WO2019001550A1 - 一种适用于点击化学反应的多臂多爪聚乙二醇衍生物 - Google Patents

一种适用于点击化学反应的多臂多爪聚乙二醇衍生物 Download PDF

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WO2019001550A1
WO2019001550A1 PCT/CN2018/093528 CN2018093528W WO2019001550A1 WO 2019001550 A1 WO2019001550 A1 WO 2019001550A1 CN 2018093528 W CN2018093528 W CN 2018093528W WO 2019001550 A1 WO2019001550 A1 WO 2019001550A1
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polyethylene glycol
integer
ethyl
group
carboxylic acid
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PCT/CN2018/093528
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English (en)
French (fr)
Inventor
熊艳丽
汪进良
冯泽旺
郑丽会
赵宣
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北京键凯科技股份有限公司
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Priority claimed from CN201810670204.4A external-priority patent/CN109206610B/zh
Application filed by 北京键凯科技股份有限公司 filed Critical 北京键凯科技股份有限公司
Publication of WO2019001550A1 publication Critical patent/WO2019001550A1/zh
Priority to US16/729,699 priority Critical patent/US11780963B2/en

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    • 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
    • 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
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/48—Polymers modified by chemical after-treatment

Definitions

  • the invention relates to a polyethylene glycol derivative suitable for click chemical reaction, in particular to a multi-arm multi-claw polyethylene glycol derivative having a terminal azide group or a terminal alkynyl group, a preparation method thereof and a preparation method thereof Its application in click chemical reactions.
  • Click chemistry is the use of a series of reliable, efficient, selective, and modular chemical reactions to form heteroatom-containing compounds that allow the attachment of carbon to heteroatoms, which are synthesized using the best chemical reactions. Achieve the desired function to avoid complex chemical reactions.
  • Click chemistry generally has the following characteristics: (1) the raw materials and reagents used are easily available; (2) the reaction conditions are simple, the reaction process is not sensitive to water and oxygen; (3) the yield is high, and there are no or harmless by-products; Good stereoselectivity; (5) simple product purification technology; (6) good product stability.
  • the reaction also includes a heterocyclic Diels-Alder reaction.
  • Polyethylene glycol is a widely used polyether polymer compound, which can be applied to many fields such as medicine, health, food, and chemical industry. PEG can be dissolved in water and many solvents, and the polymer has excellent biocompatibility, can be dissolved in the tissue fluid in the body, and can be quickly excreted by the body without any toxic side effects.
  • Non-patent literature "Synthesis and characterization of terminal azide-based polyethylene glycols” discloses the synthesis of a high molecular weight terminal azido polyethylene glycol The method, however, is a linear polyethylene glycol in the polymer prepared by the method, which has only one azide group supported on the PEG molecule.
  • Patent Document CN101787135A also discloses a method for synthesizing a polyethylene glycol having a linear structure of a terminal azide, which comprises reacting PEG with methanesulfonyl chloride and then adding sodium azide to react, which is also only on the PEG molecule. Bonding an azide group.
  • Patent document CN104497303A provides a multi-arm polyethylene glycol-azide derivative
  • patent document CN104448296A provides an alkyne-based multi-arm polyethylene glycol derivative
  • the polyethylene glycol derivatives of the above patents have A plurality of end groups, which in turn have multiple functional groups, can carry a plurality of different reactive end groups.
  • the number of azide or alkynyl groups bonded in the above polyethylene glycol derivatives is still small, and it is not sufficient to generate sufficient reaction sites when performing a click chemical reaction.
  • the present invention provides an end group azido polyethylene glycol derivative or a terminal alkynyl polyethylene glycol derivative which is particularly suitable for use in a click chemical reaction.
  • the invention provides an end group azido or terminal alkynyl polyethylene glycol derivative and a preparation method thereof, which solves the problem that the terminal azide group or the terminal alkynyl group in the polyethylene glycol derivative is less
  • the defect that the reaction site is insufficient in the click reaction is particularly suitable for bonding the drug molecule by a click reaction method.
  • the invention provides a polyethylene glycol derivative suitable for use in a click chemistry having the structure of Formula I:
  • R is a polyethylene glycol residue having a linear structure, a Y-type structure or a multi-branched structure
  • P is a terminal group of a non-azide non-alkynyl group
  • R 1 , R 2 and R 3 are a linking group independently selected from: -(CH 2 ) i -, -(CH 2 ) i O(CH 2 ) i -, -(CH 2 ) i O (CH 2 ) i CONH (CH 2) i -, - (CH 2) i NH -, - (CH 2) i OCOO -, - (CH 2) i OCONH -, - (CH 2) i NHCO -, - (CH 2 i ) one or more of NHCOO-, -(CH 2 ) i NHCONH-, -OC(CH 2 ) i COO-, -(CH 2 ) i COO- and -(CH 2 ) i CONH- Combination; i is an integer from 0 to 10 (specifically, such as 0, 1, 2, 3, 4, 6, 8, or 10);
  • D is -N 3 or -C ⁇ CH
  • l an integer selected from 1 to 20 (specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20);
  • n is selected from an integer from 0 to 19 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, 17, or 19).
  • the P is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, aldehyde, hydroxyl, amino, carboxyl, thiol, ester, maleimide
  • the R is a linear polyethylene glycol residue having the structure represented by the formula (R-1):
  • n is an integer of from 3 to 250 (specifically, such as 3, 20, 50, 100, 150, 200 or 250, etc.).
  • the R is a Y-type polyethylene glycol residue having a structure represented by the formula (R-2) or (R-3):
  • h and f are independently selected from an integer of from 3 to 250 (specifically, such as 3, 20, 50, 100, 150, 200 or 250, etc.).
  • the R is a multi-branched polyethylene glycol residue having the structure represented by the formula (R-4):
  • t is an integer from 3 to 250 (specifically, such as 3, 20, 50, 100, 150, 200 or 250, etc.)
  • j is an integer of 3-8 (specifically, 3, 4, 5, 6, 7, or 8),
  • R c is a core molecule of a multi-branched polyethylene glycol selected from the group consisting of: pentaerythritol, oligo-pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol, and polyglycerol residues;
  • the multi-branched polyethylene glycol residue has the structure:
  • p is an integer from 3 to 250 (specifically, such as 3, 20, 50, 100, 150, 200 or 250, etc.)
  • x is an integer from 1 to 18 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18);
  • the multi-branched polyethylene glycol residue has the structure:
  • q is an integer from 3 to 250 (specifically, such as 3, 20, 50, 100, 150, 200 or 250, etc.)
  • Y is an integer from 1 to 9 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, or 9);
  • the y is 1 or 3.
  • the 1 is an integer selected from 3 to 8, and more specifically, the 1 is 4, 6, or 8.
  • the m is an integer of 0 to 7, and more specifically, the m is 0.
  • the x is an integer of 1-3, and more specifically, the x is 1, 2 or 3.
  • the y is an integer of 1-3, and more specifically, the y is 1, 2 or 3.
  • the P is selected from the group consisting of -NH 2 , -COOH, -OCH 3 , with
  • the non-azide non-alkynyl group of terminal groups P and R may further include a linking group, and the linking group is selected from the group consisting of: -(CH 2 ) k -, -(CH 2 ) k O(CH 2 ) k -, -(CH 2 ) k O(CH 2 ) k CONH(CH 2 ) k -, -(CH 2 ) k NH- , - (CH 2) k OCOO -, - (CH 2) k OCONH -, - (CH 2) k NHCO -, - (CH 2) k NHCOO -, - (CH 2) k NHCONH -, - OC (CH 2) k COO -, - k CONH- combination of one or two or more of the (CH 2) - (CH 2 ) k COO- and; K is an integer of 0-10 (particularly,
  • the i is 1, 2 or 3; more specifically, the R 1 , R 2 and R 3 are independently selected from -(CH 2 ) i -, -(CH 2 ) i O(CH 2 ) i - or -(CH 2 ) i O(CH 2 ) i CONH(CH 2 ) i -; i is 1, 2 or 3.
  • the polyethylene glycol derivative has a molecular weight of 1000 to 80,000 Da; specifically, the polyethylene glycol derivative has a molecular weight of 3,000 to 40,000 Da; more specifically, the poly The molecular weight of the ethylene glycol derivative may be 3000 Da, 5000 Da, 10000 Da, 20000 Da or 40,000 Da.
  • the polyethylene glycol derivative of the invention has the structure of formula II:
  • the polyethylene glycol derivative of the invention has the structure of formula III:
  • the R 1 , R 2 and R 3 are -(CH 2 ) i -, and i is an integer of 1-10; specifically, the i is 1, 2 or 3.
  • said R 1 , R 2 and R 3 are -(CH 2 ) i O(CH 2 ) i - or -(CH 2 ) i O(CH 2 ) i CONH(CH 2 ) i -, i is an integer from 1 to 10; in particular, the i is 1, 2 or 3.
  • Another aspect of the present invention provides a method for producing the above polyethylene glycol derivative of the present invention, which comprises: (1) a polyethylene glycol or a derivative thereof having a linear structure, a Y-type structure or a multi-branched structure. Reaction with ethyl chloroformate to obtain ethyl glycol carboxylate; (2) reaction of ethyl polyethylene glycolate with tris(hydroxymethyl)aminomethane to give N-[2-hydroxy-1,1- Bis(hydroxymethyl)ethyl]-polyethylene glycol carboxylic acid amide; (3) N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-polyethylene glycol carboxylic acid amide Reaction with methanesulfonyl chloride to give N-[2-methanesulfonyloxy-1,1-bis(methanesulfonyloxymethyl)ethyl]-polyethylene glycol carboxylic acid amide; (4) N-[2 - Methanes
  • Another aspect of the present invention provides a method for producing the above polyethylene glycol derivative of the present invention, which comprises: (1) a polyethylene glycol having a linear structure, a Y-type structure or a multi-branched structure or a derivative thereof Reaction with ethyl chloroformate to obtain ethyl glycol carboxylate; (2) reaction of ethyl polyethylene glycolate with tris(hydroxymethyl)aminomethane to give N-[2-hydroxy-1,1 - bis(hydroxymethyl)ethyl]-polyethylene glycol carboxylic acid amide; (3') N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-polyethylene glycol carboxylate The acid amide is reacted with a bromoalkyne to give a terminal alkynyl polyethylene glycol derivative.
  • Another aspect of the present invention provides a method for producing the above polyethylene glycol derivative of the present invention, which comprises: (1) a polyethylene glycol having a linear structure, a Y-type structure or a multi-branched structure or a derivative thereof Reaction with ethyl chloroformate to obtain ethyl glycol carboxylate; (2) reaction of ethyl polyethylene glycolate with tris(hydroxymethyl)aminomethane to give N-[2-hydroxy-1,1 - bis(hydroxymethyl)ethyl]-polyethylene glycol carboxylic acid amide; (3") N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-polyethylene glycol carboxylate The acid amide is reacted with t-butyl bromoalkylcarboxylate to obtain a polyethylene glycol carboxylic acid amide tris(tert-butyl oxyacetate); (4") a polyethylene glycol carboxylic acid amide tris (o- oxyacetic acid tert
  • the step (1) comprises: a polyethylene glycol or a derivative thereof having a linear structure, a Y-structure or a multi-branched structure, and potassium carbonate Adding to N,N-dimethylformamide, adding ethyl chloroformate dropwise with stirring, heating to 80-100 ° C, preferably 90 ° C, stirring the reaction, after the reaction is completed, the mixture is cooled to room temperature and poured into ice water. Stir well, extract with dichloromethane, wash, dry, filter and concentrate the crystals.
  • the step (2) comprises: adding the product of the step (1), tris(hydroxymethyl)aminomethane and potassium carbonate to the dimethyl group.
  • the temperature is raised to 30-50 ° C, preferably 40 ° C, and the reaction is stirred.
  • the mixture is cooled to room temperature, poured into ice water, stirred uniformly, extracted with dichloromethane, washed with an extract, dried, and filtered. Concentrated crystals.
  • the step (3) comprises: heating the product of the step (2) and toluene to reflux, and cooling to room temperature, then adding dichloromethane and triethyl ether.
  • the amine was stirred well, cooled with ice water, and then added dropwise with methanesulfonyl chloride. After the reaction was completed, ethanol was added, stirred, filtered, and concentrated to crystallize.
  • the step (4) comprises: heating the product of the step (3), sodium azide and N,N-dimethylformamide.
  • the reaction 2-8 hours, preferably 5 hours, after cooling to room temperature add water and sodium chloride solution, stir to dissolve, extract with dichloromethane, combine the organic phase, dry, filter After concentration, the residue was cooled and precipitated with diethyl ether to give a product.
  • the step (3') comprises: dissolving the product of the step (2) in tetrahydrofuran, adding sodium hydride, reacting at room temperature for half an hour, adding The bromo alkyne and potassium iodide are heated for 1-4 hours, preferably for 1-4 hours. After cooling, water is added, the tetrahydrofuran is removed by concentration, and the residue is extracted with dichloromethane to give a final product. More preferably, the bromoalkyne is selected from the group consisting of bromoacetylene, bromopropyne and bromobutyne.
  • the step (3") comprises: dissolving the product of the step (2) in dimethylformamide (DMF), and adding sodium hydride.
  • the reaction is carried out at room temperature for 0.5-2 hours, preferably 0.5 hours, and then a reaction of t-butyl bromoalkylcarboxylate is added.
  • water is added to obtain N-(trimethylol)-tetra-arm polyethylene glycol carboxylic acid amide- More preferably, the tert-butyl bromoalkylcarboxylate is selected from the group consisting of t-butyl bromoacetate and t-butyl bromoacetate.
  • the step (4" comprises: dissolving the step (3") in methanol, adding a sodium hydroxide solution, heating the hydrolysis, preferably the hydrolysis temperature. 40-80 ° C, hydrolysis time 2-6 hours, more preferably, the hydrolysis temperature is 60 ° C, hydrolysis time 4 hours, to obtain N- (trimethylol) - four-arm polyethylene glycol carboxylic acid amide - three Alkyl carboxylic acid, N-(trimethylol)-tetra-arm polyglycol carboxylic acid amide-trialkyl carboxylic acid and N-hydroxysuccinimide are dissolved in dichloromethane, and N, N'- Dicyclohexylcarbodiimide, reacting for 2-6 hours, preferably 4 hours, is further reacted with an amine alkyne to give the final product. More preferably, the amine alkyne is selected from the group consisting of: propargyl
  • the polyethylene glycol derivative of the invention can be used in combination with proteins, peptides and pharmaceutically active small molecules to improve targeting and drug efficacy and reduce toxicity.
  • the protein, peptide, and pharmaceutically active small molecule include, but are not limited to, analgesic and anti-inflammatory agents, antacids, anthelmintics, antiarrhythmic agents, antibacterial agents, anticoagulant (blood) agents, antidepressants , antidiabetic agents, antidiarrheal agents, antiepileptic drugs, antifungal agents, anti-gout drugs, antihypertensive drugs, antimalarials, anti-migraine drugs, antimuscarinic agents, antineoplastic agents and immunosuppressants, antigens Insecticides, antirheumatic drugs, antithyroid agents, antiviral agents, anti-anxiety agents, sedatives, eye drops and tranquilizers, beta-blockers, cardiac contractions, corticosteroids, antitussives, cytotoxicity Agent
  • the method for binding a polyethylene glycol derivative of the formula I according to the present invention to a click chemical reaction with a protein, a peptide or a pharmaceutically active small molecule can be formed by a terminal group and a drug molecule as described in the patent CN102108119A. Conjugate.
  • the invention also provides the use of a polyethylene glycol derivative as described above in a click chemistry reaction.
  • the invention also provides the use of the above polyethylene glycol derivative for modifying a drug molecule.
  • the drug molecule includes amino acids, polypeptides, proteins, sugars, organic acids, alkaloids, flavonoids, terpenoids, terpenoids, phenylpropanoid phenols, steroids and steroids.
  • the drug molecule is a local anesthetic such as an amide local anesthetic, more specifically, such as lidocaine, prilocaine, bupivacaine, ropivacaine, mepivacaine Or eucaine and so on.
  • a local anesthetic such as an amide local anesthetic, more specifically, such as lidocaine, prilocaine, bupivacaine, ropivacaine, mepivacaine Or eucaine and so on.
  • the drug molecule is bupivacaine.
  • the drug molecule is an antitumor drug molecule, such as a natural plant antitumor drug, such as camptothecin and its derivatives, such as camptothecin, topotecan, and ezetidine.
  • a natural plant antitumor drug such as camptothecin and its derivatives, such as camptothecin, topotecan, and ezetidine.
  • the drug molecule is irinotecan.
  • the present invention also provides a conjugate of the above polyethylene glycol derivative and a drug molecule having a structure of the general formula IV:
  • TX is or
  • X 1 , X 2 and X 3 are linking groups independently selected from: -(CH 2 ) a -, -(CH 2 ) a NH-, -(CH 2 ) a NHCO-, -(CH 2 ) a CONH-, -(CH 2 ) a CO-, -(CH 2 ) a COO-, -(CH 2 ) a OCO-, -(CH 2 ) a SC(O)-, -(CH 2 ) a O- , -(CH 2 ) a S-, C 3 -C 10 cycloalkyl, substituted or substituted aryl and substituted or unsubstituted heterocyclic group, or a combination of two or more, a is 0- An integer of 10,
  • Q 1 , Q 2 and Q 3 are the same or different drug molecule residues.
  • the Q 1 , Q 2 and Q 3 are the same.
  • the drug molecule is a local anesthetic such as an amide local anesthetic, more specifically, eg, lidocaine, prilocaine, bupivacaine, ropivacaine, mepivaca Because of or ecicaine.
  • a local anesthetic such as an amide local anesthetic, more specifically, eg, lidocaine, prilocaine, bupivacaine, ropivacaine, mepivaca Because of or ecicaine.
  • the Q 1 , Q 2 and Q 3 are the same, both of which are bupivacaine residues, and specifically have the following structure:
  • R 0 - is an anion such as F - , Cl - , Br - , I - , mesylate, ethyl sulfonate, benzene sulfonate, citrate, lactate, succinate, fumarate, valley Lysine, citrate, salicylate and maleate.
  • said Q 1 , Q 2 and Q 3 are both local anesthetic residues, said X 1 , X 2 and X 3 being independently selected from the following structures:
  • the drug molecule is an antitumor drug molecule, such as a natural plant antitumor drug, such as camptothecin and its derivatives, such as camptothecin, topotecan, and ezetidine.
  • a natural plant antitumor drug such as camptothecin and its derivatives, such as camptothecin, topotecan, and ezetidine.
  • the Q 1 , Q 2 and Q 3 are the same, both are irinotecan residues, and specifically, have the following structure:
  • the Q 1 , Q 2 and Q 3 are anti-tumor drug molecule residues
  • the X 1 , X 2 and X 3 are independently selected from: -(CH 2 ) a - , -(CH 2 ) a NH-, -(CH 2 ) a NHCO-, -(CH 2 ) a CONH-, -(CH 2 ) a CO-, -(CH 2 ) a COO-, -(CH 2 a OCO- and -(CH 2 ) a O-, a is an integer from 0 to 5.
  • said X 1 , X 2 and X 3 are -(CH 2 ) a CO-, such as -CH 2 CO-.
  • the conjugate has the structure of Formula V:
  • x, p, TX, R 1 , R 2 , R 3 , X 1 , X 2 , X 3 , Q 1 , Q 2 and Q 3 have the above corresponding definitions of the invention.
  • the conjugate has the structure of Formula VI:
  • q, y, TX, R 1 , R 2 , R 3 , X 1 , X 2 , X 3 , Q 1 , Q 2 and Q 3 have the above corresponding definitions of the invention.
  • the above formula V the x is 1, 2, 3, 4, 5 or 6.
  • the y is 1, 2 or 3.
  • the Q 1 , Q 2 and Q 3 are the same, all of which are bupivacaine residues, such as
  • the Q 1 , Q 2 and Q 3 are the same, both are irinotecan residues, such as
  • the conjugate has the structure:
  • R 0 - is F - , Cl - , Br - or I - .
  • the conjugate has the structure:
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above conjugate of the invention and a pharmaceutically acceptable carrier or excipient.
  • the above pharmaceutical compositions will comprise from about 1 to about 99% by weight of the above composition of the invention, and from 99 to 1% by weight of a suitable carrier or pharmaceutically acceptable, depending on the mode of administration desired.
  • Shape agent Preferably, the composition comprises from about 5 to 75% by weight of the above composition of the invention, the balance being a suitable carrier or pharmaceutically acceptable excipient. More preferably, the composition comprises from about 10 to 50% by weight of the above composition of the invention, the balance being a suitable carrier or pharmaceutically acceptable excipient.
  • the above pharmaceutical composition may further comprise a small amount of auxiliary substances such as a wetting or emulsifying agent, an antioxidant, etc., for example: sorbitan monolaurate, triethanolamine oleate, butyl Base hydroxytoluene and the like.
  • auxiliary substances such as a wetting or emulsifying agent, an antioxidant, etc., for example: sorbitan monolaurate, triethanolamine oleate, butyl Base hydroxytoluene and the like.
  • the pharmaceutical composition is a tablet, a capsule, a pill, a granule, a powder, a suppository, an injection, a solution, a suspension, a paste, a patch, a lotion, a drop, and a wipe.
  • Dosage forms such as agents and sprays.
  • the above pharmaceutical compositions may be carried out using any of the accepted modes of administration or agents for similar uses.
  • the mode of administration employed may be selected by oral, intranasal, parenteral, topical, transdermal or rectal administration in the form of a solid, semi-solid or liquid pharmaceutical form, for example, tablets, suppositories, pills, Soft and hard gelatin capsules, powders, solutions, suspensions, injections and the like are preferably employed in unit dosage forms suitable for simple administration of precise dosages.
  • a pharmaceutical composition for administration in a liquid form for example, by dissolving or dispersing or dispersing or dispersing the above-mentioned composition of the present invention (about 0.5 to about 20%) and a selectively present pharmaceutical adjuvant in a carrier.
  • a solution or suspension or the like examples of the carrier are selected from the group consisting of water, saline, aqueous glucose, glycerol, ethanol, and the like.
  • the present invention also provides the use of the above conjugate of the present invention and a pharmaceutical composition thereof for the preparation of a medicament for preventing and/or treating a disease.
  • the disease is pain, in particular the pain is chronic pain, such as post-operative pain.
  • the disease is a tumor, such as colorectal cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, colon cancer, rectal cancer, liver cancer, acute and chronic myeloid leukemia, and villi.
  • a tumor such as colorectal cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, colon cancer, rectal cancer, liver cancer, acute and chronic myeloid leukemia, and villi.
  • the polyethylene glycol derivative of the invention has a plurality of azide end groups or alkynyl end groups, which can not only increase the loading rate of the active end groups, but also enhance the stability of the azide end group or the alkynyl end group. Safety, significantly increased drug loading in the application of modified drugs. Therefore, the polyethylene glycol derivative provided by the invention has greater flexibility and a wider application range, and has good application prospects in organic synthesis, drug synthesis and medical devices.
  • Alkyl refers to a hydrocarbon chain radical that is linear or branched and that does not contain an unsaturated bond, and that is linked to the rest of the molecule by a single bond.
  • Typical alkyl groups contain from 1 to about 12, from 1 to about 8, or from 1 to about 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl , tert-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, n-hexyl, isohexyl and the like.
  • Alkylene refers to a hydrocarbon chain radical that is linear or branched and contains no unsaturated bonds, and that is linked to the other two moieties of the molecule by a single bond.
  • Typical alkylene group containing 1 to about 12, 1 to about 8, or from 1 to about 6 carbon atoms, such as methylene (-CH 2 -), ethylene (-CH 2 CH 2 - , propylene (-CH 2 CH 2 CH 2 - or -CH(CH 3 )CH 2 -), and the like.
  • Alkoxy refers to a substituent formed by substituting a hydrogen in a hydroxy group with an alkyl group, such as a C1-C6 alkoxy group, such as methoxy, ethoxy, propoxy, butoxy, and the like.
  • Aryl means a monocyclic or polycyclic radical, including polycyclic radicals containing a monoaryl group and/or a fused aryl group, such as containing 1-3 monocyclic or fused rings and 6- 18 carbon ring atoms, such as an aryl group of C6-C12, such as phenyl, naphthyl, biphenylyl, fluorenyl, etc.
  • the aryl group is a phenyl group.
  • Cycloalkyl means an alicyclic hydrocarbon such as containing from 1 to 4 monocyclic and/or fused rings containing from 3 to 18 carbon atoms, preferably from 3 to 10 carbon atoms, such as cyclopropyl, cyclohexyl or Adamantyl and the like.
  • Heterocyclyl includes heteroaromatic groups and heteroalicyclic groups containing from 1 to 3 monocyclic and/or fused rings and from 3 to about 18 ring atoms, as specified, for example, from 5 to about 10 ring atoms.
  • Heteroaromatic groups and heteroalicyclic groups; in the present invention, suitable heteroaryl groups contain 1, 2 or 3 heteroatoms selected from N, O or S atoms.
  • some specific groups and chemical structures involved in the present invention correspond to the following: hydroxyl group, -OH; aldehyde group, -CHO; amino group, -NH 2 ; maleimide group, carboxyl, Ester group, or (wherein G 1 may be an alkyl group, an aryl group or a heterocyclic group such as methyl, ethyl, n-propyl, t-butyl, maleimide, succinimidyl, Etc.); sulfhydryl, -SH; acrylic, Succinimide group, Dithiopyridyl, eg, 2-pyridyldithio 4-pyridyldithio Decyl ester (wherein G 2 may be an alkyl group such as methyl, ethyl, n-propyl, t-butyl, etc.); acrylate group (acryloyloxy) Acyl hydrazino, Isocyanate group, Silyl
  • the multi-arm multi-claw polyethylene glycol and its derivatives, the combination with the drug molecule, and the pharmaceutical composition comprising the same according to the present invention are described below with reference to the examples, which are not intended to limit the present invention, and the scope of protection of the present invention is defined by the claims. Request for a limit. Unless otherwise stated, the polyethylene glycol used in the examples was supplied by Beijing Key Kai Technology Co., Ltd., and other reagents were purchased from Beijing Chemical Reagent Company.
  • N-[2-azido-1,1-bis(azidomethyl)ethyl]-tetra-arm polyethylene glycol (10K) carboxylic acid amide and bupivacaine conjugate (L1, examples 13 preparation) about 50mg, accurately weighed, placed in a 30mL volumetric flask, add 0.1mol / L hydrochloric acid 10mL dissolved, shake, tampon, cap, placed in a constant temperature drying oven at 60 ° C for 20 hours, let cool, as a supply Sample solution.
  • tert-Butyl bromoacetate (5.82 g, 30 mmol) was added to a reaction flask, dissolved with acetone (80 mL), and then a solution of sodium azide (4.55 g, 70mmoL) dissolved in water (40 mL) was added and heated to reflux overnight. The reaction mixture was evaporated to dryness. EtOAc was evaporated. This liquid was dissolved in methanol (90 mL), and then 1N sodium hydroxide solution (90 mL) was added, and the mixture was stirred and heated to reflux for 3 h.
  • Azidoacetic acid (2.5 g, 25 mmoL) and irinotecan (14.7 g, 25 mmoL) were added to the reaction flask, dissolved in dichloromethane, cooled in an ice bath, and then added with 4-dimethylaminopyridine (DMAP, 6.1 g, 50 mmoL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 10.3 g, 50 mmol) were added to the reaction flask, and stirring was continued at room temperature overnight. After the reaction mixture was concentrated, the residue was purified by column chromatography to yield 14.2 g of irinotecan acetate (Y2), yield 57%, MS m/z: 692 [M+Na] + .
  • DMAP 4-dimethylaminopyridine
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro
  • irinotecan hydrochloride 25mg, placed in a 25mL volumetric flask, dissolved in water and diluted to the scale, accurately measured 2mL in a 25mL volumetric flask, and then added 0.005mol / L sodium hydroxide solution 20mL, The mixture was heated in a water bath at 40 ° C for 30 minutes with the test solution, allowed to cool, diluted to a mark with a 0.04 mol/L hydrochloric acid solution, and shaken to obtain a standard solution.
  • the content of irinotecan in the sample L2 was 28.1% as determined by high performance liquid chromatography.

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Abstract

本发明提供一种通式Ⅰ的适用于点击化学反应的多臂多爪聚乙二醇衍生物，其中，R 为具有直链结构、Y 型结构或多分支结构的聚乙二醇残基, R1、R2和 R3 为连接基团, P 为非叠氮非炔基的端基基团, D 为- N3 或- C≡CH, l为选自1-20 的整数, m 为选自0-19 的整数. (I)

Description

一种适用于点击化学反应的多臂多爪聚乙二醇衍生物 技术领域
本发明涉及一种适用于点击化学反应的聚乙二醇衍生物，尤其是涉及一种具有端基叠氮基或端基炔基的多臂多爪聚乙二醇衍生物、其制备方法及其在点击化学反应中的应用。
背景技术
点击化学是利用一系列可靠的、高效的、选择性的而又具模块化的化学反应生成含杂原子的化合物，从而实现碳与杂原子的连接，是用最佳的化学反应合成的分子来实现期望得到的功能，从而避免复杂的化学反应。点击化学一般具备以下特点：(1)所用原料和试剂容易获得；(2)反应条件简单，反应过程对水和氧气不敏感；(3)产率高，没有或有无害的副产品；(4)立体选择性好；(5)产物净化技术简单；(6)产物稳定性好。点击反应主要有4种类型，其中最常见的是环加成反应，特别是在一价铜盐催化下的有机叠氮和炔基或叠氮和腈基之间1,3-偶极环加成反应，也包括杂环Diels-Alder反应。
聚乙二醇(PEG)是一种用途极为广泛的聚醚高分子化合物，它可应用于医药、卫生、食品、化工等众多领域。PEG能够溶解于水和许多溶剂中，且该聚合物具有优异的生物相容性，在体内能溶于组织液中，能被机体迅速排出体外而不产生任何毒副作用。
非专利文献“端叠氮基聚乙二醇的合成和表征”(王晓红等，高分子学报，2000年6月，第3期)公开了一种高分子量端叠氮基聚乙二醇的合成方法，然而该方法制备得到的聚合物中为直链聚乙二醇，其仅在PEG分子负载有一个叠氮基团。
专利文献CN101787135A同样公开了一种端叠氮基化线性结构的聚乙二醇的合成方法，包括将PEG与甲基磺酰氯反应后，加入叠氮钠反应得到，其同样仅能在PEG分子上键合一个叠氮基团。
专利文献CN104497303A提供的一种多臂聚乙二醇-叠氮衍生物，专利文献CN104448296A提供了一种炔基多臂聚乙二醇衍生物，上述专利攻来的聚乙二醇衍生物均具有多个端基，进而具有多个功能基团的引入点、可以负载多个不同的活性端基。然而上述聚乙二醇衍生物中所键合的叠氮或炔基基团的数量仍然较少，不能够满足进行点击化学反应时产生足够多的反应位点。
为克服现有技术中的缺陷，本发明提供一种特别适用于点击化学反应的端基叠氮基聚乙二醇衍生物或端基炔基聚乙二醇衍生物。
发明内容
本发明提供了一种端基叠氮基或端基炔基聚乙二醇衍生物及其制备方法，解决了聚乙二醇衍生物中端基叠氮基或端基炔基基团较少，在进行点击反应时反应位点不足的缺陷，特别适用于通过点击反应法键合药物分子。
本发明一方面提供一种适用于点击化学反应的聚乙二醇衍生物，其具有通式Ⅰ的结构：
Figure PCTCN2018093528-appb-000001
其中：
R为具有直链结构、Y型结构或多分支结构的聚乙二醇残基；
P为非叠氮非炔基的端基基团；
R 1、R 2和R 3为连接基团，独立地选自：-(CH 2) i-、-(CH 2) iO(CH 2) i-、-(CH 2) iO(CH 2) iCONH(CH 2) i-、-(CH 2) iNH-、-(CH 2) iOCOO-、-(CH 2) iOCONH-、-(CH 2) iNHCO-、-(CH 2) iNHCOO-、-(CH 2) iNHCONH-、-OC(CH 2) iCOO-、-(CH 2) iCOO-和-(CH 2) iCONH-中的一种或两种以上的组合；i为0-10的整数(具体如0、1、2、3、4、6、8或10)；
D为-N 3或-C≡CH；
l选自1-20的整数(具体2、3、4、5、6、7、8、9、10、12、14、16、18或20)；
m选自0-19的整数(具体如1、2、3、4、5、6、7、8、9、11、13、15、17或19)。
在本发明的一个实施方式，所述P选自：H、C1-C6的烷基、C1-C6的烷氧基、醛基、羟基、氨基、羧基、巯基、酯基、马来亚酰胺基、丙烯酸基、琥珀酰亚胺基、二硫吡啶基、巯酯基、丙烯酰氧基、酰肼基、异氰酸基、硅烷基、乙烯砜基和维生素H残基。
在本发明一具体实施方式中，所述R为直链聚乙二醇残基，其具有式(R-1)所示结构：
Figure PCTCN2018093528-appb-000002
其中，n为3-250的整数(具体如3、20、50、100、150、200或250等)。
在本发明一具体实施方式中，所述R为Y型聚乙二醇残基，其具有式(R-2)或(R-3)所示的结构：
Figure PCTCN2018093528-appb-000003
Figure PCTCN2018093528-appb-000004
其中，h和f独立地选自3-250的整数(具体如3、20、50、100、150、200或250等)。
在本发明一具体实施方式中，所述R为多分支聚乙二醇残基，其具有式(R-4)所示的结构：
Figure PCTCN2018093528-appb-000005
其中，t为3-250的整数(具体如3、20、50、100、150、200或250等)，
j为3-8的整数(具体如3、4、5、6、7或8)，
R c为多分支聚乙二醇的核心分子，选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基；
m和l之和等于j。
在本发明一个实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2018093528-appb-000006
其中，p是3-250的整数(具体如3、20、50、100、150、200或250等)，
x为1-18的整数(具体如，1、2、3、4、5、6、7、8、9、10、12、14、16或18)；
m和l之和等于x+2。
在本发明另一个实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2018093528-appb-000007
其中，q为3-250的整数(具体如3、20、50、100、150、200或250等)，
y为1-9的整数(具体如，1、2、3、4、5、6、7、8或9)；
m和l之和等于2y+2。
在本发明的一个实施例中，所述y为1或3。
具体地，本发明所述的聚乙二醇衍生物中，所述l为选自3-8的整数，更具体地，所述l为4、6或8。
具体地，本发明所述的聚乙二醇衍生物中，所述m为0-7的整数，更具体地，所述m为0。
具体地，本发明所述的聚乙二醇衍生物中，所述x为1-3的整数，更具体地，所述x为1、2或3。
具体地，本发明所述的聚乙二醇衍生物中，所述y为1-3的整数，更具体地，所述y为1、2或3。
具体地，本发明所述的聚乙二醇衍生物中，所述P选自以下基团：-NH 2、-COOH、-OCH 3、
Figure PCTCN2018093528-appb-000008
和
Figure PCTCN2018093528-appb-000009
具体地，本发明所述的聚乙二醇衍生物中，所述非叠氮非炔基的端基基团P与R之间还可以包括连接基团，所述的连接基团选自：-(CH 2) k-、-(CH 2) kO(CH 2) k-、-(CH 2) kO(CH 2) kCONH(CH 2) k-、-(CH 2) kNH-、-(CH 2) kOCOO-、-(CH 2) kOCONH-、-(CH 2) kNHCO-、-(CH 2) kNHCOO-、-(CH 2) kNHCONH-、-OC(CH 2) kCOO-、-(CH 2) kCOO-和-(CH 2) kCONH-中的一种或两种以上的组合；k为0-10的整数(具体如0、1、2、3、4、6、8或10)。
具体地，本发明所述的聚乙二醇衍生物中，所述i为1、2或3；更具体地，所述R 1、R 2和R 3独立的选自-(CH 2) i-、-(CH 2) iO(CH 2) i-或-(CH 2) iO(CH 2) iCONH(CH 2) i-；i为1、2或3。
在本发明具体的实施方式中，所述聚乙二醇衍生物的分子量为1000-80000Da；具体地，所述聚乙二醇衍生物的分子量为3000-40000Da；更具体地，所述的聚乙二醇衍生物的分子量可以为3000Da、5000Da、10000Da、20000Da或40000Da。
在本发明的一个实施方式中，本发明所述的聚乙二醇衍生物具有通式II的结构：
Figure PCTCN2018093528-appb-000010
在本发明的一个实施方式中，本发明所述的聚乙二醇衍生物具有通式III的结构：
Figure PCTCN2018093528-appb-000011
在本发明的一个实施方式中，当D为-N 3时，所述的R 1、R 2和R 3为-(CH 2) i-，i为1-10的整数；具体地，所述i为1、2或3。
在本发明的一个实施方式中，当D为-C≡CH时，所述的R 1、R 2和R 3为-(CH 2) iO(CH 2) i-或-(CH 2) iO(CH 2) iCONH(CH 2) i-，i为1-10的整数；具体地，所述i为1、2或3。
本发明的另一方面提供了一种本发明上述聚乙二醇衍生物的制备方法，其包括：(1)将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物与氯甲酸乙酯反应得到聚乙二醇羧酸乙酯；(2)将聚乙二醇羧酸乙酯与三(羟甲基)氨基甲烷反应得到N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺；(3)将N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺与甲磺酰氯反应得到N-[2-甲磺酰氧基-1,1-双(甲磺酰氧甲基)乙基]-聚乙二醇羧酸酰胺；(4)将N-[2-甲磺酰氧基-1,1-双(甲磺酰氧甲基)乙基]-聚乙二醇羧酸酰胺与叠氮化钠反应得到端基叠氮基聚乙二醇衍生物。
本发明的另一方面还提供了一种本发明上述聚乙二醇衍生物的制备方法，其包括：(1)将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物与氯甲酸乙酯反应得到聚乙二醇羧酸乙酯；(2)将聚乙二醇羧酸乙酯与三(羟甲基)氨基甲烷反应得到N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺；(3’)将N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺与溴代炔烃反应得到端基炔基聚乙二醇衍生物。
本发明的另一方面还提供了一种本发明上述聚乙二醇衍生物的制备方法，其包括：(1)将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物与氯甲酸乙酯反应得到聚乙二醇羧酸乙酯；(2)将聚乙二醇羧酸乙酯与三(羟甲基)氨基甲烷反应得到N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺；(3”)将N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺与溴代烷基羧酸叔丁酯反应得到聚乙二醇羧酸酰胺丁三(氧乙酸叔丁酯)；(4”)将聚乙二醇羧酸酰胺丁三(氧乙酸叔丁酯)水解，得到聚乙二醇羧酸酰胺丁三(氧乙酸)，再与胺代炔烃反应得到端基炔基聚乙二醇衍生物。
优选地，本发明所述聚乙二醇衍生物的制备方法，所述的步骤(1)包括：将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物和碳酸钾加到N,N-二甲基甲酰胺中，搅拌下滴加氯甲酸乙酯，升温到80-100℃，优选为90℃，搅拌反应，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，洗涤，干燥，过滤后浓缩结晶。
优选地，本发明所述的聚乙二醇衍生物的制备方法，所述的步骤(2)包括：将步骤(1)的产物、三(羟甲基)氨基甲烷和碳酸钾加到二甲基亚砜中，升温到30-50℃，优选为40℃，搅拌反应，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，提取液洗涤，干燥，过滤后浓缩结晶。
优选地，本发明所述的聚乙二醇衍生物的制备方法，所述的步骤(3)包括：将步骤(2)的产物和甲苯加热回流，冷到室温后加入二氯甲烷和三乙胺，搅拌均匀，用冰水冷却，再滴入甲磺酰氯反应， 反应完全后加入乙醇，搅拌后过滤，浓缩结晶。
优选地，本发明所述的聚乙二醇衍生物的制备方法，所述的步骤(4)包括：将步骤(3)的产物、叠氮化钠和N,N-二甲基甲酰胺加热到70-100℃，优选为90℃，反应2-8小时，优选为5小时，冷却到室温后加入水和氯化钠溶液，搅拌溶解，用二氯甲烷提取，合并有机相，干燥，过滤后浓缩，残分冷却后用乙醚沉淀，得到产品。
优选地，本发明所述的聚乙二醇衍生物的制备方法，所述的步骤(3’)包括：将步骤(2)的产物溶于四氢呋喃中，加入氢化钠，室温反应半小时，加入溴代炔烃和碘化钾，加热1-4小时，优选加热1-4小时，冷却后加入水，浓缩除去四氢呋喃，残分用二氯甲烷萃取，得到最终产物。更优选地，所述的溴代炔烃选自：溴乙炔、溴丙炔和溴丁炔。
优选地，本发明所述的聚乙二醇衍生物的制备方法，所述的步骤(3”)包括：将步骤(2)的产物溶于二甲基甲酰胺(DMF)中，加入氢化钠，室温反应0.5-2小时，优选0.5小时，再加入溴代烷基羧酸叔丁酯反应，反应后加入水，得到N-(三羟甲基)-四臂聚乙二醇羧酸酰胺-三烷基羧酸叔丁酯。更优选地，所述的溴代烷基羧酸叔丁酯选自：溴乙酸叔丁酯和溴乙酸叔丁酯。
优选地，本发明所述的聚乙二醇衍生物的制备方法，所述的步骤(4”)包括：将步骤(3”)溶于甲醇，加入氢氧化钠溶液，加热水解，优选水解温度40-80℃，水解时间2-6小时，更优选地，所述水解温度为60℃，水解时间4小时，得到N-(三羟甲基)-四臂聚乙二醇羧酸酰胺-三烷基羧酸，N-(三羟甲基)-四臂聚乙二醇羧酸酰胺-三烷基羧酸和N-羟基琥珀酰亚胺溶于二氯甲烷中，加入N,N’-二环己基碳二亚胺，反应2-6小时，优选为4小时，再加入胺代炔烃反应得到最终产物。更优选地，所述的胺代炔烃选自：炔丙胺和炔丁胺。
本发明所述的聚乙二醇衍生物可以用于与蛋白质、肽、药物活性小分子相结合，提高靶向性和药物疗效，降低毒性。所述的蛋白质、肽、药物活性小分子包括但不限于：镇痛剂和消炎剂、抗酸剂、驱虫药、抗心律不齐药、抗菌剂、抗凝(血)剂、抗抑郁剂、抗糖尿病剂、止泻剂、抗癫痫药、防真菌剂、抗痛风药、抗高血压药、抗疟药、抗偏头痛药、抗毒蕈碱剂、抗瘤剂和免疫抑制剂、抗原虫药、抗风湿药、抗甲状腺剂、抗病毒剂、抗焦虑剂、镇静剂、安眼药和安定药、β状受体阻断剂、心脏收缩剂、皮质类甾醇、镇咳剂、细胞毒性剂、减充血剂、利尿剂、酶、抗帕金森氏症药、胃肠道药、组胺受体拮抗剂、油脂调节剂、局部麻醉剂、神经肌肉阻断剂、硝酸酯和抗心绞痛药、营养剂、麻醉性镇痛剂、口服疫苗、蛋白、肽和重组药物、性激素和避孕药、杀精子剂、和刺激剂。本发明所述的通式Ⅰ的适用于点击化学反应的聚乙二醇衍生物与蛋白质、肽、药物活性小分子结合方式，可以如专利CN102108119A所描述，通过其端基与药物分子所形成的结合物。
本发明还提供了一种上述聚乙二醇衍生物在点击化学反应中的应用。
本发明还提供一种上述聚乙二醇衍生物在修饰药物分子中的应用。
所述的药物分子包括氨基酸、多肽、蛋白质、糖类、有机酸、生物碱、黄酮类、醌类、萜类、苯丙素酚类、甾体和甙类等药物。
在本发明的一个实施方式中，所述药物分子为局部麻醉剂，如酰胺类局部麻醉剂，更具体地，如利多卡因、丙胺卡因、布比卡因、罗哌卡因、甲哌卡因或依替卡因等。
在本发明的一个实施例中，所述药物分子为布比卡因。
在本发明的另一个实施方式中，所述药物分子为抗肿瘤药物分子，如天然植物类抗肿瘤药物，具体如喜树碱及其衍生物，如喜树碱、拓扑替康、依喜替康(DX-8951f)、伊立替康(CPT-11)、9-氨基喜树碱、卢比替康(9-硝基喜树碱)、勒托替康(GG-211)、CKD-601、吉马替康(ST1481)、BNP-1350或BN-80915等。
在本发明的一个实施例中，所述药物分子为伊立替康。
本发明还提供一种上述聚乙二醇衍生物与药物分子的偶联物，其具有通式Ⅳ结构：
Figure PCTCN2018093528-appb-000012
其中，所述P、R、R 1、R 2、R 3、l和m具有本发明上述相应定义，
TX为
Figure PCTCN2018093528-appb-000013
或
Figure PCTCN2018093528-appb-000014
X 1、X 2和X 3为连接基团，独立地选自：-(CH 2) a-、-(CH 2) aNH-、-(CH 2) aNHCO-、-(CH 2) aCONH-、-(CH 2) aCO-、-(CH 2) aCOO-、-(CH 2) aOCO-、-(CH 2) aSC(O)-、-(CH 2) aO-、-(CH 2) aS-、C 3-C 10环烷基、取代或为取代的芳基和取代或未取代的杂环基中的一种或两种以上的组合，a为0-10的整数，
Q 1、Q 2和Q 3为相同或不相同的药物分子残基。
在本发明的一个实施方式中，所述Q 1、Q 2和Q 3相同。
在本发明的一个实施方式中，所述药物分子为局部麻醉剂，如酰胺类局部麻醉剂，更具体地，如，利多卡因、丙胺卡因、布比卡因、罗哌卡因、甲哌卡因或依替卡因等。
在本发明的一个具体实施方式中，所述Q 1、Q 2和Q 3相同，均为布比卡因残基，具体地，其具有如下结构：
Figure PCTCN2018093528-appb-000015
其中，R 0 -为阴离子，如，F -、Cl -、Br -、I -、甲磺酸根、乙基磺酸根、苯磺酸根、枸橼酸根、乳酸根、琥珀酸根、富马酸根、谷氨酸根、柠檬酸根、水杨酸根和马来酸根等。
在本发明的一个实施例中，所述Q 1、Q 2和Q 3均为局部麻醉剂残基，所述X 1、X 2和X 3独立地选自如下结构：
Figure PCTCN2018093528-appb-000016
在本发明的另一个实施方式中，所述药物分子为抗肿瘤药物分子，如天然植物类抗肿瘤药物，具体如喜树碱及其衍生物，如喜树碱、拓扑替康、依喜替康(DX-8951f)、伊立替康(CPT-11)、9-氨基喜树碱、卢比替康(9-硝基喜树碱)、勒托替康(GG-211)、CKD-601、吉马替康(ST1481)、BNP-1350或BN-80915等。
在本发明的一个具体实施方式中，所述Q 1、Q 2和Q 3相同，均为伊立替康残基，具体地，其具有如下结构：
Figure PCTCN2018093528-appb-000017
在本发明的一个实施方式中，所述Q 1、Q 2和Q 3均为抗肿瘤药物分子残基，所述X 1、X 2和X 3独立地选自：-(CH 2) a-、-(CH 2) aNH-、-(CH 2) aNHCO-、-(CH 2) aCONH-、-(CH 2) aCO-、-(CH 2) aCOO-、-(CH 2) aOCO-和-(CH 2) aO-，a为0-5的整数。
在本发明的一个实施例中，所述X 1、X 2和X 3为-(CH 2) aCO-，如-CH 2CO-。
在本发明的一个实施方式中，所述偶联物具有通式Ⅴ的结构：
Figure PCTCN2018093528-appb-000018
其中，所述x、p、TX、R 1、R 2、R 3、X 1、X 2、X 3、Q 1、Q 2和Q 3具有本发明上述相应定义。
在本发明的另一个实施方式中，所述偶联物具有通式Ⅵ的结构：
Figure PCTCN2018093528-appb-000019
其中，所述q、y、TX、R 1、R 2、R 3、X 1、X 2、X 3、Q 1、Q 2和Q 3具有本发明上述相应定义。在本发明的一个实施例中，上式Ⅴ，所述x为1、2、3、4、5或6。
在本发明的一个实施例中，上式Ⅵ中，所述y为1、2或3。
在本发明的一个实施例中，上式Ⅴ和Ⅵ中，所述Q 1、Q 2和Q 3相同，均为布比卡因残基，如
Figure PCTCN2018093528-appb-000020
在本发明的一个实施例中，上式Ⅴ和Ⅵ中，所述Q 1、Q 2和Q 3相同，均为伊立替康残基，如
Figure PCTCN2018093528-appb-000021
在本发明的一个实施例中，所述偶联物具有如下结构：
Figure PCTCN2018093528-appb-000022
其中，B为
Figure PCTCN2018093528-appb-000023
具体地，所述R 0 -为F -、Cl -、Br -或I -。
在本发明的另一个实施例中，所述偶联物具有如下结构：
Figure PCTCN2018093528-appb-000024
其中，B为
Figure PCTCN2018093528-appb-000025
本发明还提供一种包含本发明上述偶联物及药学上可接受的载体或赋形剂的药物组合物。
在本发明的一些实施方式中，根据所需给药方式，上述药物组合物将包含约1至约99重量％的本发明上述组合物、以及99至1重量％的适宜的载体或药用赋形剂。优选组合物包含约5至75重量％的本发明上述组合物，其余为适宜的载体或药用赋形剂。更优选组合物包含约10至50重量％的本发明上述组合物，其余为适宜的载体或药用赋形剂。
在本发明的一些实施方式中，上述药物组合物还可包含少量的辅助物质，如润湿剂或乳化剂、抗氧化剂等，例如：脱水山梨醇单月桂酸酯、三乙醇胺油酸酯、丁基化羟基甲苯等。
在本发明的一些实施方式中，上述药物组合物为片剂、胶囊剂、丸剂、颗粒剂、散剂、栓剂、注射剂、溶液剂、混悬剂、膏剂、贴剂、洗剂、滴剂、擦剂、喷雾剂等剂型。
在本发明的一些实施方式中，上述药物组合物可采用任何可接受的给药方式或用于类似的用途的试剂进行。因此，采用的给药方式可选择通过口、鼻内、非肠道、局部、透皮或直肠方式，其形式为固体、半固体或液体药剂形式给药，例如，片剂、栓剂、丸剂、软和硬明胶胶囊剂、散剂、溶液剂、混悬剂和注射剂等，优选采用适用于精确剂量的简单给药的单元剂量形式。
可采用液体形式给药的药物组合物，例如，通过溶解或分散等手段将本发明上述组合物(约0.5至约20％)和选择性存在的药用辅剂溶解或分散于载体中形成的溶液剂或混悬剂等，所述载体的实例选自水、盐水、含水葡萄糖、甘油和乙醇等。
本发明还提供一种本发明上述偶联物及其药物组合物在制备预防和/或治疗疾病的药物中的应用。
在本发明的一个实施方式中，所述疾病为疼痛，具体地，所述疼痛为慢性疼痛，如术后疼痛。
在本发明的一个实施方式中，所述疾病为肿瘤，具体如大肠癌、肺癌、乳腺癌、胰腺癌、胃癌、食管癌、结肠癌、直肠癌、肝癌、急性和慢性粒细胞性白血病、绒毛膜上皮癌、膀胱癌、卵巢癌和黑色素瘤等。
本发明所述的聚乙二醇衍生物具有多个叠氮端基或炔基端基，不但能够提高活性端基的负载率，同时可以增强叠氮端基或炔基端基的稳定性与安全性，在修饰药物的应用中可显著提高载药量。因此，本发明提供的聚乙二醇衍生物具有更强的灵活性和更大的应用范围，在有机合成、药物合成及医疗器械等方面均有较好的应用前景。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义，如：
“烷基”指的是直链或支链的且不含不饱和键的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烷基基团含有1至约12个、1至约8个、或者1至约6个碳原子，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、叔戊基、正己基、异己基等。
“亚烷基”指的是直链或支链的且不含不饱和键的烃链自由基，且该烃链自由基以单键与分子其它两个部分连接。典型的亚烷基基团含有1至约12个、1至约8个、或者1至约6个碳原子，如亚甲基(-CH 2-)、亚乙基(-CH 2CH 2-)、亚丙基(-CH 2CH 2CH 2-或-CH(CH 3)CH 2-)等。
“烷氧基”指的是羟基中的氢被烷基取代后形成的取代基，如C1-C6的烷氧基，具体如甲氧基、乙氧基、丙氧基、丁氧基等。
“芳基”指的是单环或多环自由基，包括含单芳基基团和/或稠芳基基团的多环自由基，如包含1-3个单环或稠环及6-18个碳环原子，具体如C6-C12的芳基，如苯基、萘基、联苯基、茚基等，在本发明的一个实施例中，所述芳基为苯基。
“环烷基”指的是脂环烃，如含1至4个单环和/或稠环、含3-18个碳原子，优选3-10个碳原子，如环丙基、环己基或金刚烷基等。
“杂环基”包括含1至3个单环和/或稠环及3至约18个环原子的杂芳香族基团和杂脂环基团，具体如含5至约10个环原子的杂芳香族基团和杂脂环基团；本发明中，合适的杂芳基含1、2或3种杂原子，所述杂原子选自N、O或S原子。
上述基团可以在一个或多个可用的位置被一个或多个合适的基团所取代，所述基团如：OR'、＝O、SR'、SOR'、SO 2R'、OSO 2R'、OSO 3R'、NO 2、NHR'、N(R') 2、＝N-R'、N(R')COR'、N(COR') 2、N(R')SO 2R'、N(R')C(＝NR')N(R')R'、N 3、CN、卤素、COR'、COOR'、OCOR'、OCOOR'、OCONHR'、OCON(R') 2、CONHR'、CON(R') 2、CON(R')OR'、CON(R')SO 2R'、PO(OR') 2、PO(OR')R'、PO(OR')(N(R')R')、C 1-C 12烷基、C 3-C 10环烷基、C 2-C 12烯基、C 2-C 12炔基、芳基和杂环基，其中每个R'基团各自独立地选自：氢、OH、NO 2、NH 2、SH、CN、卤素、COH、CO烷基、COOH、C 1-C 12烷基、C 3-C 10环烷基、C 2-C 12烯基、C 2-C 12炔基、芳基和杂环基。
另外，本发明中涉及的部分具体基团及其化学结构对应如下：羟基，-OH；醛基，-CHO；氨基，-NH 2；马来亚酰胺基，
Figure PCTCN2018093528-appb-000026
羧基，
Figure PCTCN2018093528-appb-000027
酯基，
Figure PCTCN2018093528-appb-000028
或
Figure PCTCN2018093528-appb-000029
(其中G 1可为烷基、芳基或杂环基，如甲基、乙基、正丙基、叔丁基、马来酰亚胺基、琥珀酰亚胺基、
Figure PCTCN2018093528-appb-000030
等)；巯基，-SH；丙烯酸基，
Figure PCTCN2018093528-appb-000031
琥珀酰亚胺基，
Figure PCTCN2018093528-appb-000032
二硫吡啶基，如，2-吡啶二硫基
Figure PCTCN2018093528-appb-000033
4-吡啶二硫基
Figure PCTCN2018093528-appb-000034
巯酯基
Figure PCTCN2018093528-appb-000035
(其中G 2可为烷基，如甲基、乙基、正丙基、叔丁基等)；丙烯酸酯基(丙烯酰氧基)
Figure PCTCN2018093528-appb-000036
酰肼基，
Figure PCTCN2018093528-appb-000037
异氰酸基，
Figure PCTCN2018093528-appb-000038
硅烷基，
Figure PCTCN2018093528-appb-000039
(其中G 3、G 4和G 5可为相同或不同的烷基或烷氧基，如甲基、乙基、丙基、丁基、戊基、甲氧基、乙氧基、丙氧基、丁氧基等；具体地，所述G 3、G 4和G 5相同，均为甲基、 乙基、正丙基、甲氧基、乙氧基、正丙氧基等)；乙烯砜基，
Figure PCTCN2018093528-appb-000040
维生素H残基，
Figure PCTCN2018093528-appb-000041
下面结合实例描述本发明的多臂多爪聚乙二醇及其衍生物、与药物分子的结合物以及包含该结合物的药物组合物，其并不限制本发明，本发明的保护范围由权利要求限定。除非另有说明，实施例中所用的聚乙二醇由北京键凯科技股份有限公司提供，其他试剂均购自北京化学试剂公司。
实施例1四臂聚乙二醇(10K)羧酸乙酯的制备
Figure PCTCN2018093528-appb-000042
将四臂聚乙二醇(10K)和碳酸钾加到40mL干燥N,N-二甲基甲酰胺(DMF)中，搅拌下滴加氯甲酸乙酯，滴完后升温到90℃搅拌过夜，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，提取液分别用稀盐酸和饱和盐水洗涤，干燥。过滤后浓缩，残分用异丙醇结晶，得白色固体。 1H NMR:(DMSO):1.23(t,12H),3.53(m,900H)，3.66(s,8H),4.25(m,8H)，4.37(s,8H)。
实施例2N-[2-羟基-1,1-双(羟甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺的制备
Figure PCTCN2018093528-appb-000043
将化合物1(实施例1制备)、三(羟甲基)氨基甲烷和碳酸钾加到二甲基亚砜(DMSO)中，升温到40℃搅拌过夜，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，提取液分别用稀盐酸和饱和盐水洗涤，干燥。过滤后浓缩，残分用异丙醇结晶，得白色固体。 1H NMR:(DMSO):3.46(s,24H),3.53(m,900H),3.66(s,8H),4.37(s,8H),8.20(s,4H)。
实施例3N-[2-甲磺酰氧基-1,1-双(甲磺酰氧甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺的制备
Figure PCTCN2018093528-appb-000044
将化合物2(60g，3mmol，实施例2制备)和甲苯(600mL)加到反应瓶中，加热回流，蒸出120mL甲苯后停止加热，冷到室温后加入二氯甲烷(60mL)和三乙胺(5.4mL)，搅拌均匀，用冰水冷却，再滴入甲磺酰氯(MsCl，2.8mL)，滴完后撤去冷浴，自然升到室温，反应过夜。次日，往体系中加入乙醇(6mL)，搅拌后过滤，浓缩，残分中加入异丙醇，加热溶清后冷却析晶。过滤，干燥后得产品59g。 1H NMR:(DMSO):3.16(s,36H),3.53(m,900H),3.66(s,8H),3.81(s,24H),4.37(s,8H),8.20(s,4H)。
实施例4N-[2-叠氮基-1,1-双(叠氮甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺的制备
Figure PCTCN2018093528-appb-000045
将化合物3(58.9g，2.94mmol，实施例3制备)和叠氮化钠(3.1g，47mmol)加到反应瓶中，加入N,N-二甲基甲酰胺(295mL)，加热到90℃反应5小时，停止加热，冷却到室温后向体系中加入水(295mL)和15％的氯化钠溶液，搅拌溶解，用二氯甲烷提取(240mL+180mL+120mL)，合并有机相， 干燥。过滤后浓缩，残分冷却后用乙醚沉淀，抽滤，干燥后得产品52g。 1H NMR:(DMSO):3.41(s,24H),3.53(m,900H),3.66(s,8H),4.37(s,8H),8.20(s,4H)。
实施例5八臂聚乙二醇(20K)羧酸乙酯的制备
Figure PCTCN2018093528-appb-000046
将八臂聚乙二醇(20K)和碳酸钾加到40mL干燥的DMF中，搅拌下滴加氯甲酸乙酯，滴完后升温到90℃搅拌过夜，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，提取液分别用稀盐酸和饱和盐水洗涤，干燥。过滤后浓缩，残分用异丙醇结晶，得白色固体。 1H NMR:(DMSO):1.25(t,24H),3.53(m,1800H),3.82(s,16H),4.28(m,16H)，4.46(s,16H)。
实施例6N-[2-羟基-1,1-双(羟甲基)乙基]-八臂聚乙二醇(20K)羧酸酰胺的制备
Figure PCTCN2018093528-appb-000047
将化合物5(实施例5制备)、三(羟甲基)氨基甲烷和碳酸钾加到DMSO中，升温到40℃搅拌过夜，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，提取液分别用稀盐酸和饱和盐水洗涤，干燥。过滤后浓缩，残分用异丙醇结晶，得白色固体。 1H NMR:(DMSO):3.53(m,1800H),3.63(s,48H),3.82(s,16H),4.28(m,16H),8.20(s,8H)。
实施例7N-[2-甲磺酰氧基-1,1-双(甲磺酰氧甲基)乙基]-八臂聚乙二醇(20K)羧酸酰胺的制备
Figure PCTCN2018093528-appb-000048
将化合物6(60g，3mmol，实施例6制备)和甲苯(600mL)加到反应瓶中，加热回流，蒸出120mL甲苯后停止加热，冷到室温后加入二氯甲烷(60mL)和三乙胺(5.4mL)，搅拌均匀，用冰水冷却，再滴入甲磺酰氯(2.8mL)，滴完后撤去冷浴，自然升到室温，反应过夜。次日，往体系中加入乙醇(6mL)，搅拌后过滤，浓缩，残分中加入异丙醇，加热溶清后冷却析晶。过滤，干燥后得产品59g。 1H NMR:(DMSO):3.16(s,72H),3.53(m,1800H),3.63(s,48H),3.82(s,16H),4.28(m,16H),8.20(s,8H)。
实施例8N-[2-叠氮基-1,1-双(叠氮甲基)乙基]-八臂聚乙二醇(20K)羧酸酰胺的制备
Figure PCTCN2018093528-appb-000049
将化合物7(58.9g，2.94mmol，实施例7制备)和叠氮化钠(3.1g，47mmol)加到反应瓶中，加入N,N-二甲基甲酰胺(295mL)，加热到90℃反应5小时，停止加热，冷却到室温后向体系中加入水(295mL)和15％的氯化钠溶液，搅拌溶解，用二氯甲烷提取(240mL+180mL+120mL)，合并有机相，干燥。过滤后浓缩，残分冷却后用乙醚沉淀，抽滤，干燥后得产品52g。 1H NMR:(DMSO):3.53(m,1800H),3.61(s,48H),3.82(s,16H),4.28(m,16H),8.20(s,8H)。
实施例9四臂聚乙二醇(10K)羧酸酰胺丁三(炔丙醚)的制备
Figure PCTCN2018093528-appb-000050
将化合物2(10g，0.5mmol，实施例2制备)溶于四氢呋喃(150mL)中，氮气保护下冷却到0℃，加入氢化钠(0.48g)，室温反应半小时，加入溴丙炔22.4mL和碘化钾0.09克，避光加热回流2小时，冷却后加入水100mL，浓缩除去四氢呋喃，残份用二氯甲烷萃取，提取液洗涤，干燥，过滤，浓缩，残液用乙醚沉淀，过滤后真空干燥，得产品8.4g。 1H NMR:(DMSO):3.34(s,12H),3.53(m,900H),3.66(s,8H),4.19(s,24H),4.37(s,8H),8.20(s,4H)。
实施例10四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸叔丁酯)的制备
Figure PCTCN2018093528-appb-000051
将化合物2(10g，0.5mmol，实施例2制备)溶于DMF(30mL)中，氮气保护下冷却到0℃，加入氢化钠(0.48g)，室温反应半小时，再加入溴乙酸叔丁酯，室温反应过夜，冷却后加入水100mL，用二氯甲烷提取，提取液洗涤，干燥，过滤，浓缩，残液用异丙醇沉淀，过滤后真空干燥，得产品8.8g。 1H NMR:(DMSO):1.43(s,108H),3.53(m,900H),3.66(s,8H),4.35(s,8H),4.41(s,24H),8.20(s,4H)。
实施例11四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸)的制备
Figure PCTCN2018093528-appb-000052
将化合物10(实施例10制备)溶于甲醇(100mL)中，加入1.0M氢氧化钠溶液(100mL)，加热到60℃反应5小时，浓缩除去甲醇，残分用稀盐酸酸化，二氯甲烷提取，提取液洗涤，干燥，过滤，浓缩，残液用异丙醇沉淀，过滤后真空干燥，得产品7.6g。 1H NMR:(DMSO):3.53(m,900H),3.61(s,8H),4.33(s,8H),4.42(s,24H),8.20(s,4H)。
实施例12四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸炔丙酰胺)的制备
Figure PCTCN2018093528-appb-000053
将化合物11(实施例11制备)和N-羟基琥珀酰亚胺(0.6g)溶于二氯甲烷100mL中，加入N,N’-二环己基碳二亚胺(1.5g)，反应4小时，再加入炔丙胺1mL，室温反应过夜，过滤，反应液浓缩，残分用异丙醇沉淀，抽滤，所得固体真空干燥得产品7.2g。 1H NMR:(DMSO):3.09(s,12H),3.53(m,900H),3.66(s,8H),4.25(s,24H),4.33(m,32H),8.20(s,12H),8.23(s,4H)。
实施例13N-[2-叠氮基-1,1-双(叠氮甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺与布比卡因偶联物(L1)的制备
Figure PCTCN2018093528-appb-000054
N-[2-叠氮基-1,1-双(叠氮甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺(10K，10g，1mmol，实施例4制备)，化合物Y1(9.3g，15mmol)、维生素C(2.2g，125mmol)加到N,N-二甲基甲酰胺(200mL)中，快速搅拌使其溶解，然后加入五水合硫酸铜(0.38g，15mmol)的水溶液(44mL)，室温反应过夜，用异丙醇沉淀得到8.6g产物。 1H NMR:(DMSO)：0.92(t,36H),1.33(m,48H),1.79(m,48H),2.15(s,72H),2.21(m,24H),2.34(s,36H),2.60(s,12H),3.23(m,36H),3.53(m,900H),3.66(s,8H),3.91(s,24H),4.35(s,8H),4.59(m,36H),6.89(m,24H),7.08(m,48H),7.69(s,12H),8.20(m,4H),9.82(s,12H)。
实施例14N-[2-叠氮基-1,1-双(叠氮甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺与布比卡因偶联物(L1)载药量的确定
取N-[2-叠氮基-1,1-双(叠氮甲基)乙基]-四臂聚乙二醇(10K)羧酸酰胺与布比卡因偶联物(L1，实施例13制备)约50mg，精密称定，置30mL量瓶中，加入0.1mol/L盐酸10mL溶解，摇匀，压塞，轧盖，置60℃的恒温干燥箱中20小时，放冷，作为供试品溶液。另精密称取盐酸布比卡因对照品25mg，置于10mL量瓶中，用0.1mol/L盐酸溶解并稀释至刻度，精密量取5mL置于25mL量瓶中，加入0.1mol/L盐酸稀释至刻度，作为对照品溶液。用高效液相色谱法测得样品L1中布比卡因的含量为19.7％。
实施例15叠氮乙酸的制备
Figure PCTCN2018093528-appb-000055
将溴乙酸叔丁酯(5.82g，30mmoL)加到反应瓶中，用丙酮(80mL)溶解，再加入叠氮化钠(4.55g，70mmoL)溶于水(40mL)的溶液，加热回流过夜。反应液蒸去丙酮，残分用乙醚提取，提取液用饱和盐水洗涤，干燥，减压浓缩后得油状液体。将此液体用甲醇(90mL)溶解，再加入1N氢氧化钠溶液(90mL)，搅拌，加热回流3h。冷却后减压蒸去甲醇，残液用冰浴冷却，加6N盐酸将pH调至2，再用乙醚提取，提取液加水洗涤，干燥，浓缩后得叠氮乙酸，MS m/z:124[M+Na] +。
实施例16叠氮乙酸伊立替康酯(Y2)的制备
Figure PCTCN2018093528-appb-000056
将叠氮乙酸(2.5g，25mmoL)和伊立替康(14.7g，25mmoL)加到反应瓶中，用二氯甲烷溶解，冰浴冷却，再加入4-二甲氨基吡啶(DMAP，6.1g，50mmoL)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI，10.3g，50mmoL)加到反应瓶中，加完后继续在室温下搅拌过夜。反应液浓缩后残分用柱层析纯化，得叠氮乙酸伊立替康酯(Y2)14.2g，收率57％，MS m/z:692[M+Na] +。
实施例17四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸炔丙酰胺)与叠氮乙酸伊立替康酯偶联物(L2)的制备
Figure PCTCN2018093528-appb-000057
四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸炔丙酰胺)(10K，10g，1mmol，实施例12制备)，化合物Y2(10.0g，15mmol)、维生素C(2.2g，125mmol)加到N,N-二甲基甲酰胺(200mL)中，快速搅拌使其溶解，然后加入五水合硫酸铜(0.38g，15mmol)的水溶液(44mL)，室温反应过夜，用异丙醇沉淀得到8.9g产物。 1H NMR:(DMSO)：0.92(t,12H),1.22(t,12H),1.52(m,24H),1.86(m,24H),2.32(s,8H),2.56(m,20H),3.53(m,900H),3.66(s,8H),4.16(s,24H),4.23(s,8H),4.28(s,8H),4.33(s,24H),4.79(s,8H),6.81(s,4H),7.50(m,12H),8.20(s,4H),8.41(s,12H),8.67(s,12H)。
实施例18四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸炔丙酰胺)与叠氮乙酸伊立替康酯偶联物(L2)载药量的确定
取四臂聚乙二醇(10K)羧酸酰胺丁三(氧乙酸炔丙酰胺)与叠氮乙酸伊立替康酯偶联物(L2，实施例17制备)约25mg，精密称定，置25mL量瓶中，加水2mL，再加入0.005mol/L的氢氧化钠溶液20mL溶解，摇匀，置40℃的水浴中加热30分钟，放冷，用0.04mol/L的盐酸溶液稀释至刻度，摇匀，作为供试品溶液。另精密称取盐酸伊立替康标准品25mg，置于25mL量瓶中，用水溶解并稀释至刻度，精密量取2mL置于25mL量瓶中，再加入0.005mol/L的氢氧化钠溶液20mL，与供试品溶液同置40℃的水浴中加热30分钟，放冷，用0.04mol/L盐酸溶液稀释至刻度，摇匀，作为标准品溶液。用高效液相色谱法测得样品L2中伊立替康的含量为28.1％。

Claims (22)

  1. 一种通式Ⅰ的聚乙二醇衍生物，
    Figure PCTCN2018093528-appb-100001
    其中：
    R为具有直链结构、Y型结构或多分支结构的聚乙二醇残基；
    P为非叠氮非炔基的端基基团；
    R 1、R 2和R 3独立的选自：-(CH 2) i-、-(CH 2) iO(CH 2) i-、-(CH 2) iO(CH 2) iCONH(CH 2) i-、-(CH 2) iNH-、-(CH 2) iOCOO-、-(CH 2) iOCONH-、-(CH 2) iNHCO-、-(CH 2) iNHCOO-、-(CH 2) iNHCONH-、-OC(CH 2) iCOO-、-(CH 2) iCOO-和-(CH 2) iCONH-中的一种或两种以上的组合；i为0-10的整数；
    D为-N 3或-C≡CH；
    l选自1-20的整数；
    m选自0-19的整数。
  2. 如权利要求1所述的聚乙二醇衍生物，其中，所述R为直链聚乙二醇残基，其具有式(R-1)所示结构：
    Figure PCTCN2018093528-appb-100002
    其中， n为3-250的整数；或，
    所述R为Y型聚乙二醇残基，其具有式(R-2)或(R-3)所示的结构：
    Figure PCTCN2018093528-appb-100003
    其中，h和f独立地选自3-250的整数；或，
    所述R为多分支聚乙二醇残基，其具有式(R-4)所示的结构：
    Figure PCTCN2018093528-appb-100004
    其中，t为3-250的整数，
    j为3-8的整数，
    R c为多分支聚乙二醇的核心分子，选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基，
    m和l之和等于j。
  3. 如权利要求2所述的聚乙二醇衍生物，其中，所述多分支聚乙二醇残基具有如下结构：
    Figure PCTCN2018093528-appb-100005
    其中，p是3-250的整数，
    x为1-18的整数，
    m和l之和等于x+2；或，
    所述多分支聚乙二醇残基具有如下结构：
    Figure PCTCN2018093528-appb-100006
    其中，q为3-250的整数，
    y为1-9的整数，
    m和l之和等于2y+2。
  4. 如权利要求1-3任一项所述的聚乙二醇衍生物，其中，所述的l为选自3-8的整数，所述的m为0；优选地，所述的l为4、6或者8。
  5. 如权利要求1-4任一项所述的聚乙二醇衍生物，其中，所述的P选自以下基团：H、C1-C6的烷基、C1-C6的烷氧基、醛基、羟基、氨基、羧基、巯基、酯基、马来亚酰胺基、丙烯酸基、琥珀酰亚胺基、二硫吡啶基、巯酯基、丙烯酰氧基、酰肼基、异氰酸基、硅烷基、乙烯砜基和维生素H残基；优选地，所述P选自：NH 2、-COOH、-OCH 3、
    Figure PCTCN2018093528-appb-100007
    和/或，
    所述i为1、2或3。
  6. 如权利要求1-5任一项所述的聚乙二醇衍生物，所述P与R之间还包括连接基团，所述的连接基团选自：-(CH 2) k-、-(CH 2) kO(CH 2) k-、-(CH 2) kO(CH 2) kCONH(CH 2) k-、-(CH 2) kNH-、-(CH 2) kOCOO-、-(CH 2) kOCONH-、-(CH 2) kNHCO-、-(CH 2) kNHCOO-、-(CH 2) kNHCONH-、-OC(CH 2) kCOO-、-(CH 2) kCOO-和-(CH 2) kCONH-中的一种或两种以上的组合；k为0-10的整数。
  7. 如权利要求1-6任一项所述的聚乙二醇衍生物，所述的聚乙二醇衍生物的分子量为1000-80000Da。
  8. 如权利要求3所述的聚乙二醇衍生物，所述的聚乙二醇衍生物具有通式II或III所示结构：
    Figure PCTCN2018093528-appb-100008
    Figure PCTCN2018093528-appb-100009
  9. 如权利要求1-8任一项所述的聚乙二醇衍生物，所述的D为-N 3，所述的R 1、R 2和R 3为-(CH 2) i-，i为1-10的整数；或者，
    所述的D为-C≡CH，所述的R 1、R 2和R 3为-(CH 2) iO(CH 2) i-或-(CH 2) iO(CH 2) iCONH(CH 2) i-，i为1-10的整数。
  10. 一种权利要求1-9任一项所述的聚乙二醇衍生物的制备方法，包括：
    (1)将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物与氯甲酸乙酯反应得到聚乙二醇羧酸乙酯；
    (2)将聚乙二醇羧酸乙酯与三(羟甲基)氨基甲烷反应得到N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺；
    (3)将N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺与甲磺酰氯反应得到N-[2-甲磺酰氧基-1,1-双(甲磺酰氧甲基)乙基]-聚乙二醇羧酸酰胺；
    (4)将N-[2-甲磺酰氧基-1,1-双(甲磺酰氧甲基)乙基]-聚乙二醇羧酸酰胺与叠氮化钠反应得到端基叠氮基聚乙二醇衍生物，
    或者，
    (1)将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物与氯甲酸乙酯反应得到聚乙二醇羧酸乙酯；
    (2)将聚乙二醇羧酸乙酯与三(羟甲基)氨基甲烷反应得到N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺；
    (3’)将N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺与溴代炔烃反应得到端基炔基聚乙二醇衍生物，
    或者
    (1)将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物与氯甲酸乙酯反应得到聚乙二醇羧酸乙酯；
    (2)将聚乙二醇羧酸乙酯与三(羟甲基)氨基甲烷反应得到N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺；
    (3”)将N-[2-羟基-1,1-双(羟甲基)乙基]-聚乙二醇羧酸酰胺与溴代烷基羧酸叔丁酯反应得到聚乙二醇羧酸酰胺丁三(氧乙酸叔丁酯)；
    (4”)将聚乙二醇羧酸酰胺丁三(氧乙酸叔丁酯)水解，得到聚乙二醇羧酸酰胺丁三(氧乙酸)，再与胺代炔烃反应得到端基炔基聚乙二醇衍生物。
  11. 如权利要求10所述的制备方法，所述的步骤(1)包括：将直链结构、Y型结构或多分支结构的聚乙二醇或其衍生物和碳酸钾加到N,N-二甲基甲酰胺中，搅拌下滴加氯甲酸乙酯，升温到80-100℃，搅拌反应，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，洗涤，干燥，过 滤后浓缩结晶；所述的步骤(2)包括：将步骤(1)的产物、三(羟甲基)氨基甲烷和碳酸钾加到二甲基亚砜中，升温到30-50℃，搅拌反应，反应完成后混合物冷到室温，倒入冰水中，搅拌均匀，用二氯甲烷提取，提取液洗涤，干燥，过滤后浓缩结晶；所述的步骤(3)包括：将步骤(2)的产物和甲苯加热回流，冷到室温后加入二氯甲烷和三乙胺，搅拌均匀，用冰水冷却，再滴入甲磺酰氯反应，反应完全后加入乙醇，搅拌后过滤，浓缩结晶；所述的步骤(4)包括：将步骤(3)的产物、叠氮化钠和N,N-二甲基甲酰胺加热到70-100℃，反应2-8小时，冷却到室温后加入水和氯化钠溶液，搅拌溶解，用二氯甲烷提取，合并有机相，干燥，过滤后浓缩，残分冷却后用乙醚沉淀，得到产品。
  12. 如权利要求10所述的制备方法，所述的步骤(3’)包括：将步骤(2)的产物溶于四氢呋喃中，加入氢化钠，室温反应半小时，加入溴代炔烃和碘化钾，加热1-4小时，冷却后加入水，浓缩除去四氢呋喃，残分用二氯甲烷萃取，得到最终产物。
  13. 如权利要求10所述的制备方法，所述的步骤(3”)包括：将步骤(2)的产物溶于二甲基甲酰胺中，加入氢化钠，室温反应0.5-2小时，再加入溴代烷基羧酸叔丁酯反应，反应后加入水，得到N-(三羟甲基)-四臂聚乙二醇羧酸酰胺-三烷基羧酸叔丁酯；
    所述的步骤(4”)包括：将步骤(3”)溶于甲醇，加入氢氧化钠溶液，加热水解，得到聚乙二醇羧酸酰胺丁三(氧乙酸)，聚乙二醇羧酸酰胺丁三(氧乙酸)和N-羟基琥珀酰亚胺溶于二氯甲烷中，加入N,N’-二环己基碳二亚胺，反应2-6小时再加入胺代炔烃反应得到最终产物。
  14. 如权利要求1-9任一项所述的聚乙二醇衍生物在用于点击化学反应中的应用。
  15. 一种权利要求1-9任一项所述的聚乙二醇衍生物与药物分子的偶联物，其具有通式Ⅳ所示结构：
    Figure PCTCN2018093528-appb-100010
    其中：
    R为具有直链结构、Y型结构或多分支结构的聚乙二醇残基；
    P为非叠氮非炔基的端基基团；
    R 1、R 2和R 3独立的选自：-(CH 2) i-、-(CH 2) iO(CH 2) i-、-(CH 2) iO(CH 2) iCONH(CH 2) i-、-(CH 2) iNH-、-(CH 2) iOCOO-、-(CH 2) iOCONH-、-(CH 2) iNHCO-、-(CH 2) iNHCOO-、-(CH 2) iNHCONH-、-OC(CH 2) iCOO-、-(CH 2) iCOO-和-(CH 2) iCONH-中的一种或两种以上的组合；i为1-10的整数；
    D为-N 3或-C≡CH；
    l选自1-20的整数；
    m选自0-19的整数；
    TX为
    Figure PCTCN2018093528-appb-100011
    X 1、X 2和X 3为连接基团，独立地选自：-(CH 2) a-、-(CH 2) aNH-、-(CH 2) aNHCO-、-(CH 2) aCONH-、-(CH 2) aCO-、-(CH 2) aCOO-、-(CH 2) aOCO-、-(CH 2) aSC(O)-、-(CH 2) aO-、-(CH 2) aS-、C 3-C 10环烷基、取代或为取代的芳基和取代或未取代的杂环基中的一种或两种以上的组合， a为0-10的整数，
    Q 1、Q 2和Q 3为相同或不相同的药物分子残基。
  16. 如权利要求15所述的偶联物，其中，所述偶联物具有通式Ⅴ的结构：
    Figure PCTCN2018093528-appb-100012
    其中，p是3-250的整数，
    x为1-18的整数；或，
    所述偶联物具有通式Ⅵ的结构：
    Figure PCTCN2018093528-appb-100013
    其中，q为3-250的整数，
    y为1-9的整数。
  17. 如权利要求15或16所述的偶联物，其中，所述药物分子为局部麻醉剂，优选自：利多卡因、丙胺卡因、布比卡因、罗哌卡因、甲哌卡因和依替卡因。
  18. 如权利要求15或16所述的偶联物，其中，所述药物分子为抗肿瘤药物分子，优选为天然植物类抗肿瘤药物，更优选自：喜树碱、拓扑替康、依喜替康、伊立替康、9-氨基喜树碱、卢比替康、勒托替康、CKD-601、吉马替康、BNP-1350和BN-80915。
  19. 如权利要求15或16所述的偶联物，其中，所述Q 1、Q 2和Q 3相同，均为布比卡因残基，其具有如下结构：
    Figure PCTCN2018093528-appb-100014
    其中，R 0 -为阴离子，如，F -、Cl -、Br -、I -、甲磺酸根、乙基磺 酸根、苯磺酸根、枸橼酸根、乳酸根、琥珀酸根、富马酸根、谷氨酸根、柠檬酸根、水杨酸根和马来酸根；或，
    所述Q 1、Q 2和Q 3相同，均为伊立替康残基，具体地，其具有如下结构：
    Figure PCTCN2018093528-appb-100015
  20. 如权利要求19所述的偶联物，其中，所述偶联物具有如下结构：
    Figure PCTCN2018093528-appb-100016
    其中，B为
    Figure PCTCN2018093528-appb-100017
    或，
    所述偶联物具有如下结构：
    Figure PCTCN2018093528-appb-100018
    其中，B为
    Figure PCTCN2018093528-appb-100019
  21. 一种包含权利要求15-20任一项所述的偶联物及药学上可接受的载体或赋形剂的药物组合物。
  22. 一种权利要求15-20任一项所述的偶联物或权利要求21所述的药物组合物在制备预防和/或治疗疾病的药物中的应用。
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Citations (4)

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Publication number Priority date Publication date Assignee Title
CN104448296A (zh) * 2013-12-02 2015-03-25 北京键凯科技有限公司 炔基多臂聚乙二醇衍生物
CN104497303A (zh) * 2013-12-02 2015-04-08 天津键凯科技有限公司 多臂聚乙二醇-叠氮衍生物
WO2016183359A1 (en) * 2015-05-12 2016-11-17 Blinkbio, Inc. Silicon based drug conjugates and methods of using same
WO2017214491A1 (en) * 2016-06-09 2017-12-14 Blinkbio, Inc. Silanol based therapeutic payloads

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104448296A (zh) * 2013-12-02 2015-03-25 北京键凯科技有限公司 炔基多臂聚乙二醇衍生物
CN104497303A (zh) * 2013-12-02 2015-04-08 天津键凯科技有限公司 多臂聚乙二醇-叠氮衍生物
WO2016183359A1 (en) * 2015-05-12 2016-11-17 Blinkbio, Inc. Silicon based drug conjugates and methods of using same
WO2017214491A1 (en) * 2016-06-09 2017-12-14 Blinkbio, Inc. Silanol based therapeutic payloads

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