Fmoc-Asp(OtBu)OH、脱除Fmoc，DMF洗涤、DCM洗涤、甲醇洗涤后干燥，加入裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体化合物9。
化合物10的制备
2L的三口烧瓶中加入14.0g化合物9(1.0eq)，加入1L DMF，降温至0℃，加入9.2g碳酸氢钠(8.0eq)，溶清后加入15.1g DPPA(4.0eq)，保温过夜，TLC反应完毕后倒入5L水中，EA萃取2次，水洗，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得类白色固体化合物10 11.5g
化合物11的制备
1L的氢化釜中加入11.5g化合物10，1L甲醇，2.5gPd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物11 11.0g
实施例4 连有保护基的靶向分子iRGD(化合物20)的制备
Figure PCTCN2018083746-appb-000048
利用Fmoc-Sieber Resin，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测法，依次将下列保护氨基酸连接到树脂上：Fmoc-Cys(Acm)-OH、Fmoc-Asp(Alloc)OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Cys(Acm)-OH、DMF洗涤后加入三氟乙酸铊(2.0eq)搅拌18小时后DMF洗涤，脱除Fmoc，缩合Fmoc-Cys(Trt)-OH，DMF洗涤、脱除Fmoc，加入醋酐 吡啶反应20min，DMF洗涤，加入3eq的Pd(PPh 3) 4的CHCl 3:AcOH:NMM(18:1:0.5)的溶液，反应2h，脱除Alloc，随后用氯仿(6*20ml)洗涤，20％HOAc的DCM溶液、DCM和DMF洗涤，加DMF洗涤、DCM洗涤、甲醇洗涤后干燥，加入1％TFA/DCM，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体化合物20。
实施例5 连有保护基的靶向分子tLyP-1(化合物30)的制备
Figure PCTCN2018083746-appb-000049
利用2Cl-Trt Resin，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测法，依次将下列保护氨基酸连接到树脂上：Fmoc-Arg(Pbf)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Boc-Cys(Trt)-OH，加入裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体化合物30。
实施例6 连有保护基的靶向分子RPARPAR(化合物40)的制备
Figure PCTCN2018083746-appb-000050
利用2Cl-Trt Resin，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测法，依次将下列保护氨基酸连接到树脂上：Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、 Boc-Arg(Pbf)-OH加入裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体化合物40。
实施例7 连有保护基的靶向分子Angiopep-2(化合物50)的制备
Figure PCTCN2018083746-appb-000051
Angiopep-2的序列为TFFYGGSRGKRNNFKTEEY
利用2Cl-Trt Resin，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测法，依次将下列保护氨基酸连接到树脂上：Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Phe-OH、Boc-Thr(tBu)-OH加入裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体化合物50。
实施例8 连有保护基的靶向分子GE11(化合物60)的制备
Figure PCTCN2018083746-appb-000052
GE11的序列为YHWYGYTPQNVI
利用2Cl-Trt Resin，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测法，依次将下列保护氨基酸连接到树脂上：Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Pro-OH、Fmoc-Thr(tBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Trp(Boc)-OH、Fmoc-His(Trt)-OH、Boc-Tyr(tBu)-OH、加入裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体60。
实施例9 化合物a和化合物A的制备
Figure PCTCN2018083746-appb-000053
Figure PCTCN2018083746-appb-000054
化合物12的制备
5ml反应瓶中加入480mg化合物11(1.0eq)，380mg化合物8(1.1eq)，1ml DMF，203mgDIEA(3.0eq)，128mg DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒 入10mL水中，EA萃取2次，稀盐酸洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得果冻状固体化合物12 0.8g，直接进行下一步反应。
化合物13的制备
200ml的氢化釜中加入0.8g化合物10，30ml甲醇，0.28gPd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物13 0.66g
化合物14的制备
100ml反应瓶中加入6.60g化合物13(1.0eq)，3.59g化合物3(1.05eq)，66ml DMF，1.16gDIEA(3.0eq)，1.10g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入700ml TBME中，打浆后抽滤，固体用150DCM溶解后倒入1.5L TBME中，打浆抽滤，干燥灰色粉末化合物14 9.0g，直接进行下一步反应。
化合物15的制备
250ml反应瓶中，加入9.0g化合物14，裂解试剂92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化、冻干，得淡黄色絮状物5.0g化合物15。
化合物a的制备
反应瓶中加入2.3g化合物15(4.5eq)，6.0g 4ARM-PEG20K-SCM(1.0eq)，60ml DMF，0.27gTEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入1000ml TBME中，打浆、抽滤，干燥得类色粉末粗品a 7.6g，HPLC纯化后脱盐，浓缩除去有机溶剂，冻干得类白色粉末化合物a 3.4g。
化合物A的制备
得到化合物a的粉末粗品后，HPLC纯化后脱盐，浓缩除去有机溶剂，用稀盐酸调节pH＝5～6，冻干得黄绿色粉末化合物A 3.4g。
MALDI-TOF检测分子量为25480.27。
实施例10 化合物b和化合物B的制备
Figure PCTCN2018083746-appb-000055
Figure PCTCN2018083746-appb-000056
化合物21的制备
100ml反应瓶加入5.00g化合物20(1.0eq)，2.36g化合物17(1.1eq)，50ml DMF，1.11g DIEA(3.0eq)，0.71g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入300mL水中，EA萃取2次，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得淡黄色固体21 7.18g，直接进行下一步反应。
化合物22的制备
200ml的氢化釜中加入7.00g化合物21，120ml甲醇，0.35g Pd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物22 7.05g
化合物23的制备
100ml反应瓶中加入7.00g化合物22(1.0eq)，2.22g化合物3(1.05eq)，70ml DMF，1.10gDIEA(3.0eq)，0.70g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入700ml TBME中，打浆后抽滤，固体用100DCM溶解后倒入1.0L TBME中，打浆抽滤，干燥得灰色粉末化合物23 8.60g，直接进行下一步反应。
化合物24的制备
8.60g化合物23加入200ml裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得并HPLC纯化得2.10类白色固体24。
化合物25的制备
50ml反应瓶中加入1.23g化合物24(4.5eq)，2.00g 4ARM-PEG20K-SCM(1.0eq)，20ml DMF，0.09gTEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入400ml TBME中，打浆、抽滤，干燥得25 3.05g，直接进行下一步反应。
化合物b的制备
50ml反应瓶中，加入3.0g化合物25，30ml裂解试剂：92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，冻干得类白色粉末化合物b 1.02g。
化合物B的制备
化合物b粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，用稀盐酸调节pH＝5～6，冻干得黄绿色粉末化合物B 1.02g。
MALDI-TOF检测分子量为29013.19。
实施例11 化合物c和化合物C的制备
Figure PCTCN2018083746-appb-000057
Figure PCTCN2018083746-appb-000058
化合物31的制备
100ml反应瓶中加入5.60g化合物30(1.0eq)，2.39g化合物17(1.1eq)，60ml DMF，1.03gDIEA(3.0eq)，0.65g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入300mL水中，EA萃取2次，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得淡黄色固体化合物31 7.08g，直接进行下一步反应。
化合物32的制备
200ml的氢化釜中加入7.05g化合物31，150ml甲醇，0.35gPd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物32 6.95g。
化合物33的制备
100ml反应瓶中加入6.80g化合物32(1.0eq)，1.89g化合物3(1.05eq)，70ml DMF，0.94gDIEA(3.0eq)，0.59g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入700ml TBME中，打浆后抽滤，固体用100DCM溶解后倒入1.0L TBME 中，打浆抽滤，干燥得灰色粉末化合物33 8.20g，直接进行下一步反应。
化合物34的制备
8.20g化合物33加入160ml裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得并HPLC纯化得5.6g类白色固体化合物网34。
化合物35的制备
50ml反应瓶中加入0.75g化合物34(4.5eq)，1.00g 4ARM-PEG20K-SCM(1.0eq)，10ml DMF，0.05gTEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入200ml TBME中，打浆、抽滤，干燥得化合物35 1.69g，直接进行下一步反应。
化合物c的制备
50ml反应瓶中，加入1.65g化合物25，20ml裂解试剂：92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，冻干得类白色粉末化合物c 0.84g。
化合物C的制备
化合物c粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，用稀盐酸调节pH＝5～6，冻干得黄绿色粉末化合物C 0.84g。
MALDI-TOF检测分子量为28076.21。
实施例12 化合物d和化合物D的制备
Figure PCTCN2018083746-appb-000059
Figure PCTCN2018083746-appb-000060
化合物41的制备
100ml反应瓶中加入6.20g化合物40(1.0eq)，3.30g化合物17(1.1eq)，62ml DMF，1.43gDIEA(3.0eq)，0.90g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入310mL水中，EA萃取2次，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得淡黄色固体化合物41 8.84g，直接进行下一步反应。
化合物42的制备
200ml的氢化釜中加入8.80g化合物41，150ml甲醇，0.44gPd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物42 8.84g。
化合物43的制备
100ml反应瓶中加入8.50g化合物42(1.0eq)，2.77g化合物3(1.05eq)，85ml DMF，1.38g DIEA(3.0eq)，0.87g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入850ml TBME中，打浆后抽滤，固体用110DCM溶解后倒入1.1L TBME中，打浆抽滤，干燥得灰色粉末化合物43 9.86g，直接进行下一步反应。
化合物44的制备
9.80g化合物43加入200ml裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得并HPLC纯化得5.92g类白色固体化合物44。
化合物45的制备
50ml反应瓶中加入0.60g化合物34(4.5eq)，1.00g 4ARM-PEG20K-SCM(1.0eq)，10ml DMF，0.05gTEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入200ml TBME中，打浆、抽滤，干燥得化合物45 1.45g，直接进行下一步反应。
化合物d的制备
50ml反应瓶中，加入1.45g化合物45，15ml裂解试剂：92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，冻干得类白色粉末化合物d 0.57g。
化合物D的制备
化合物d粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，用稀盐酸调节pH＝5～6，冻干得黄绿色粉末化合物D 0.57g。
MALDI-TOF检测分子量为27963.54。
实施例13 化合物e和化合物E的制备
Figure PCTCN2018083746-appb-000061
Figure PCTCN2018083746-appb-000062
Figure PCTCN2018083746-appb-000063
化合物51的制备
100ml反应瓶中加入6.20g化合物50(1.0eq)，1.39g化合物17(1.1eq)，62ml DMF，0.60gDIEA(3.0eq)，0.38g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入10mL水中，EA萃取2次，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得果冻状固体化合物51 6.5g，直接进行下一步反应。
化合物52的制备
200ml的氢化釜中加入6.53g化合物51，150ml甲醇，0.33gPd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物52 6.50g
化合物53的制备
100ml反应瓶中加入6.50g化合物52(1.0eq)，1.08g化合物3(1.05eq)，65ml DMF，0.54gDIEA(3.0eq)，0.34g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入650ml TBME中，打浆后抽滤，固体用100ml DCM溶解后倒入1.0L TBME中，打浆抽滤，干燥灰色粉末53 6.71g，直接进行下一步反应。
化合物54的制备
2.5g化合物53加入50ml裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得并HPLC纯化得0.97g类白色固体化合物54。
化合物55的制备
50ml反应瓶中加入2.91g化合物54(4.5eq)，3.00g 4ARM-PEG20K-SCM(1.0eq)，30ml DMF，0.13gTEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入300ml TBME中，打浆、抽滤，干燥得55 5.83g，直接进行下一步反 应。
化合物e的制备
50ml反应瓶中，加入2.04g化合物55，30ml裂解试剂：92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，冻干得类白色粉末E 0.42g。
化合物E的制备
化合物e粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，用稀盐酸调节pH＝5～6，冻干得黄绿色粉末E 0.42g。
MALDI-TOF检测分子量为33812.65。
实施例14 化合物f和化合物F的制备
Figure PCTCN2018083746-appb-000064
Figure PCTCN2018083746-appb-000065
化合物61的制备
100ml反应瓶中加入5.00g化合物60(1.0eq)，1.66g化合物17(1.1eq)，50ml DMF，0.72gDIEA(3.0eq)，0.45g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入10mL水中，EA萃取2次，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得果冻状固体61 6.14g，直接进行下一步反 应。
化合物62的制备
200ml的氢化釜中加入6.10g化合物61，150ml甲醇，0.31gPd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物62 5.98g
化合物63的制备
100ml反应瓶中加入5.95g化合物62(1.0eq)，1.36g化合物3(1.05eq)，60ml DMF，0.68gDIEA(3.0eq)，0.43g DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入600ml TBME中，打浆后抽滤，固体用100ml DCM溶解后倒入1.0L TBME中，打浆抽滤，干燥灰色粉末化合物63 6.53g，直接进行下一步反应。
化合物64的制备
6.50g化合物63加入130ml裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得并HPLC纯化得3.63g类白色固体化合物64。
化合物65的制备
50ml反应瓶中加入2.06g化合物64(4.5eq)，3.00g 4ARM-PEG20K-SCM(1.0eq)，30ml DMF，0.13gTEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入300ml TBME中，打浆、抽滤，干燥得化合物65 4.67g，直接进行下一步反应。
化合物f的合成
200ml反应瓶中，加入4.60g化合物65，100ml裂解试剂：92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，冻干得类白色粉末化合物f 1.34g。
化合物F的合成
化合物f粗品经反相HPLC纯化后脱盐，浓缩除去有机溶剂，用稀盐酸调节pH＝5～6，冻干得黄绿色粉末化合物F 1.34g。
MALDI-TOF检测分子量为30907.82。
本发明实施例15-19中所用到的供试品及试剂、仪器等来源如下：
伊立替康(原料药)系购买所得。
nktr-102的制备方法参考CN102711837A所公开的方法，如下：
将实施例1中的化合物3(829mg,4.5eq)添加到250mL的反应瓶中，加 入DCM(50mL)，三乙胺(221mg,9.0eq)，溶解后加入4ARM-PEG20K-SCM(5.00g,1.0eq)添加到该反应瓶中。HPLC监控反应没有明显进展后，减压蒸馏出去约20mL DCM，将溶液倒入300mL TBME中搅拌沉淀，过滤，得到5.4g粗品，粗品经HPLC制备纯化，脱盐，用稀盐酸调节pH至5-6，冻干得到2.71g淡绿色粉末nktr-102。
生理盐水购自上海华源长富药业(集团)有限公司。1ml无菌注射器购自上海康德莱企业发展集团股份有限公司(上海，中国)。MDA-MB-231培养于DMEM培养基(GIBCO,美国)，含10％胎牛血清FBS(GIBCO，美国)，培养于含5％CO 2的37℃培养箱。基质胶(BD Matrigel)Matrigel购自美国BD公司。
生物安全柜(型号：AC2-6E1)，购自ESCO；CO 2隔水细胞培养箱(型号：3111)，购自Thermo Scientific Forma；倒置显微镜(型号：CKX41SF)，购自Olympus；电动吸引器(型号YX930D)，购自上海医疗器械工业(集团)有限公司；天平(梅特勒-托利多AB135-S)，购自梅特勒-托利多；低速离心机(型号LD5-2A)，购自北京雷勃尔离心机有限公司；电子数显卡尺(型号：SF2000)，购自桂林广陆数字测控股份有限公司。
在本发明实施例15-19中，所有动物实验操作严格遵守动物使用和管理规范。肿瘤相关参数的计算参考中国CFDA《细胞毒类抗肿瘤药物非临床研究技术指导原则》，根据中国SFDA《细胞毒类抗肿瘤药物非临床研究技术指导原则》(2006年11月)，％T/C≤40％并经统计学分析P<0.05为有效。
肿瘤体积(TV)计算公式如下：TV(mm 3)＝l×w 2/2
其中，l表示肿瘤长径(mm)；w表示肿瘤短径(mm)。
相对肿瘤体积(RTV)的计算公式为：RTV＝TV t/TV initial
其中，TV initial为分组给药时测量到的肿瘤体积；TV t为给药期间每一次测量时的肿瘤体积。
相对肿瘤增殖率(％T/C)的计算公式为：％T/C＝100％×(RTV T/RTV C)
其中，RTV T表示治疗组RTV；RTV C表示溶剂对照组RTV。
肿瘤生长抑制率TGI(％)的计算公式为：TGI＝100％×[1－(TV t(T)－TV initial(T))/(TV t(C)－TV initial(C))]
其中，TV t(T)表示治疗组每次测量的肿瘤体积；TV initial(T)表示分组给药时治疗组的肿瘤体积；TV t(C)表示溶剂对照组每次测量的肿瘤体积；TV initial(C)表示分 组给药时溶剂对照组的肿瘤体积。
动物体重下降率的计算公式为：动物体重下降率＝100％×(BW initial－BW t)/BW initial
其中，BW t表示给药期间每次测量的动物体重；BW initial表示分组给药时的动物体重。
瘤重抑瘤率IR(％)的计算公式为：IR(％)＝100％×(W C－W T)/W C
其中，W C表示对照组瘤重；W T表示治疗组瘤重。
试验数据用Microsoft Office Excel 2007软件进行计算和相关统计学处理。数据除特别说明外，用均数±标准误(Mean±SE)表示，组间比较采用t-检验，P<0.05为显著性差异。
实施例15 系列化合物在人结肠癌HT-29细胞株裸鼠移植瘤模型体内药效评价
供试品：伊立替康、nktr-102、本发明12个化合物。
试剂：McCoy’s 5A培养液，胎牛血清(FBS)，胰蛋白酶，青-链双抗，注射用水，生理盐水，乳酸，山梨糖醇。
实验动物：雌性BALB/c裸小鼠(只数：150只；周龄：6～7周)从北京维通利华实验动物技术有限公司购买，饲养于SPF动物房，温度20～25℃，相对湿度40％～70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
移植性肿瘤瘤株：人结肠癌细胞HT-29，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验室液氮冻存)。
HT-29细胞培养：在5％CO 2、37℃培养条件下，HT-29细胞在含10％胎牛血清McCoy’s 5A培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代2到3次，传代比例为1:4到1:6。
动物模型制备：收取对数生长期HT-29细胞，细胞计数后重悬于无血清McCoy’s 5A培养基中，调整细胞浓度至4×10 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(4×10 6细胞/只)，建立HT-29裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤 体积达到100～300mm 3，挑选健康状况良好、肿瘤体积相近的动物90只，采用随机区组法分为15组(n＝6)。实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
溶剂配制：称取0.5g山梨糖醇装入50mL离心管中，在离心管中加入50mL注射用水，涡旋振荡使固体物质完全溶解，配制成浓度1％的山梨糖醇水溶液(w/v)，保存于4℃冰箱备用。
伊立替康给药制剂配制：称取12.0mg的伊立替康，加入0.15mL的1％乳酸，涡旋振荡使药物完全溶解，再分别加入2.85mL的1％山梨糖醇水溶液，涡旋振荡混合均匀，溶液中1％乳酸、1％山梨糖醇水溶液的比例约为5:95(v/v)。溶液中伊立替康有效浓度为4.0mg·mL -1。
nktr-102给药制剂配制：每次给药前，准确称量101.5mg的nktr-102，加入2.5mL的生理盐水，涡旋振荡使药物完全溶解，溶液中伊立替康有效浓度为4.0mg·mL -1。
本发明化合物给药制剂配制：每次给药前，分别准称量120.3mg的化合物a和化合物A、137.0mg的化合物b和化合物B、132.6mg的化合物c和化合物C、132.0mg的化合物d和化合物D、159.6mg的化合物e和化合物E、145.9mg的化合物f和化合物F，加入2.5mL的生理盐水，涡旋振荡，超声(如需要)使药物完全溶解，溶液中伊立替康有效浓度为4.0mg·mL -1。
动物分组及给药方案：于分组当天开始第一次给药，21天左右后结束实验，给药体积均为10mL·kg -1。折合伊立替康的有效剂量均为40mg·kg -1。第一组为溶剂对照组，尾静脉注射给予生理盐水，每4天1次，共给药3次(Q4D×3)。第2-15组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a、化合物A、化合物b、化合物B、化合物c、化合物C、化合物d、化合物D、化合物e、化合物E、化合物f和化合物F，均为每4天给药一次，Q4D×3。
实验最后一天，称量体重、测量瘤径后动物安乐死(CO 2)。剥取肿瘤组织并称重。针对人癌异体移植瘤模型，推荐采用相对肿瘤增殖率(％T/C)作为试验评价指标，相对肿瘤增殖率越低，说明抑制肿瘤效果越良好，结果见表1。
表1 相对肿瘤增值率(％T/C)
Figure PCTCN2018083746-appb-000066
Figure PCTCN2018083746-appb-000067
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，本发明化合物对人结肠癌HT-29裸鼠移植瘤模型肿瘤体内生长有良好抑制作用，且优于伊立替康和nktr-102。
实施例16 人乳腺癌MDA-MB-231裸鼠异种移植模型的抑制作用
供试品：伊立替康、nktr-102、本发明12个化合物。
实验动物：雌性BALB/c裸鼠，接种150只，实验用90只，年龄为6-8周，体重20-22g±20％体重均值，动物来源为上海西普尔-必凯实验动物有限公司(BK)，许可证号SCXK(沪)2008-0016。所有实验动物均饲养在SPF级别实验室。实验人员负责日常护理和实验研究。每个鼠笼均佩挂有实验编号、实验组别、实验人员姓名、小鼠品种和性别等信息的身份卡片，小鼠用耳钉标记。
随机分组：当肿瘤体积达到150-200mm 3后用随机区组法分为15组，每组6只小鼠，保证各组间肿瘤体积和小鼠体重均一。各组肿瘤体积的均值与所有实验动物肿瘤体积的均值差异不超过±10％。
饲养条件：居住条件：IVC系统，每笼6只；温度：20℃-26℃；湿度：40％±70％；光照：12小时昼夜交替。辐照大小鼠饲料，购自北京科澳协力饲料有 限公司。自由进食。饮水为城市自来水，经过滤高压灭菌后饮用。垫料为玉米芯，上海茂生衍生物科技有限公司，高压灭菌后使用。每周换两次垫料。实验前给予小鼠最短一周环境适应期。
其他化学试剂和材料：人乳腺癌MDA-MB-231购于上海中科院细胞生物研究所。
建立人乳腺癌MDA-MB-231裸鼠皮下移植瘤模型，每只接种1×10 6个细胞。给药体积均为10mL·kg -1。折合伊立替康的有效剂量均为40mg·kg -1。第一组为溶剂对照组，尾静脉注射给予生理盐水，每4天1次，共给药3次(Q4D×3)。第2-15组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a、化合物A、化合物b、化合物B、化合物c、化合物C、化合物d、化合物D、化合物e、化合物E、化合物f和化合物F，均为每4天给药一次，Q4D×3。
供试品的配制见实施例15。单次给药需要的体积3mL。
实验方法：MDA-MB-231细胞培养于DMEM，含10％胎牛血清FBS(GIBCO，美国)。细胞放置于5％CO 2培养箱37℃培养。细胞接种法建立肿瘤裸鼠皮下移植模型：收集对数生长期的肿瘤细胞，计数后重悬于1×PBS，调整细胞悬液浓度至1×10 7/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞，1×10 6/0.1ml/鼠。在肿瘤体积达到100-200mm 3时，将动物按随机区组法进行随机分组，分为15组，使各组肿瘤差异小于均值的10％，每组6只，分组当日作为Day1，分组当天给药。实验周期进行3周，实验期间每周测定两次动物体重和肿瘤大小。每日观察记录临床症状。实验最后一天处死动物，称量体重，剥离肿瘤，称重并拍照记录。结果见表2。
表2 相对肿瘤增殖率(％T/C)
Figure PCTCN2018083746-appb-000068
Figure PCTCN2018083746-appb-000069
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，本发明化合物对人乳腺癌MDA-MB-231裸鼠移植瘤有良好抑制作用，且优于伊立替康和nktr-102。
实施例17 对人胰腺癌MIA Paca-2裸鼠异种移植模型的抑制作用
供试品：伊立替康、nktr-102、本发明12个化合物。
实验动物：雌性BALB/c裸鼠，接种150只，实验用90只，年龄为6-8周，体重20-22g±20％体重均值，动物来源为上海西普尔-必凯实验动物有限公司(BK)，许可证号SCXK(沪)2008-0016。所有实验动物均饲养在SPF级别实验室。实验人员负责日常护理和实验研究。每个鼠笼均佩挂有实验编号、实验组别、实验人员姓名、小鼠品种和性别等信息的身份卡片，小鼠用耳钉标记。当肿瘤体积达到150-200mm 3后用随机区组法分为15组，每组6只小鼠，保证各组间肿瘤体积和小鼠体重均一。各组肿瘤体积的均值与所有实验动物肿瘤体积的均值差异不超过±10％。
饲养条件：居住条件为IVC系统，每笼6只；温度：20℃-26℃；湿度：40％±70％；光照：12小时昼夜交替。辐照大小鼠饲料，购自北京科澳协力饲料有限公司。自由进食。饮水为城市自来水，经过滤高压灭菌后饮用。垫料为玉米芯，上海茂生衍生物科技有限公司，高压灭菌后使用。每周换两次垫料。实验前给予小鼠最短一周环境适应期。
其他化学试剂和材料：人胰腺癌MIA Paca-2购于上海中科院细胞生物研究 所。
建立人胰腺癌MIA Paca-2裸鼠皮下移植瘤模型，每只接种3×10 6个细胞。给药体积均为10mL·kg -1。折合伊立替康的有效剂量均为40mg·kg -1。第一组为溶剂对照组，尾静脉注射给予生理盐水，每4天1次，共给药3次(Q4D×3)。第2-15组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a、化合物A、化合物b、化合物B、化合物c、化合物C、化合物d、化合物D、化合物e、化合物E、化合物f和化合物F，均为每4天给药一次，Q4D×3。
供试品的配制见实施例15。单次给药需要的体积3mL。
实验方法：MIA Paca-2细胞培养于DMEM，含10％胎牛血清FBS(GIBCO，美国)和2.5％HS。细胞放置于5％CO 2培养箱37℃培养。
细胞接种法建立肿瘤裸鼠皮下移植模型：收集对数生长期的肿瘤细胞，计数后重悬于1×PBS，调整细胞悬液浓度至3×10 7/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞，3×10 6/0.1ml/鼠。
在肿瘤体积达到100-200mm 3时，将动物按随机区组法进行随机分组，分为15组，使各组肿瘤差异小于均值的10％，每组6只，分组当日作为Day1，分组当天给药。
实验周期进行3周，实验期间每周测定两次动物体重和肿瘤大小。每日观察记录临床症状。实验最后一天处死动物，称量体重，剥离肿瘤，称重并拍照记录。结果见表3。
表3 相对肿瘤增殖率(％T/C)
Figure PCTCN2018083746-appb-000070
Figure PCTCN2018083746-appb-000071
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，本发明化合物对人胰腺癌MIA Paca-2裸鼠移植瘤有良好抑制作用，且优于伊立替康和nktr-102。
实施例18 对人胃癌NCI-N87细胞株裸鼠移植瘤模型肿瘤体内生长的抑制作用。
供试品：伊立替康、nktr-102、本发明12个化合物。
试剂：RPMI-1640培养液，胎牛血清(FBS)，胰蛋白酶，青-链双抗，生理盐水。
实验动物：雌性BALB/c裸小鼠(只数：150只；周龄：6～8周)从北京维通利华实验动物技术有限公司购买，饲养于苏州圣苏新药开发有限公司SPF动物房，温度20～25℃，相对湿度40％～70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
移植性肿瘤瘤株：人胃癌细胞NCI-N87，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验室液氮冻存)。
实验方法
NCI-N87细胞培养：在5％CO 2、37℃培养条件下，NCI-N87细胞在含10％胎牛血清RPMI-1640培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代1到2次，传代比例为1:2到1:6。
动物模型制备：收取对数生长期NCI-N87细胞，细胞计数后重悬于无血清的RPMI-1640培养基中，调整细胞浓度至5×10 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细 胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(5×10 6细胞/只)，建立NCI-N87裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤体积达到100～300mm 3，挑选健康状况良好、肿瘤体积相近的动物90只，采用随机区组法分为15组(n＝6)。实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
供试品的配制见实施例15。单次给药需要的体积3mL。
动物分组及给药：于分组当天开始第一次给药，21天后结束实验，给药体积均为10mL·kg -1。第1组为溶剂对照组，尾静脉注射给予空白溶剂，每4天1次，共给药3次(Q4D×3)。第2-15组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a、化合物A、化合物b、化合物B、化合物c、化合物C、化合物d、化合物D、化合物e、化合物E、化合物f和化合物F，给药剂量均为40mg·kg -1(以伊立替康含量计算)，Q4D×3。
实验结束后，称量体重、测量瘤径后动物安乐死(CO 2)。剥取肿瘤组织并称重。结果见表4。
表4 相对肿瘤增殖率(％T/C)
Figure PCTCN2018083746-appb-000072
Figure PCTCN2018083746-appb-000073
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，本发明化合物对人胃癌NCI-N87细胞株裸鼠移植瘤模型肿瘤生长有良好抑制作用，且优于伊立替康和nktr-102。
实施例19 对U87MG裸鼠脑原位模型生存率的影响。
供试品：伊立替康、nktr-102、本发明12个化合物。
试剂：RPMI-1640培养液，胰蛋白酶，青-链双抗，生理盐水。
实验动物：雌性BALB/c裸小鼠(只数：150只；周龄：6～8周)从北京维通利华实验动物技术有限公司购买，饲养于SPF动物房，温度20～25℃，相对湿度40％～70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
移植性肿瘤瘤株：脑胶质瘤细胞U87MG，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验室液氮冻存)。
实验方法：
NCI-N87细胞培养：在5％CO 2、37℃培养条件下，NCI-N87细胞在RPMI-1640培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代1到2次，传代比例为1:2到1:6。
动物模型制备：收取对数生长期NCI-N87细胞，细胞计数后重悬于无血清的RPMI-1640培养基中，调整细胞浓度至1×10 8细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细胞悬液，借助动物立体定向仪的引导，采用微注射方法将体外培养人脑胶质瘤细胞U87MG细胞1μL(1×10 5细胞/只)，建立U87MG脑胶质瘤原位模型，接种后定期观察动物状态。接种后第12天，挑选动物90只，采用随机区组法分为15组(n＝6)。
给药制剂配制：供试品的配制见实施例15。单次给药需要的体积3mL。
动物分组及给药：于分组当天开始第一次给药，21天后结束实验，给药体积均为10mL·kg -1。第1组为溶剂对照组，尾静脉注射给予空白溶剂，每4天1 次，共给药3次(Q4D×3)。第2-15组分别尾静脉注射给予受试样品伊立替康、nktr-102、受试化合物，给药剂量均为40mg·kg -1(以伊立替康含量计算)，Q4D×3。
数据记录、计算公式：记录动物生存时间。试验数据用Microsoft Office Excel2007软件进行计算和相关统计学处理。两组间比较采用t检验。结果见表5
表5 动物生存时间(天)
Figure PCTCN2018083746-appb-000074
*与空白溶剂、伊立替康和nktr-102组的中位生存时间相比，P＜0.05
实验结果显示，本发明化合物对脑胶质瘤有良好抑制作用，且优于伊立替康和nktr-102。

Claims (17)

  1. 具有如下结构式(Ⅰ)的多支链药物偶联物或其药学上可接受的盐：
    Figure PCTCN2018083746-appb-100001
    R为有机中心，POLY为聚合物，L为多价接头，T为靶向分子，D为活性剂，q为3-8之间的任一整数，其中L为：
    Figure PCTCN2018083746-appb-100002
    符号“*”代表多价接头L与靶向分子T的连接点，“#”代表多价接头L与活性剂D的连接点，“％”代表多价接头L与POLY的连接点，其中，l为2-20之间的任一整数，m、n分别为0-10之间的任一整数；
    T为含有“精氨酸-甘氨酸-天冬氨酸”序列的RGD肽、tLyp-1、Lyp-1、RPARPAR、Angiopep2或GE11；
    D为如式(Ⅱ)所示的喜树碱类药物：
    Figure PCTCN2018083746-appb-100003
    R 1-R 5相互独立地选自于以下基团：氢、卤素、酰基、烷基、取代烷基、烷氧基、取代烷氧基、烯基、炔基、环烷基、羟基、氰基、硝基、叠氮基、酰胺基、肼、胺基、取代胺基、羟基羰基、烷氧羰基、烷氧羰氧基、氨基甲酰氧基、芳基磺酰氧基、烷基磺酰氧基；R 6为H或OR 8，R 8为烷基、烯基、环烷基、卤代烷基或羟烷基；R 7为羟基。
  2. 如权利要求1所述的多支链药物偶联物或其药学上可接受的盐，T为cRGD或iRGD。
  3. 如权利要求1所述的多支链药物偶联物或其药学上可接受的盐，POLY为聚乙二醇。
  4. 如权利要求3所述的多支链药物偶联物或其药学上可接受的盐，POLY为
    Figure PCTCN2018083746-appb-100004
    其中，
    Figure PCTCN2018083746-appb-100005
    代表原子的连接处，标“&”号的氧原子为与有机中心“R”连接的原子，k为50～200范围内的一个整数，r为1-10之间的任一整数。
  5. 如权利要求4所述的多支链药物偶联物或其药学上可接受的盐，其为结构式(Ⅲ)、(Ⅳ)、或(Ⅴ)所示：
    Figure PCTCN2018083746-appb-100006
    Figure PCTCN2018083746-appb-100007
  6. 如权利要求5所述的多支链药物偶联物或其药学上可接受的盐，POLY为
    Figure PCTCN2018083746-appb-100008
  7. 如权利要求6所述的多支链药物偶联物或其药学上可接受的盐，其为：
    Figure PCTCN2018083746-appb-100009
  8. 如权利要求7所述的多支链药物偶联物或其药学上可接受的盐，k具有的平均值为113。
  9. 如权利要求8所述的多支链药物偶联物或其药学上可接受的盐，L为：
    Figure PCTCN2018083746-appb-100010
  10. 如权利要求9所述的多支链药物偶联物或其药学上可接受的盐，D为伊立替康、SN-38、10-羟基喜树碱或鲁比替康。
  11. 如权利要求10所述的多支链药物偶联物或其药学上可接受的盐，其为：
    Figure PCTCN2018083746-appb-100011
    Figure PCTCN2018083746-appb-100012
    Figure PCTCN2018083746-appb-100013
  12. 如权利要求11所述的多支链药物偶联物或其药学上可接受的盐，其为：
    Figure PCTCN2018083746-appb-100014
    Figure PCTCN2018083746-appb-100015
    Figure PCTCN2018083746-appb-100016
  13. 一种药学上可接受的组合物，其包含权利要求1-12任一所述的多支链药物偶联物，以及药学上可接受的赋形剂。
  14. 如权利要求1-12任一所述的多支链药物偶联物在制备用于治疗癌症的药物中的应用。
  15. 如权利要求1-12任一所述的多支链药物偶联物在制备用于治疗结肠癌、肺癌、乳腺癌、卵巢癌、胰腺癌、胃癌、脑胶质瘤及乳房、卵巢、结肠、肾、胆管、肺和脑的恶性肉瘤、癌和淋巴瘤的药物中的应用。
  16. 如权利要求13所述的组合物在制备用于治疗癌症的药物中的应用。
  17. 如权利要求13所述的组合物在制备用于治疗结肠癌、肺癌、乳腺癌、卵巢癌、胰腺癌、胃癌、脑胶质瘤及乳房、卵巢、结肠、肾、胆管、肺和脑的恶性肉瘤、癌和淋巴瘤的药物中的应用。
PCT/CN2018/083746 2017-04-21 2018-04-19 多臂靶向抗癌偶联物 WO2018192550A1 (zh)

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