WO2016045505A1 - 一种喜树碱类磷脂化合物、其药物组合物及应用 - Google Patents

一种喜树碱类磷脂化合物、其药物组合物及应用 Download PDF

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Publication number
WO2016045505A1
WO2016045505A1 PCT/CN2015/089181 CN2015089181W WO2016045505A1 WO 2016045505 A1 WO2016045505 A1 WO 2016045505A1 CN 2015089181 W CN2015089181 W CN 2015089181W WO 2016045505 A1 WO2016045505 A1 WO 2016045505A1
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camptothecin
compound
ion
phospholipid
phospholipid compound
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PCT/CN2015/089181
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English (en)
French (fr)
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李新松
方硕
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东南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings

Definitions

  • the invention relates to a camptothecin-based phospholipid compound having anti-tumor effect, a pharmaceutical composition thereof and use thereof, and relates to the technical field of medicine.
  • Camptothecin is an alkaloid with strong cytotoxic activity and can be used to treat a variety of malignant tumors such as gastric cancer, liver cancer, bladder cancer and leukemia.
  • Camptothecin compounds are the most classical specific inhibitors of Topo I. Topo I has become one of the hottest targets for designing new anticancer drugs. Topo I inhibitors have been listed as one of the six major classes of anti-tumor drugs by the National Cancer Institute (NCI) Drug Mechanism Analysis Network System.
  • Camptothecin and its analogs can be extracted from plants or chemically synthesized. Such camptothecin and its analogs exert serious anti-tumor activities as well as serious side effects such as myelosuppression, vomiting and diarrhea.
  • the biggest drawback of camptothecin and its analogues for the treatment of tumors is poor water solubility, making it difficult to obtain high concentrations of solutions.
  • many research institutions and enterprises have modified the chemical structure of camptothecin and synthesized hundreds of camptothecin derivatives.
  • camptothecin derivatives have been approved for clinical oncology treatment, namely irinotecan irinotecan and topotecan topotecan, but their water solubility is still low.
  • 9-AC, 9-NC, G7147211, DX-8951f, Bay38-3441, BN80915 and CKD-602 are in clinical trials, respectively.
  • More camptothecin analogs have not been used. Part of the reason is that the water solubility is too poor, and camptothecin and its analogues have disadvantages such as large side effects in the treatment of tumors.
  • camptothecin and its analogs contain four six-membered rings and one five-membered ring, wherein the ring E is an alpha-hydroxy lactone and has a chiral center in the 20S configuration. E-ring carbonyl activity is high, and it is easy to open the ring under the attack of nucleophile. Because camptothecin has a balance between the closed lactone form and the ring-opened carboxylate form in the body, the open-loop form depends on the pH and the animal species.
  • human serum albumin preferentially binds to the open-loop form of camptothecin to form a stable complex, which shifts the equilibrium to the open-loop form, so that the lactone content of the antitumor activity in the body is too low, resulting in the human body.
  • the anti-tumor activity is very low.
  • camptothecin maximal tolerated dose of MTD 12mg/kg in mice
  • camptothecin maximal tolerated dose of MTD 12mg/kg in mice
  • the open-loop form of camptothecin is tightly bound to HSA when the drug enters the human body, so that the balance of camptothecin in the body From the closure of the lactone form to the open-loop form, camptothecin is rapidly opened (half-life is less than half an hour).
  • camptothecin and 10-hydroxycamptothecin are unmodified natural products, they are highly toxic and have low antitumor activity, which limits their clinical application. Furthermore, long-term use of these drugs is prone to drug resistance. There is an urgent need to improve the water solubility of camptothecin or its analogs, and to find high-efficiency and low-toxic camptothecin antitumor drugs.
  • camptothecin and its derivatives it is necessary to modify the structure of camptothecin and its derivatives to improve drug solubility, targeting, prevent lactone ring opening, improve efficacy and reduce toxicity. It has also been a research hotspot in the field of drugs.
  • Liposomes are novel pharmaceutical preparations with targeted drug delivery functions and are extremely widely used in the field of anticancer drug research.
  • Phospholipid is a constituent lipid
  • the main chemical component of the plastid consists of a hydrophobic tail composed of a substituted gene having a phosphate-linked gene and a hydrophobic tail composed of a long hydrocarbon chain.
  • the hydrophobic tails of the molecules tend to clump together, avoiding the aqueous phase, while the hydrophilic head is exposed to the aqueous phase, forming a closed vesicle having a bilayer structure, forming a liposome.
  • the hydrophobic tail of natural phospholipids contains unsaturated bonds, it has a great influence on the stability of liposomes. Therefore, structural modification of phospholipid hydrophobic tails to improve their stability has become a research hotspot.
  • the hydrophobic drug is usually in the double lipid layer of the liposome, and the hydrophilic drug is in the aqueous phase of the liposome. Due to the fluidity of the liposome membrane, the drug is easily leaked out, making it difficult for the wrapped drug to exert good pharmacological effects.
  • the invention utilizes two molecules of camptothecin and its analog chemical structure as a hydrophobic tail and a phospholipid hydrophilic head to form a camptothecin-like phospholipid compound by covalent bond, and the solubility is significantly better than camptothecin and its derivatives.
  • the liposome nanoparticle can be prepared alone or in combination with a phospholipid; the present invention has a camptothecin-based phospholipid compound having properties capable of forming a liquid preparation, a solid preparation, a semisolid preparation, a sterilization preparation, and a sterile preparation;
  • the camptothecin-based phospholipid compound is prepared into nanoparticles, has liposome characteristics, and has characteristics of forming a liquid preparation, a solid preparation, a semi-solid preparation, a sterilization preparation, and a sterile preparation, and is used for tumor treatment.
  • the camptothecin-like phospholipid compound is degraded by an esterase in the body, releasing the drug, not only a prodrug, but also a novel drug release carrier of the camptothecin drug; the camptothecin-like phospholipid compound of the invention has a lower
  • the toxic side effects of liposomes have a targeting function.
  • An object of the present invention is to provide a camptothecin-based phospholipid compound which improves the solubility of camptothecin or the like, and a pharmaceutical composition based on the camptothecin-based phospholipid compound, and the camptothecin-based phospholipid The use of compounds in the preparation of antitumor drugs.
  • the parent rings of all camptothecins and analogs thereof or phospholipid compounds thereof according to the invention are all defined according to the position in formula (A), and the carbon atom at position 20 is in the S configuration.
  • camptothecin-based phospholipid compound of the present invention is a pharmaceutically acceptable salt of a compound of the following formula (1) or a compound of the formula (1) and a counter ion:
  • R 1 and R 2 are a spacer arm which is an alkylene group, and the alkylene group is defined as a linear or branched alkylene group/alkenylene group having 1 to 20 carbon atoms or a carbon number. a 6 to 10 aromatic hydrocarbon group; L represents 2-amino-2-carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation or 2,3-dihydroxypropyl;
  • R x and R y are a 20-position substituent of a camptothecin compound (20S) having the following formula (2), a 10-position substituent of a camptothecin compound (20S) of the formula (3) or a formula (4) Camptothecin-like compound (20S) 7-methylene substituent:
  • R 7 , R 9 , R 10 , R 11 and R 12 are each of the following groups: hydrogen, halogen, hydroxy, C 1 -C 6 straight Chain or branched alkyl/alkoxy, cyano, nitro, amino, C 1 -C 6 straight or branched haloalkyl/haloalkoxy, C 1 -C 6 straight or branched trisubstituted silyl group, C straight or branched chain of 1 -C 6 alkene group, oxymethylene group, C linear or branched alkyl ester group of 1 -C 6, C 1 -C 6 straight chain or Branched alkylcarbonyl, C 1 -C 6 straight or branched hydroxyalkyl, C 1 -C 6 linear or branched alkoxymethylene, phenoxymethylene, benzyl ester Methylidene, dimethylaminomethyl, dimethylaminoethyl, having a piperaz
  • R represents a C 1 - C 18 linear/branched alkyl group or a C 6 - C 10 aromatic hydrocarbon group.
  • R x and R y are preferably a hydroxyl substituent at position 20 of camptothecin (CPT), a hydroxyl group substituent at position 20 of 10-hydroxycamptothecin, a hydroxyl group substituent at position 10 of 10-hydroxycamptothecin, irinotecan ( CPT11, bipermidine base) 20-hydroxyl substituent, topological Likang (9-dimethylamino-10-hydroxycamptothecin) 10-hydroxyl substituent, topological Likang (9-dimethylamino-10 -hydroxycamptothecin) 20-hydroxyl substituent, 7-ethyl-10-hydroxycamptothecin 20-position hydroxy substituent, 7-ethyl-10-hydroxycamptothecin 10-position hydroxy substituent,
  • the counter ion is a cationic proton, a sodium ion, a potassium ion, a calcium ion, a ferric ion, a magnesium ion, an ammonium ion, a zinc ion, an anionic chloride ion, a sulfate ion, a sulfate group.
  • a combination of the two is preferably a proton, a sodium ion, or a chloride ion.
  • the method for preparing the above camptothecin-based phospholipid compound of the present invention firstly reacts a hydroxyl group of camptothecin or camptothecin analog with a dianhydride or a carboxylic acid to prepare an intermediate containing a carboxyl group, and then further the intermediate
  • the camptothecin-like phospholipid compound is obtained by performing a condensation reaction with glycerol phosphate choline under the action of a condensation reagent.
  • a condensation reagent is preferably used, preferably 1,3 dicyclohexylcarbodiimide, dipyridine carbonate, 1-(3-dimethylaminopropyl)-3-ethylcarbimide salt.
  • One of the acid salt, 1,3-diisopropylcarbimide or N,N-carbonyldiimidazole and a tertiary amine, and the tertiary amine is preferably 4-dimethylaminopyridine or 4-pyrrolidinopyridine.
  • the pharmaceutical composition of the present invention comprises the above camptothecin-based phospholipid compound or the camptothecin-based phospholipid compound and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention is a liquid preparation, a solid preparation, a semisolid preparation, a capsule, a granule, a gel, an injection, a sustained release preparation or a controlled release preparation.
  • the pharmaceutical composition is a liposome nanoparticle having a particle diameter of 10 to 1000 nm, and an auxiliary agent is further included in the pharmaceutical composition.
  • the adjuvant is a phospholipid.
  • the compound of the present invention may exist in the form of an isomer, and generally, the "compound of the present invention” includes an isomer of the compound.
  • the compounds of the invention may exist in an asymmetric center having an S configuration or an R configuration, and the invention includes all possible stereoisomers as well as mixtures of two or more isomers.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient a compound of the invention or a compound of the invention and a conventional pharmaceutical adjuvant.
  • the pharmaceutical compositions of the invention contain from 0.1 to 100% by weight of a compound of the invention.
  • compositions of the compounds of the invention can be prepared according to methods well known in the art.
  • the compound of the invention or a compound of the invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to provide a suitable form of administration for use as a human medicament. Or dosage form.
  • the compound of the present invention or a pharmaceutical composition containing the same may be administered in a unit dosage form, which may be enterally or parenterally, such as orally, muscle, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.
  • the administration route of the compound of the present invention or the pharmaceutical composition containing the same may be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and acupoint injection.
  • the dosage form can be a liquid dosage form or a solid dosage form.
  • the liquid dosage form may be a true solution type, a colloid type, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form.
  • Other dosage forms such as tablets, capsules, pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powders, and the like.
  • the compounds of the present invention can be formulated into common preparations, sustained release preparations, controlled release preparations, targeted preparations, and various microparticle delivery systems.
  • carriers In order to form a unit dosage form into tablets, various carriers well known in the art can be widely used.
  • carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid.
  • wetting agent and binder such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethyl cellulose , shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.
  • disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium bicarbonate and tannic acid, calcium carbonate, polyoxyethylene sorbes Sugar alcohol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.
  • absorption enhancer For example, quaternary ammonium salts, sodium lauryl sulfate, and the like
  • lubricants such as talc, silica, corn
  • various carriers known in the art can be widely used.
  • the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, Gelatin, ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, and the like.
  • the active ingredient compound of the present invention is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or soft capsule.
  • Active Ingredient The compound of the present invention can also be formulated into a microcapsule, suspended in an aqueous medium to form a suspension, or can be enclosed in a hard capsule or used as an injection.
  • the compound of the present invention is formulated into an injectable preparation, such as a solution, a suspension solution, an emulsion, or a lyophilized powder injection.
  • the preparation may be aqueous or non-aqueous, and may contain one and/or more pharmacodynamics.
  • the diluent may be selected from the group consisting of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like.
  • an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional solubilizer, a buffer, a pH adjuster or the like may be added. These excipients are commonly used in the art.
  • Coloring agents preservatives, perfumes, flavoring agents, sweeteners or other materials may also be added to the pharmaceutical preparations if desired.
  • Liposomal nanoparticles are prepared from the compounds of the invention having a particle size of from 10 to 1000 nanometers.
  • the liposomal nanoparticles are prepared from the compound of the invention and the auxiliary agent, the particle size is 10-1000 nm, and the auxiliary agent is dimyristoyl phosphatidylcholine DMPC, distearoylphosphatidylcholine DSPC, dipalmitoyl phospholipid Acetylcholine DPPC, L- ⁇ -phosphatidylcholine- ⁇ -arachidonic acid- ⁇ -stearyl, didecanoylglycerol-3-phosphocholine DDPC, 1,2-dioleoylphosphatidylcholine DOPC, 2-oleoyl-1-palmitoylglycerol-3-phosphocholine POPC, 1-stearoyl -2-oleoylglycerol-3-phosphocholine SOPC, 1-stearoyl-2-palmitoylglycerol-3-phosphocholine SPPC, 1-myristoyl-2-stearoylphosphatidylcholine MSPC, 1-palmito
  • composition liposome nanoparticles of the present invention are liquid preparations, solid preparations, semisolid preparations, capsules, granules, gels, injections, sustained release preparations or controlled release preparations.
  • the compounds of the present invention exhibit good antitumor activity from the in vitro antitumor activity screening. Tests have shown that the compounds of the invention have less in vivo toxicity than camptothecin and topotecan. It can therefore be used as an anti-tumor drug in animals, preferably in mammals, especially humans.
  • the liposome nanoparticles of the present compounds exhibit good antitumor activity. Tests have shown that the compounds of the invention are much less toxic in vivo than camptothecin and topotecan. It can therefore be used as an anti-tumor drug in animals, preferably in mammals, especially humans.
  • the method for preparing the camptothecin-based phospholipid compound liposome nanoparticles of the present invention is a mixture of a camptothecin-based phospholipid compound of the present invention or a compound of the present invention and an auxiliary agent, by a film dispersion method, a reverse phase evaporation method, and a freeze drying method. It is prepared by a method such as a method, an ultrasonic dispersion method, a spray drying method, a film extrusion method, or a high pressure homogenization method.
  • the present invention utilizes 2 molecules of camptothecin or camptothecin analog as a hydrophobic tail and a phospholipid hydrophilic head to form a camptothecin-like phospholipid compound by covalent bond, and the solubility is significantly better than that of camptothecin or camptothecin.
  • the present invention prepares camptothecin-based phospholipid compounds into nanoparticles, has the characteristics of liposome, and has the characteristics of forming liquid preparations, solid preparations, semi-solid preparations, sterilization preparations and sterile preparations, and is used for tumor treatment. .
  • the camptothecin-like phospholipid compound nanoparticle is not only a prodrug, but also a novel drug release carrier of camptothecin drugs, having a targeting function; the camptothecin-based phospholipid compound of the present invention and liposomal nanoparticles thereof It is also a new prodrug of camptothecin drugs, and it is also a drug carrier, which exerts pharmacological effects by esterase hydrolysis in the body and has low toxic and side effects.
  • camptothecin-based phospholipid compound of the formula (1) of the present invention and the pharmaceutically acceptable salt-containing salt formed by the compound have good water solubility, have an antitumor effect, and can prolong the drug release half-life, and Has low toxic side effects.
  • camptothecin-based phospholipid compound of the present invention has the following structural formula (1):
  • R 1 and R 2 are a spacer arm and are an alkylene group, and the alkylene group is defined as a linear or branched alkylene/alkenylene group having 1 to 20 carbon atoms or a carbon number of 6 to 10 Aromatic hydrocarbon group;
  • L represents 2-amino-2-carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation, N-polyethylene glycol-aminoethyl group having a molecular weight of 200-2000 or 2, 3-dihydroxypropyl
  • R x and R y are substituents of camptothecin or camptothecin analogs, as described in the technical scheme.
  • the structure of formula (1) contains a hydrophilic head portion, which makes the phospholipid compound water-soluble significantly superior to camptothecin, irinotecan (CPT11, bipermidine), and topotecan (9-dimethylamino-10- Hydroxycamptothecin), 7-ethyl-10-hydroxycamptothecin, has low toxicity and excellent antitumor activity;
  • R x and R y are a substituent of a camptothecin or a camptothecin analog, which are linked to a hydrophilic head of a phospholipid via an ester bond, and the ester bond is hydrolyzed by an esterase in the living body, a molecule of hi-tree
  • the alkali phospholipid compound releases two molecules of camptothecin or camptothecin analog, thereby allowing the antitumor efficacy of the camptothecin or camptothecin analog phospholipid compound to reach the camptothecin or camptothecin analog 2
  • the R x and R y substituents have a hydrophobic interaction and synergistically form a stable liposome, which is part of the structure of the liposome, which overcomes the disadvantage that the drug is easily leaked when the universal liposome encapsulates the hydrophobic drug, and simultaneously improves The efficiency of drug packaging.
  • R 1 and R 2 are spacer arms, and the spacer arms and the adjacent two ester bonds have the function of enhancing the hydrophobicity of the hydrophobic head, and neatly arranging and self-assembling the lipids of the camptothecin-like phospholipid compound.
  • the plastid nanoparticles are very advantageous to form stable liposome particles, thereby prolonging the release and anti-tumor action time of camptothecin or the like drugs, and at the same time, the camptothecin-like phospholipid compounds are easily added to the cells through the liposome form, Play a drug.
  • camptothecin-like phospholipid compound structure of the present invention contains two molecules of camptothecin or camptothecin analog active molecule, and the antitumor efficacy is camptothecin, irinotecan (CPT11, bipermazine), topologically Kang (9-dimethylamino-10-hydroxycamptothecin), 7-ethyl-10-hydroxycamptothecin about twice;
  • the camptothecin-based phospholipid compound of the present invention can be easily self-assembled into liposome nanoparticles by a thin film method or the like, and has a particle diameter of 10 to 1000 nm.
  • the camptothecin-based phospholipid compound of the present invention can be combined with a phospholipid complex system, and can be easily self-assembled into a liposome nanoparticle by a thin film method or the like, and has a particle diameter of 10 to 1000 nm.
  • the liposome structure of the camptothecin-like phospholipid compound liposome nanoparticles of the present invention has similarity to the cell membrane structure, is easily phagocytized by cells, and releases the active drug molecule camptothecin or camptothecin analog to exert antitumor effect. ;
  • camptothecin-like phospholipid liposome nanoparticles of the present invention have passive targeting, which is a common feature of liposome drug delivery systems; and camptothecin, irinotecan (CPT11, bipermazine), Topological Likang (9-dimethylamino-10-hydroxycamptothecin), 7-ethyl-10-hydroxycamptothecin, etc. Alkali analogues do not have this property;
  • camptothecin-based phospholipid compound of the present invention and the liposome nano-particle thereof are a novel release carrier of camptothecin drugs, and are also a prodrug;
  • the compound of the present invention or a pharmaceutical composition of the compound of the present invention and a conventional pharmaceutical carrier contains 0.1 to 100% by weight of the compound of the present invention, and has low toxicity and excellent antitumor activity.
  • the camptothecin-based phospholipid compound of the invention and the liposome nano-particle thereof can be used as a liquid preparation, a solid preparation, a semi-solid preparation, a sterilization preparation and a sterile preparation, and can be used in water, phosphate buffer, citrate buffer Forming a liposome under an aqueous phase;
  • camptothecin-based phospholipid compound liposome of the invention binds to a promoter containing a targeting group and has an active targeting effect
  • camptothecin-based phospholipid compound of the formula (1) and the liposome nano-particle thereof according to the invention is simple;
  • camptothecin-based phospholipid compound of the formula (1) and the liposome nanoparticle thereof of the invention are also novel prodrugs of camptothecin drugs, which exert pharmacological effects by esterase hydrolysis in the body and can prolong drug release.
  • Half-life, with low toxic side effects, anti-tumor effect is significantly better than camptothecin, irinotecan (CPT11, dipyridamidine), topotecan (9-dimethylamino-10-hydroxycyanaceae a camptothecin analog such as 7-ethyl-10-hydroxycamptothecin.
  • Figure 1 is a synthetic route diagram of a camptothecin-20-phospholipid compound.
  • Figure 2 is a synthetic route diagram of camptothecin-20-acetate-10-phospholipid compound.
  • Figure 3 is a synthetic route diagram of irinotecan-20-phospholipid compound.
  • Figure 4 is a synthetic route diagram of a camptothecin-7-methylene phospholipid compound.
  • Figure 5 is a synthetic route diagram of a 9-nitrocamptothecin-20-phospholipid compound.
  • Figure 6 is a synthetic route diagram of a hexacyclocamptothecin (Lurtotecan)-20-phospholipid compound.
  • Figure 7 is a synthetic route diagram of difluorocamptothecin (BN80915)-20-phospholipid compound.
  • Figure 8 is a synthetic route diagram of CKD-602-20-phospholipid compound.
  • Figure 9 is a synthetic route diagram of a 9-nitrocamptothecin-20-phosphatidylethanolamine compound.
  • Figure 10 is a synthetic route of a 9-nitrocamptothecin-20-phosphatidylethanolamine-N-polyethylene glycol compound.
  • Figure 11 is a synthetic route diagram of a camptothecin-20-phosphatidylserine compound.
  • Figure 12 is a synthetic route diagram of a camptothecin-20-phosphatidylglycerol compound.
  • Figure 13 is a synthetic route diagram of a 9-nitrocamptothecin-20-phthalate-phosphatidylcholine compound.
  • Figure 14 is a synthetic route diagram of a 9-aminocamptothecin-20-phospholipid compound.
  • Figure 15 is a synthetic route diagram of a camptothecin analog DX-8951f-20-phospholipid compound.
  • Figure 16 is a graph showing the particle size distribution of camptothecin-20-phospholipid compound liposomes.
  • Figure 17 is a diagram showing the morphology (transmission electron micrograph) of camptothecin-20-phospholipid compound liposome nanoparticles.
  • Fig. 18 is a schematic view showing the particle size distribution of the topographical Lik-10 phospholipid compound liposome.
  • a camptothecin-based phospholipid compound according to the present invention is a pharmaceutically acceptable salt of a compound of the following formula (1) or a compound of the formula (1) and a counter ion:
  • R 1 and R 2 are a spacer arm which is an alkylene group, and the alkylene group is defined as a linear or branched alkylene group/alkenylene group having 1 to 20 carbon atoms or a carbon number. a 6 to 10 aromatic hydrocarbon group; L represents 2-amino-2-carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation or 2,3-dihydroxypropyl;
  • R x and R y are a 20-position substituent of a camptothecin compound (20S) having the following formula (2), a 10-position substituent of a camptothecin compound (20S) of the formula (3) or a formula (4) Camptothecin-like compound (20S) 7-methylene substituent:
  • R 7 , R 9 , R 10 , R 11 and R 12 are each of the following groups: hydrogen, halogen, hydroxy, C 1 -C 6 straight Chain or branched alkyl/alkoxy, cyano, nitro, amino, C 1 -C 6 straight or branched haloalkyl/haloalkoxy, C 1 -C 6 straight or branched trisubstituted silyl group, C straight or branched chain of 1 -C 6 alkene group, oxymethylene group, C linear or branched alkyl ester group of 1 -C 6, C 1 -C 6 straight chain or Branched alkylcarbonyl, C 1 -C 6 straight or branched hydroxyalkyl, C 1 -C 6 linear or branched alkoxymethylene, phenoxymethylene, benzyl ester Methylidene, dimethylaminomethyl, dimethylaminoethyl, having a piperaz
  • R 7 , R 9 , R 10 , R 11 and R 12 may be the same group or different groups, or a part thereof may be the same group, and the other part may be a different group. .
  • R represents a C 1 - C 18 linear/branched alkyl group or a C 6 - C 10 aromatic hydrocarbon group.
  • the carbon atom at position 20 is in the S configuration.
  • R x and R y are preferably camptothecin (CPT) 20-hydroxyl substituent, 10-hydroxycamptothecin 20-position hydroxy substituent, 10-hydroxycamptothecin 10-position hydroxy substituent, irinotecan (CPT11, hydrazine) Camptothecin) 20-hydroxyl substituent, topological Likang (9-dimethylamino-10-hydroxycamptothecin) 10-hydroxyl substituent, topological Likang (9-dimethylamino-10-hydroxycime tree ⁇ ) 20-position hydroxy substituent, 7-ethyl-10-hydroxycamptothecin 20-position hydroxy substituent, 7-ethyl-10-hydroxycamptothecin 10-position hydroxy substituent, 9-aminocamptothecin 20 position Hydroxy substituent, hydroxyl residue at position 20 of 9-nitrocamptothecin, hydroxyl group at position 20 of difluorocamptothecin (BN80915), hydroxy substituent at position 20 of Lurtotec
  • Counter ions are cationic protons, sodium ions, potassium ions, calcium ions, iron ions, magnesium ions, ammonium ions, zinc ions, anion chloride ions, sulfate ions, sulfate ions, nitrate ions, carboxylate ions, carbonate ions , bromide ion, phosphate ion, formate, acetate, citric acid, lactate, fumarate, tartrate, gluconate ion or any combination of the two, preferably proton, sodium ion, chloride ion .
  • a method for preparing the camptothecin-based phospholipid compound which firstly reacts a hydroxyl group of camptothecin or a camptothecin analog with an acid anhydride or a carboxylic acid to prepare an intermediate containing a carboxyl group.
  • the intermediate is subjected to a condensation reaction with glycerylphosphocholine to obtain a camptothecin-like phosphatidylcholine compound; or the intermediate is condensed with monohydroxy-protected glycerol, deprotected, and trichloro
  • the oxyphosphorus reaction it is condensed with glycerol acetal, and the acetone is used to obtain camptothecin-like phosphatidylglycerol; or the intermediate is condensed with monohydroxy-protected glycerol, deprotected, and reacted with phosphorus oxychloride.
  • a condensation reaction with a hydroxyl group of a tert-butoxycarbonyl Boc-protected aminoethanol is carried out to obtain a camptothecin-like phosphatidylethanolamine by deprotection; or the intermediate is condensed with a monohydroxy-protected glycerol, deprotected, and After reacting with phosphorus oxychloride, the hydroxyl group of the serine protected by both the amino group and the carboxyl group is condensed, and the camptothecin-like phosphatidylserine is obtained by deprotecting the group.
  • Another method is to condense the hydroxyl group of camptothecin or camptothecin analog with the two carboxyl groups of the monohydroxy-protected glyceryl dicarboxylic acid di-monoester, deprotect the hydroxyl group, further activate the reaction with phosphorus oxychloride, and then Or condensation reaction with amino-protected aminoethanol hydroxy group, deprotection group, to obtain camptothecin-like phosphatidylethanolamine; or condensation reaction with glycerol acetal, deacetone, to obtain camptothecin-like phosphatidylglycerol; Or reacting with dimethylaminoethanol and methylating to obtain a camptothecin-like phosphatidylcholine compound; or reacting with a hydroxyl group of a serine protected by an amino group and a carboxyl group, and deprotecting the group to obtain a camptothecin group. Phosphatidylserine.
  • the condensation reagent is preferably 1,3 dicyclohexylcarbodiimide, dipyridine carbonate, 1-(3-dimethylaminopropyl)-3-ethylcarbimide hydrochloride, 1,3-diiso One of propyl carboimide or N,N-carbonyldiimidazole and a tertiary amine, and the tertiary amine is preferably 4-dimethylaminopyridine or 4-pyrrolidinopyridine.
  • the pharmaceutical composition of the present invention is a liquid preparation, a solid preparation, a semisolid preparation, a capsule, a granule, a gel, an injection, a sustained release preparation or a controlled release preparation.
  • the pharmaceutical composition of the present invention is a liposome nanoparticle having a particle diameter of 10 to 1000 nm and is composed of a self-assembly of a camptothecin-based phospholipid compound having a structure of the formula (1).
  • camptothecin-based phospholipid compound having the structure of the general formula (1) and the phospholipid are co-assembled to form liposome nanoparticles having a particle diameter of 10 to 1000 nm.
  • camptothecin-based phospholipid compound of the present invention for the preparation of an antitumor drug is characterized in that the method comprises the camptothecin-based phospholipid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier Prepared as a medicament.
  • the compound of the invention or a compound of the invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to provide a suitable administration form or dosage form for use as a human.
  • the pharmaceutical compositions of the invention contain from 0.1 to 100% by weight of a compound of the invention.
  • the compound of the present invention or a pharmaceutical composition containing the same may be administered enterally or parentally, such as orally, muscle, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum. It can be administered by injection, including intravenous, intramuscular, subcutaneous, intradermal and acupoint injection.
  • the dosage form can be a liquid dosage form or a solid dosage form.
  • the liquid dosage form may be a true solution type, a colloid type, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form.
  • Other dosage forms such as tablets, capsules, pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powders, and the like.
  • the compounds of the present invention can be formulated into common preparations, sustained release preparations, controlled release preparations, targeted preparations, and various microparticle delivery systems.
  • the carrier of the pharmaceutical composition of the present invention is, for example, a diluent and an absorbent such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystals.
  • a diluent and an absorbent such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystals.
  • wetting agent and binder such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin pulp, carboxy Methylcellulose sodium, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.
  • disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium hydrogencarbonate and tannic acid, calcium carbonate , polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil Etc., absorption enhancers such as quaternary ammonium salts, sodium lauryl sulfate, and the like
  • lubricants such as talc,
  • the compound of the present invention is formulated into an injectable preparation, such as a solution, a suspension solution, an emulsion, or a lyophilized powder injection.
  • the preparation may be aqueous or non-aqueous, and may contain one and/or more pharmacodynamics.
  • the diluent may be selected from the group consisting of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like.
  • an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional solubilizer, a buffer, a pH adjuster or the like may be added. These excipients are commonly used in the art.
  • the liposomal nanoparticles are prepared from the compound of the invention and the auxiliary agent, the particle size is 10-1000 nm, and the auxiliary agent is dimyristoyl phosphatidylcholine DMPC, distearoylphosphatidylcholine DSPC, dipalmitoyl phospholipid Acetylcholine DPPC, L- ⁇ -phosphatidylcholine- ⁇ -arachidonic acid- ⁇ -stearyl, didecanoylglycerol-3-phosphocholine DDPC, 1,2-dioleoylphosphatidylcholine DOPC, 2-oleoyl-1-palmitoylglycerol-3-phosphocholine POPC, 1-stearoyl-2-oleoylglycerol-3-phosphocholine SOPC, 1-stearoyl-2-palmitoylglycerol -3-phosphocholine SPPC, 1-myristoyl-2-stearoylphosphatidylcholine MSPC, 1-palmito
  • the compounds of the present invention exhibit good antitumor activity from the in vitro antitumor activity screening. Tests have shown that the compounds of the invention have less in vivo toxicity than camptothecin and topotecan. It can therefore be used as an anti-tumor drug in animals, preferably in mammals, especially humans.
  • the liposome nanoparticles of the present compounds exhibit good antitumor activity. Tests have shown that the compounds of the invention are much less toxic in vivo than camptothecin and topotecan. It can therefore be used as an anti-tumor drug in animals, preferably in mammals, especially humans.
  • the method for preparing the camptothecin-based phospholipid compound liposome nanoparticles of the present invention is a mixture of a camptothecin-based phospholipid compound of the present invention or a compound of the present invention and an auxiliary agent, by a film dispersion method, a reverse phase evaporation method, and a freeze drying method. It is prepared by a method such as a method, an ultrasonic dispersion method, a spray drying method, a film extrusion method, or a high pressure homogenization method.
  • the present invention utilizes 2 molecules of camptothecin or camptothecin analog as a hydrophobic tail and a phospholipid hydrophilic head to form a camptothecin-like phospholipid compound by covalent bond, and the solubility is significantly better than that of camptothecin or camptothecin.
  • the present invention prepares camptothecin-based phospholipid compounds into nanoparticles, has the characteristics of liposome, and has the characteristics of forming liquid preparations, solid preparations, semi-solid preparations, sterilization preparations and sterile preparations, and is used for tumor treatment. .
  • the camptothecin-like phospholipid compound nanoparticle is not only a prodrug, but also a novel drug release carrier of camptothecin drugs, having a targeting function; the camptothecin-based phospholipid compound of the present invention and liposomal nanoparticles thereof It is also a new prodrug of camptothecin drugs, and it is also a drug carrier. It can exert pharmacological effects by esterase hydrolysis in the body, which can prolong the half-life of drug release and has low toxic and side effects.
  • 2-hydroxycamptothecin 2g acetic acid 2g, EDCI 2g, HOBT 1g, adding dichloromethane or chloroform as reaction solvent, reacting at 40 ° C for 12h; washing with dilute hydrochloric acid three times, filtering dilute hydrochloric acid layer, taking filter cake; The methanol was dissolved, placed at 4 ° C for 12 h, filtered, and the cake was taken and dried to give 10-hydroxycamptothecin-20-acetate.
  • 2-ethyl-10-hydroxycamptothecin 2g acetic acid 3g, EDCI 2g, HOBT 1g, adding dichloromethane or chloroform as reaction solvent, reacting at 40 ° C for 12h; washing with dilute hydrochloric acid three times, filtering dilute hydrochloric acid layer, The filter cake was taken; dissolved in methanol, placed at 4 ° C for 12 h, filtered, and the filter cake was taken and dried to give 7-ethyl-10-hydroxycamptothecin-20-acetate.
  • Irinotecan (CPT11, bipermazine)-20-phospholipid compound is dissolved in 0.1M calcium lactate aqueous solution, and lyophilized to obtain irinotecan (CPT11, bipercamtoline) containing counterion calcium ion and lactate ion. ) 20-phospholipid compound solid powder.
  • the 9-nitrocamptothecin-20-phospholipid compound was dissolved in physiological saline and lyophilized to obtain a solid powder of 9-nitrocamptothecin-20-phospholipid compound containing a counter ion sodium ion and a chloride ion.
  • the Lurtotecan-20-phospholipid compound was dissolved in 0.1 M aqueous sodium phosphate solution, and lyophilized to obtain a solid powder of Lurtotecan-20-phospholipid compound containing a counter ion sulfate and sodium ion.
  • the difluorocamptothecin (BN80915)-20-phospholipid compound was dissolved in 0.1 M aqueous acetic acid solution, and lyophilized to obtain a solid powder of difluorocamptothecin (BN80915)-20-phospholipid compound containing an acetate counter ion.
  • Solvent was added; dilute acetic acid was added and reacted at room temperature for 2 h to remove solvent; 1 g of phosphorus oxychloride was added, 0.8 g of triethylamine was added, and dichloromethane and trichlorobenzene were added.
  • reaction solvent Methane or THF as the reaction solvent, react at 10 ° C for 24 h, and evaporate dry solvent; N-tert-butoxycarbonyl-aminoethanol 1 g, triethylamine 0.3 g, add dichloromethane, chloroform or DMSO as reaction solvent, 10 ° C After reacting for 24 hours, the solvent was evaporated to dryness; isopropyl alcohol and aqueous acetic acid were added, and the reaction was heated for 24 h. The obtained reaction liquid was purified by column chromatography to give the product 9-nitroc-cyanine-20-phosphatidylethanolamine compound 1.4 g.
  • reaction solvent Take mPEG20002g, CDI 0.2g, dichloromethane, chloroform or DMSO as the reaction solvent, react at room temperature for 2h; add 9g of 9-nitrocamptothecin-20-phosphatidylethanolamine compound in Example 11, DBU 1g, heat Reaction for 24h.
  • the obtained reaction liquid was purified by column chromatography to give the product 9-nitroc-cyanine-20-phosphatidylethanolamine-N-polyethylene glycol compound 1.8 g.
  • 2-nitrocamptothecin 2g phthalic anhydride 2g, DMAP 0.5g, triethylamine 2g, adding dichloromethane or chloroform as the reaction solvent, adding reflux reaction, reaction 12h; dilute hydrochloric acid washing three times, The dilute hydrochloric acid layer was filtered, and the filter cake was taken; dissolved in methanol, placed at 4 ° C for 12 h, filtered, and the filter cake was taken and dried to obtain an intermediate product of 9-nitrocamptothecin-20-phthalic acid monoester.
  • the particle size analysis results are shown in Fig. 16 below, and the average particle diameter was 35.40 nm.
  • the morphology of the nanoparticles measured by transmission electron microscopy is shown in Fig. 17.
  • Particle size analysis showed an average particle diameter of 380 nm.
  • camptothecin-20-phospholipid compound 0.5mmol, distearoylphosphatidylcholine DSPC 0.5mmol, distearoylphosphatidylethanolamine-polyethylene Alcohol DSPE-PEG-folic acid (polyethylene glycol molecular weight 400-3000) 0.1mmol, adding 20ml of chloroform, steaming dry solvent at 60 ° C; adding 20ml PBS (pH 7.4) at 60 ° C to form a camptothecin phospholipid compound Liposomal nanoparticle solution. Particle size analysis showed an average particle diameter of 620 nm. The camptothecin-20-phospholipid compound liposome nanoparticle solution was freeze-dried to obtain powdery nanoparticles.
  • Calf serum is a product of Nanjing Shengxing Biotechnology Co., Ltd.
  • DMSO is pure
  • RPMI1640 is a GIBCO product.
  • Collect well-grown tumor cells prepare 1 ⁇ 10-4/mL cell suspension with RPMI1640 medium containing 10% calf serum, and inoculate in a 96-well culture plate, each 100 ⁇ L (containing 1000 tumor cells). After being cultured at 37 ° C in a 5% CO 2 incubator for 24 hours, the drug was added. The experiment was set up with a blank control and a solvent control. The test sample was set to 4 concentrations, 3 parallel holes per concentration, 37 ° C, 5% CO 2 incubator. The culture was carried out for 4 days. The culture solution was discarded, and 100 ⁇ L of MTT solution (0.4 mg/mL, prepared in RPMI1640) was added to each well, and incubated at 37 ° C for 4 hours.
  • MTT solution 0.4 mg/mL, prepared in RPMI1640
  • the OD value was measured with a 550-type microplate reader at a detection wavelength of 540 nm and a reference wavelength of 450 nm.
  • the antitumor activity results of the derivatives of the present invention on human tumor cell lines are shown in Table 1. From the screening of in vitro antitumor activity, the half inhibitory concentration of the compounds of the present invention was less than that of irinotecan, indicating that the activity was superior to that of irinotecan. The half inhibitory concentration of the compound of the present invention is less than that of camptothecin, which is about half of camptothecin, probably because the camptothecin-based phospholipid compound contains two camptothecin molecular structural units.
  • MCF-7 human breast cancer cells
  • Hep G2 human liver cancer cells
  • A2780 human ovarian cancer cells
  • HCT-8 human colon cancer cells
  • A549 human lung adenocarcinoma cells
  • EC-304 human vascular endothelial normal cells
  • ND Not tested.
  • mice ICR mice, male, 18-22 g, purchased from Vitallihua Laboratory Animal Technology Co., Ltd.
  • the compound of the invention camptothecin-20-phospholipid compound, 10-hydroxycamptothecin-10-phospholipid compound, 7-methylene camptothecin phospholipid compound, 7-ethyl-10-hydroxycamptothecin-10-phospholipid
  • the results of the in vivo toxicity test of the compound showed that the maximum tolerated dose was greater than 100 mg/kg, indicating that the toxicity was much less than that of camptothecin and topotecan.
  • Camptothecin 12 Topotecan ⁇ 15 Camptothecin-20-phospholipid compound >100 Camptothecin-20-acetate-10-phospholipid compound >100 Camptothecin-7-methylene phospholipid compound >100 7-ethyl-camptothecin-20-acetate-10-phospholipid compound >100

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Abstract

一种喜树碱类磷脂化合物其制备方法及药物组合物与用途。药物组合物为喜树碱类磷脂化合物和药效学上可接受的载体的组合物，是液体制剂、固体制剂、半固体制剂、胶囊剂、颗粒剂、凝胶剂、注射剂。该药物组合物是喜树碱类磷脂化合物或喜树碱类磷脂化合物和助剂制成的脂质体纳米颗粒，粒径10-1000纳米。

Description

一种喜树碱类磷脂化合物、其药物组合物及应用 技术领域
本发明是一种具有抗肿瘤作用的喜树碱类磷脂化合物及其药物组合物与用途，涉及医药技术领域。
背景技术
喜树碱(Camptothecin，CPT)是一种生物碱，具有较强的细胞毒活性,可用于治疗多种恶性肿瘤，如胃癌、肝癌、膀胱癌和白血病等。科学家发现喜树碱类化合物独特的抗癌机制：通过形成喜树碱-拓扑异构酶I-DNA稳定的三联体络合物,使DNA双螺旋结构破裂后不能复制。喜树碱类化合物是最经典的Topo I的特异性抑制剂。Topo I已成为设计新型抗癌药物的最热门靶点之一。Topo I抑制剂被美国国立癌症研究所(NCI)药物机制分析网络系统列为重点研究的六大类抗肿瘤药物之一。
喜树碱及其类似物可以从植物中提取，也可进行化学合成。这类喜树碱及其类似物等在发挥其抗肿瘤活性的同时还产生骨髓抑制、呕吐和腹泻等严重副作用。喜树碱及其类似物用于肿瘤治疗的最大缺陷是水溶性差，难以获得高浓度的溶液。为了改善喜树碱的缺陷，许多研究机构和企业对喜树碱的化学结构进行了改性，合成了数百种喜树碱衍生物。至今已经有2种喜树碱衍生物被批准用于临床肿瘤治疗，分别是依替立康irinotecan和拓扑立康topotecan，但是其水溶性仍然较低。此外，9-AC，9-NC，G7147211，DX-8951f，Bay38-3441，BN80915和CKD-602等分别处于临床试验阶段。更多的喜树碱类似物没有得到应用。部分原因是因为水溶性太差，喜树碱及其类似物用于肿瘤治疗存在副反应大等缺陷。
喜树碱及其类似物的分子中含有四个六元环和一个五元环，其中E环是一个α-羟基内酯，且有一个手性中心，为20S构型。E环羰基活性高，在亲核试剂的进攻下，很容易开环。因为喜树碱在体内存在着关闭的内酯形式和开环的羧酸盐形式之间的平衡，而开环形式取决于pH值和动物种类。特别是人血清白蛋白(HSA)优先与喜树碱的开环形式结合，形成稳定的复合物，使平衡向开环形式位移，使得体内具有抗肿瘤活性的内酯含量太低，导致在人体中的抗肿瘤活性很低。据称，喜树碱的严重毒性(小鼠最大耐受剂量MTD 12mg/kg)主要是因为当药物进入人体内后喜树碱的开环形式与HSA紧密结合，使喜树碱在体内的平衡从关闭内酯形式向开环形式转移，喜树碱很快开环(半衰期小于半小时)。
由于喜树碱和10-羟基喜树碱是未经修饰的天然产物，毒性较大，抗肿瘤活性低，限制了它们在临床的应用；再者，病人长期使用这些药物易产生耐药性，迫切需要提高喜树碱或其类似物的水溶性，寻找高效低毒的喜树碱类抗肿瘤药物。
因此有必要通过对喜树碱及其衍生物进行结构修饰，以达到提高药物溶解性、靶向性，防止内酯开环，提高疗效，降低毒性的目的，也一直是药物领域的研究热点。
脂质体是一种具有靶向给药功能的新型药物制剂，在抗癌药物研究领域具有极其广泛的应用。磷脂是构成脂 质体的主要化学成分，由具有磷酸相连的取代基因构成的亲水头和长烃基链构成的疏水尾组成。当分散于水相时，分子的疏水尾部倾向于聚集在一起，避开水相，而亲水头部暴露在水相，形成具有双分子层结构的的封闭囊泡，形成脂质体。由于天然磷脂疏水尾部多含有不饱和键，对脂质体的稳定性造成了很大的影响，因此对磷脂疏水尾部进行结构修饰以提高其稳定性逐渐成为研究热点。作为载体时，疏水性药物通常在脂质体的双脂质层中，亲水性药物在脂质体内的水相中。由于脂质体膜的流动性，导致药物易于渗漏出来，使包裹的药物难以发挥良好的药效。
本发明利用2分子的喜树碱及其类似物化学结构部分作为疏水尾和磷脂亲水头通过共价键相连，制备喜树碱类磷脂化合物，溶解性显著优于喜树碱及其衍生物，可以单独或者与磷脂混合制成脂质体纳米颗粒；本发明将喜树碱类磷脂化合物具有可形成液体制剂、固体制剂、半固体制剂、灭菌制剂和无菌制剂的特性；本发明将喜树碱类磷脂化合物制备成纳米颗粒，具有脂质体的特性，具有可形成液体制剂、固体制剂、半固体制剂、灭菌制剂和无菌制剂的特性，用于肿瘤治疗。这种喜树碱类磷脂化合物在体内被酯酶降解，释放药物，不仅是一种前药，也是喜树碱类药物全新的药物释放载体；本发明的喜树碱类磷脂化合物，具有较低的毒副作用，脂质体具有靶向功能。
发明内容
技术问题：本发明的目的在于提供一种提高喜树碱或其类似物溶解性的喜树碱类磷脂化合物，同时基于该喜树碱类磷脂化合物的药物组合物，以及该喜树碱类磷脂化合物在制备抗肿瘤药物中的应用。
技术方案：为了对化合物命名和定位，定义喜树碱各原子的位置如下：
Figure PCTCN2015089181-appb-000001
本发明涉及的所有喜树碱及其类似物或其磷脂化合物的母环均按照式(A)中的位置进行定义，且20位碳原子为S构型。
本发明的喜树碱类磷脂化合物，为下列通式(1)的化合物或所述通式(1)的化合物与抗衡离子所形成的在药学上可接受的盐：
Figure PCTCN2015089181-appb-000002
式(1)中，R1和R2为间隔臂，是亚烃基，所述亚烃基定义为碳原子数为1～20的直链或支链的亚烷烃基/亚烯烃基或碳原子数为6～10的芳烃基；L代表2-氨基-2-羧基乙基、2-氨基乙基、2-三甲基胺基乙基阳离子或2、3-二羟基丙基；
Rx和Ry是结构为下列式(2)的喜树碱类化合物(20S)20位取代基、式(3)的喜树碱类化合物(20S)10位取代基或式(4)的喜树碱类化合物(20S)7位亚甲基取代基：
Figure PCTCN2015089181-appb-000003
式(2)和(3)和(4)中，R7、R9、R10、R11和R12均为以下任一种基团：氢、卤素、羟基、C1-C6的直链或支链的烷基/烷氧基、氰基、硝基、氨基、C1-C6的直链或支链的卤代烷基/卤代烷氧基、C1-C6的直链或支链烷基的三取代硅烷基、C1-C6的直链或支链烯烃基、甲醛基、C1-C6的直链或支链烷基酯基、C1-C6的直链或支链烷基羰基、C1-C6的直链或支链的羟基烷基、C1-C6的直链或支链烷氧基亚甲基、苯氧基亚甲基、苯甲酯基亚甲基、二甲胺基甲基、二甲基胺基乙基、具有哌嗪结构
Figure PCTCN2015089181-appb-000004
的取代基、具有结构
Figure PCTCN2015089181-appb-000005
的取代基、具有联哌羧基结构
Figure PCTCN2015089181-appb-000006
的取代基和-CH2NR13R14，所述-CH2NR13R14中，R13和R14为C1-C6的直链烷基、C1-C6的支链烷基、或者R11和R10形成结构
Figure PCTCN2015089181-appb-000007
的取代基；
式(3)和(4)中R代表C1-C18的直链/支链烷基或C6-C10的芳烃基。
式(2)和(3)和(4)中，20位碳原子均为S构型。
本发明中，Rx和Ry优选喜树碱(CPT)20位羟基取代基、10-羟基喜树碱20位羟基取代基、10-羟基喜树碱10位羟基取代基、伊立替康(CPT11，联哌喜树碱)20位羟基取代基、拓扑立康(9-二甲基氨基-10-羟基喜树缄)10位羟基取代基、拓扑立康(9-二甲基氨基-10-羟基喜树缄)20位羟基取代基、7-乙基-10-羟基喜树碱20位羟基 取代基、7-乙基-10-羟基喜树碱10位羟基取代基，
本发明喜树碱类磷脂化合物的优选方案中，抗衡离子是阳离子质子、钠离子、钾离子、钙离子、铁离子、镁离子、铵离子、锌离子、阴离子氯离子、硫酸根离子、硫酸根离子、硝酸根离子、羧酸根离子、碳酸根离子、溴离子、磷酸根离子、甲酸根、乙酸根、柠檬酸更、乳酸根、富马酸根、酒石酸根、葡萄糖酸根离子的任一种或任两种的组合，优选质子、钠离子、氯离子。
本发明的制备上述喜树碱类磷脂化合物的方法，首先将喜树碱或喜树碱类似物的羟基与二酸酐或羧酸反应，制备得到含有羧基的中间体，然后进一步将所述中间体与甘油磷酸胆碱进行在缩合试剂作用下缩合反应，即得到喜树碱类磷脂化合物。
本发明制备方法的优选方案中，使用缩合试剂优选1，3二环己基碳二亚胺、二吡啶碳酸酯、1-(3-二甲胺丙基)-3-乙基碳酰亚胺盐酸盐、1，3-二异丙基碳酰亚胺或N,N-羰基二咪唑中的一种和三级胺，三级胺优选4-二甲氨基吡啶或4-吡咯烷基吡啶。
本发明的药物组合物，包括上述的喜树碱类磷脂化合物或所述的喜树碱类磷脂化合物与药效学上可接受的载体。
本发明的药物组合物是液体制剂、固体制剂、半固体制剂、胶囊剂、颗粒剂、凝胶剂、注射剂、缓释制剂或控释制剂。
本发明药物组合物的优选方案中，药物组合物为粒径10-1000纳米的脂质体纳米颗粒，该药物组合物中还包括助剂。
本发明药物组合物的上述优选方案中，助剂是磷脂。
本发明的喜树碱类磷脂化合物在制备抗肿瘤药物中的应用，将喜树碱类磷脂化合物或其在药学上可接受的盐，与药效学上可接受的载体制备成药剂。
本发明化合物可以异构体的形式存在，而且通常所述的“本发明化合物”包括该化合物的异构体。
本发明化合物可存在不对称中心具有S构型或R构型，本发明包括所有可能的立体异构体以及两种或多种异构体的混合物。
本发明还涉及含有作为活性成份的本发明化合物或本发明化合物和常规药物助剂的药物组合物。通常本发明药物组合物含有0.1-100重量％的本发明化合物。
本发明化合物的药物组合物可根据本领域公知的方法制备。用于此目的时，如果需要，可将本发明化合物或本发明化合物与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人药使用的适当的施用形式或剂量形式。
本发明化合物或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、肌肉、皮下、鼻腔、口腔粘膜、皮肤、腹膜或直肠等。
本发明化合物或含有它的药物组合物的给药途径可为注射给药，包括静脉注射、肌肉注射、皮下注射、皮内注射和穴位注射等。给药剂型可以是液体剂型、固体剂型。如液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂等。
本发明化合物可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂，如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。
为了将给药单元制成胶囊，将有效成分本发明化合物与上述的各种载体混合，并将由此得到的混合物置于硬的明胶胶囊或软胶囊中。也可将有效成分本发明化合物制成微囊剂，混悬于水性介质中形成混悬剂，亦可装入硬胶囊中或制成注射剂应用。
将本发明化合物制成注射用制剂，如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂，这种制剂可以是含水或非水的，可含一种和/或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、1，3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。这些辅料是本领域常用的。
如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
由本发明化合物制成脂质体纳米颗粒，颗粒粒径10-1000纳米。
由本发明化合物和助剂制成脂质体纳米颗粒，粒径10-1000纳米，使用的助剂是二肉豆蔻酰磷脂酰胆碱DMPC，二硬脂酰磷脂酰胆碱DSPC，二棕榈酰磷脂酰胆碱DPPC，L-α-磷脂酰胆碱-β-花生四烯酰-γ-硬脂酰，二癸酰基甘油-3-磷酸胆碱DDPC，1,2-二油酰基磷脂酰胆碱DOPC，2-油酰-1-棕榈酰甘油-3-磷酸胆碱POPC，1-硬脂酰 -2-油酰甘油-3-磷酸胆碱SOPC，1-硬脂酰-2-棕榈酰甘油-3-磷酸胆碱SPPC，1-肉豆蔻酰基-2-硬脂酰基磷脂酰胆碱MSPC，1-棕榈酰基-2-肉豆蔻酰基磷脂酰胆碱PMPC，1-棕榈酰基-2-硬脂酰基磷脂酰胆碱PSPC，1-硬脂酰基-2-肉豆蔻酰基磷脂酰胆碱SMPC，1-肉豆蔻酰基-2-油酰基磷脂酰胆碱MOPC，1-棕榈酰基-2-油酰基磷脂酰胆碱POPC，1-油酰-2-棕榈酰甘油-3-磷酸胆碱OPPC，1-油酰-2-硬脂酰甘油-3-磷酸胆碱OSPC，1,2-二月桂酰磷脂酰胆碱DLPC，1,2-二花生四烯酰基磷脂酰胆碱DAPC，1-棕榈酰-2-肉豆蔻酰基-甘油-3-磷酸胆碱PMPC，1-肉豆蔻酰基-2-硬脂酰甘油-3-磷酸胆碱MSPC，1-肉豆蔻酰基-2-棕榈酰甘油-3-磷酸胆碱MPPC，1-棕榈酰-2花生四烯酰-甘油-3-磷酸胆碱PAPC，1-硬脂酰-2-花生四烯酰甘油-3-磷酸胆碱SAPC，1-油酰-2-肉豆蔻酰基甘油-3-磷酸胆碱OMPC，1-棕榈酰基-2-硬脂酰甘油-3-磷酸胆碱HSPC，二芥酰基甘油-3-磷酸胆碱DEPC，1,2-二月桂酰磷脂酰乙醇胺,二肉豆蔻酰基磷脂酰乙醇胺DMPE，二棕榈酰基磷脂酰乙醇胺DPPE，二硬脂酰基磷脂酰乙醇胺DSPE，二油酰基磷脂酰乙醇胺DOPE，二芥酰基磷脂酰乙醇胺DEPE，1-棕榈酰基-2-油酰基基磷脂酰乙醇胺DLPE，1-棕榈酰基-2-油酰基基磷脂酰乙醇胺POPE，二硬脂酰基磷脂酰乙醇胺-聚乙二醇DSPE-PEG2000(聚乙二醇分子量400-3000)，二硬脂酰磷脂酰聚乙二醇单甲醚(聚乙二醇分子量400-3000)或胆固醇中的一种或多种。助剂还含有一种靶向基团叶酸、半乳糖、抗体、生物素或多肽。
本发明的药物组合物脂质体纳米颗粒，是液体制剂、固体制剂、半固体制剂、胶囊剂、颗粒剂、凝胶剂、注射剂、缓释制剂或控释制剂。
从体外抗肿瘤活性筛选来看，本发明化合物表现良好的抗肿瘤活性。试验表明本发明化合物的体内毒性，小于喜树碱、拓扑替康。因此可作为抗肿瘤药物用于动物，优选用于哺乳动物，特别是人。
从体外抗肿瘤活性筛选来看，本发明化合物脂质体纳米颗粒表现良好的抗肿瘤活性。试验表明本发明化合物的体内毒性远小于喜树碱、拓扑替康。因此可作为抗肿瘤药物用于动物，优选用于哺乳动物，特别是人。
本发明的喜树碱类磷脂化合物脂质体纳米颗粒的制备方法，是由本发明化合物喜树碱类磷脂化合物或本发明化合物与助剂的混合物，通过薄膜分散法、逆相蒸发法、冷冻干燥法、超声波分散法、喷雾干燥法、膜挤压法、或高压均质法等方法制备。
本发明利用2分子的喜树碱或喜树碱类似物作为疏水尾和磷脂亲水头通过共价键相连，制备喜树碱类磷脂化合物，溶解性显著优于喜树碱或喜树碱类似物；本发明将喜树碱类磷脂化合物制备成纳米颗粒，具有脂质体的特性，具有可形成液体制剂、固体制剂、半固体制剂、灭菌制剂和无菌制剂的特性，用于肿瘤治疗。这种喜树碱类磷脂化合物纳米颗粒不仅是一种前药，也是喜树碱类药物全新的药物释放载体，具有靶向功能；本发明的喜树碱类磷脂化合物及其脂质体纳米颗粒也是一种全新的喜树碱类药物的前药，也是药物载体，在体内经酯酶水解发挥药效，具有低毒副作用。
本发明的通式(1)的喜树碱类磷脂化合物以及这些化合物所形成的药学上可接受的含有抗衡离子的盐，具有良好的水溶性，具有抗肿瘤作用，能延长药物释放半衰期，并具有较低的毒副作用。
有益效果：本发明与现有技术相比，具有以下优点：
本发明的喜树碱类磷脂化合物具有如下结构通式(1)：
Figure PCTCN2015089181-appb-000008
其中，R1和R2为间隔臂，是亚烃基，所述亚烃基定义为碳原子数为1～20的直链或支链的亚烷烃基/亚烯烃基或碳原子数为6～10的芳烃基；L代表2-氨基-2-羧基乙基、2-氨基乙基、2-三甲基胺基乙基阳离子、分子量为200-2000的N-聚乙二醇-氨基乙基或2、3-二羟基丙基，Rx和Ry是喜树碱或喜树碱类似物的取代基，具体见技术方案。
式(1)结构中含有亲水头部分，使磷脂化合物水溶性显著优于喜树碱、伊立替康(CPT11，联哌喜树碱)、拓扑立康(9-二甲基氨基-10-羟基喜树缄)、7-乙基-10-羟基喜树碱，具有低毒性和优异的抗肿瘤活性；
式(1)中Rx和Ry是喜树碱或喜树碱类似物的取代基，通过酯键与磷脂亲水头相连，在生物体中由酯酶水解该酯键，一分子喜树碱类磷脂化合物释放出两个分子的喜树碱或喜树碱类似物，从而使喜树碱或喜树碱类似物磷脂化合物的抗肿瘤药效达到喜树碱或喜树碱类似物的2倍；Rx和Ry取代基具有疏水作用，相互协同，直接构成稳定的脂质体，是脂质体结构的一部分，克服了通用脂质体包裹疏水药物时药物易于泄漏的缺点，同时提高药物包裹的效率。
式(1)中R1和R2为间隔臂，该间隔臂和相邻的两个酯键具有增强疏水头部疏水性的作用，对喜树碱类磷脂化合物的整齐排列并自组装成脂质体纳米颗粒非常有利，形成稳定的脂质体颗粒，从而延长喜树碱或类似物药物的释放和抗肿瘤作用时间，同时使喜树碱类磷脂化合物通过脂质体形式易于加入细胞内，发挥药效。
本发明的喜树碱类磷脂化合物结构中含两分子喜树碱或喜树碱类似物活性分子，抗肿瘤药效是喜树碱、伊立替康(CPT11，联哌喜树碱)、拓扑立康(9-二甲基氨基-10-羟基喜树缄)、7-乙基-10-羟基喜树碱的2倍左右；
本发明的喜树碱类磷脂化合物可以采用薄膜法等非常容易地自组装成脂质体纳米颗粒，粒径10-1000纳米，
本发明的喜树碱类磷脂化合物可以与磷脂组成复合体系，采用薄膜法等非常容易地自组装成脂质体纳米颗粒，粒径10-1000纳米，
本发明的喜树碱类磷脂化合物脂质体纳米颗粒的脂质体结构与细胞膜结构具有相似性，易于被细胞吞噬，并释放活性药物分子喜树碱或喜树碱类似物，发挥抗肿瘤作用；
本发明的喜树碱类磷脂化合物脂质体纳米颗粒具有被动靶向作用，这是脂质体释药系统的共同特征；而喜树碱、伊立替康(CPT11，联哌喜树碱)、拓扑立康(9-二甲基氨基-10-羟基喜树缄)、7-乙基-10-羟基喜树碱等喜树 碱类似物均不具备这种特性；
本发明的喜树碱类磷脂化合物及其脂质体纳米颗粒是一种全新的喜树碱类药物的释放载体，也是一种前药；
本发明的化合物或本发明的化合物和常规药物载体的药物组合物，含有0.1-100重量％的本发明化合物，具有低毒性和优异的抗肿瘤活性。
本发明的喜树碱类磷脂化合物及其脂质体纳米颗粒，可用作液体制剂、固体制剂、半固体制剂、灭菌制剂和无菌制剂，可在水、磷酸缓冲液、柠檬酸缓冲液等水相体系下形成脂质体；
本发明的喜树碱类磷脂化合物脂质体结合含有靶向基团的助剂，具有主动靶向作用；
本发明所涉及式(1)的喜树碱类磷脂化合物及其脂质体纳米颗粒制备工艺简单；
本发明所涉及式(1)的喜树碱类磷脂化合物及其脂质体纳米颗粒也是一种全新的喜树碱类药物的前药，在体内经酯酶水解发挥药效，能延长药物释放半衰期，并具有较低的毒副作用，抗肿瘤作用显著优于喜树碱、伊立替康(CPT11，联哌喜树碱)、拓扑立康(9-二甲基氨基-10-羟基喜树缄)、7-乙基-10-羟基喜树碱等喜树碱类似物。
附图说明
图1为喜树碱-20-磷脂化合物合成路线图。
图2为喜树碱-20-乙酸酯-10-磷脂化合物的合成路线图。
图3为伊立替康-20-磷脂化合物合成路线图。
图4为喜树碱-7-亚甲基磷脂化合物合成路线图。
图5为9-硝基喜树碱-20-磷脂化合物的合成路线图。
图6为六环喜树碱(Lurtotecan)-20-磷脂化合物的合成路线图。
图7为二氟喜树碱(BN80915)-20-磷脂化合物的合成路线图。
图8为CKD-602-20-磷脂化合物的合成路线图。
图9为9-硝基喜树碱-20-磷脂酰乙醇胺化合物的合成路线图。
图10为9-硝基喜树碱-20-磷脂酰乙醇胺-N-聚乙二醇化合物的合成路线。
图11为喜树碱-20-磷脂酰丝氨酸化合物的合成路线图。
图12为喜树碱-20-磷脂酰甘油化合物的合成路线图。
图13为9-硝基喜树碱-20-邻苯二甲酸酯-磷脂酰胆碱化合物的合成路线图。
图14为9-氨基喜树碱-20-磷脂化合物的合成路线图。
图15为喜树碱类似物DX-8951f-20-磷脂化合物的合成路线图。
图16为喜树碱-20-磷脂化合物脂质体的粒径分布示意图。
图17为喜树碱-20-磷脂化合物脂质体纳米颗粒的形态(透射电镜图)。
图18为拓扑立康-10-磷脂化合物脂质体的粒径分布示意图。
具体实施方式
下面结合说明书附图和实施例对本发明的的技术方案做进一步详细说明。
本发明所涉及的一种喜树碱类磷脂化合物，是下列通式(1)的化合物或所述通式(1)的化合物与抗衡离子所形成的在药学上可接受的盐：
Figure PCTCN2015089181-appb-000009
式(1)中，R1和R2为间隔臂，是亚烃基，所述亚烃基定义为碳原子数为1～20的直链或支链的亚烷烃基/亚烯烃基或碳原子数为6～10的芳烃基；L代表2-氨基-2-羧基乙基、2-氨基乙基、2-三甲基胺基乙基阳离子或2、3-二羟基丙基；
Rx和Ry是结构为下列式(2)的喜树碱类化合物(20S)20位取代基、式(3)的喜树碱类化合物(20S)10位取代基或式(4)的喜树碱类化合物(20S)7位亚甲基取代基：
Figure PCTCN2015089181-appb-000010
式(2)和(3)和(4)中，R7、R9、R10、R11和R12均为以下任一种基团：氢、卤素、羟基、C1-C6的直链或支链的烷基/烷氧基、氰基、硝基、氨基、C1-C6的直链或支链的卤代烷基/卤代烷氧基、C1-C6的直链或支链烷基的三取代硅烷基、C1-C6的直链或支链烯烃基、甲醛基、C1-C6的直链或支链烷基酯基、C1-C6的直链或支链烷基羰基、C1-C6的直链或支链的羟基烷基、C1-C6的直链或支链烷氧基亚甲基、苯氧基亚甲基、苯甲酯基亚甲基、二甲胺基甲基、二甲基胺基乙基、具有哌嗪结构
Figure PCTCN2015089181-appb-000011
的取代基、具有结构
Figure PCTCN2015089181-appb-000012
的取代基、具有联哌羧基结构
Figure PCTCN2015089181-appb-000013
的取代基和-CH2NR13R14，所述-CH2NR13R14中，R13和R14为C1-C6的直链烷基、C1-C6的支链烷基、或者R11和R10形成结构
Figure PCTCN2015089181-appb-000014
的取代基；
R7、R9、R10、R11和R12可以是相同的基团，也可以是互不相同的基团，还可以是其中一部分为相同的基 团，而另一部分为不同的基团。
式(3)和(4)中R代表C1-C18的直链/支链烷基或C6-C10的芳烃基。
式(2)和(3)和(4)中，20位碳原子为S构型。
Rx和Ry优选喜树碱(CPT)20位羟基取代基、10-羟基喜树碱20位羟基取代基、10-羟基喜树碱10位羟基取代基、伊立替康(CPT11，联哌喜树碱)20位羟基取代基、拓扑立康(9-二甲基氨基-10-羟基喜树缄)10位羟基取代基、拓扑立康(9-二甲基氨基-10-羟基喜树缄)20位羟基取代基、7-乙基-10-羟基喜树碱20位羟基取代基、7-乙基-10-羟基喜树碱10位羟基取代基、9-氨基喜树碱20位羟基取代基、9-硝基喜树碱20位羟基取代基、二氟喜树碱(BN80915)20位羟基取代基、六环喜树碱(Lurtotecan)-20位羟基取代基、喜树碱类似物DX-8951f-20位羟基取代基、喜树碱类似物CKD-602-20位羟基取代基。
抗衡离子是阳离子质子、钠离子、钾离子、钙离子、铁离子、镁离子、铵离子、锌离子、阴离子氯离子、硫酸根离子、硫酸根离子、硝酸根离子、羧酸根离子、碳酸根离子、溴离子、磷酸根离子、甲酸根、乙酸根、柠檬酸更、乳酸根、富马酸根、酒石酸根、葡萄糖酸根离子的任一种或任两种的组合，优选质子、钠离子、氯离子。
制备所述的喜树碱类磷脂化合物的方法，该方法首先将喜树碱或喜树碱类似物的羟基与酸酐或羧酸反应，制备得到含有羧基的中间体。然后，将所述中间体与甘油磷酸胆碱进行缩合反应，即得到喜树碱类磷脂酰胆碱化合物；或者，将所述中间体与单羟基保护甘油缩合，经脱保护，并与三氯氧磷反应后，与甘油缩丙酮缩合反应，经脱丙酮，即得到喜树碱类磷脂酰甘油；或者，所述中间体与单羟基保护甘油缩合，经脱保护，并与三氯氧磷反应后，与叔丁氧羰基Boc保护的氨基乙醇的羟基缩合反应，经脱保护基，即得到喜树碱类磷脂酰乙醇胺；或者，所述中间体与单羟基保护甘油缩合，经脱保护，并与三氯氧磷反应后，与氨基和羧基均保护的丝氨酸的羟基缩合反应，经脱保护基，即得到喜树碱类磷脂酰丝氨酸。
另一方法是将喜树碱或喜树碱类似物的羟基与单羟基保护的甘油基二羧酸二单酯的二个羧基缩合，脱羟基保护基，进一步与三氯氧磷反应活化，然后，或者与氨基保护的氨基乙醇的羟基缩合反应，经脱保护基，即得到喜树碱类磷脂酰乙醇胺；或者与甘油缩丙酮缩合反应，经脱丙酮，即得到喜树碱类磷脂酰甘油；或者与二甲基氨基乙醇反应，并甲基化，即得到喜树碱类磷脂酰胆碱化合物；或者与氨基和羧基均保护的丝氨酸的羟基反应，经脱保护基，即得到喜树碱类磷脂酰丝氨酸。
使用缩合试剂优选1，3二环己基碳二亚胺、二吡啶碳酸酯、1-(3-二甲胺丙基)-3-乙基碳酰亚胺盐酸盐、1，3-二异丙基碳酰亚胺或N,N-羰基二咪唑中的一种和三级胺，三级胺优选4-二甲氨基吡啶或4-吡咯烷基吡啶。
本发明的药物组合物是液体制剂、固体制剂、半固体制剂、胶囊剂、颗粒剂、凝胶剂、注射剂、缓释制剂或控释制剂。
本发明的药物组合物，是粒径10-1000纳米的脂质体纳米颗粒，由具有通式(1)结构的喜树碱类磷脂化合物自组装构成。
由具有通式(1)结构的喜树碱类磷脂化合物与磷脂共同自组装构成粒径10-1000纳米的脂质体纳米颗粒。
本发明的喜树碱类磷脂化合物在制备抗肿瘤药物中的应用其特征在于，该方法将所述喜树碱类磷脂或其在药学上可接受的盐，与药效学上可接受的载体制备成药剂。可将本发明化合物或本发明化合物与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人药使用的适当的施用形式或剂量形式。通常本发明药物组合物含有0.1-100重量％的本发明化合物。
本发明化合物或含有它的药物组合物给药途径可为肠道或非肠道，如口服、肌肉、皮下、鼻腔、口腔粘膜、皮肤、腹膜或直肠等。可为注射给药，包括静脉注射、肌肉注射、皮下注射、皮内注射和穴位注射等。给药剂型可以是液体剂型、固体剂型。如液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂等。
本发明化合物可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
本发明的药物组合物采用的载体是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
将本发明化合物制成注射用制剂，如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂，这种制剂可以是含水或非水的，可含一种和/或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、1，3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。这些辅料是本领域常用的。
由本发明化合物和助剂制成脂质体纳米颗粒，粒径10-1000纳米，使用的助剂是二肉豆蔻酰磷脂酰胆碱DMPC，二硬脂酰磷脂酰胆碱DSPC，二棕榈酰磷脂酰胆碱DPPC，L-α-磷脂酰胆碱-β-花生四烯酰-γ-硬脂酰，二癸酰基甘油-3-磷酸胆碱DDPC，1,2-二油酰基磷脂酰胆碱DOPC，2-油酰-1-棕榈酰甘油-3-磷酸胆碱POPC，1-硬脂酰-2-油酰甘油-3-磷酸胆碱SOPC，1-硬脂酰-2-棕榈酰甘油-3-磷酸胆碱SPPC，1-肉豆蔻酰基-2-硬脂酰基磷脂酰胆碱MSPC，1-棕榈酰基-2-肉豆蔻酰基磷脂酰胆碱PMPC，1-棕榈酰基-2-硬脂酰基磷脂酰胆碱PSPC，1-硬脂酰基-2-肉豆蔻酰基磷脂酰胆碱SMPC，1-肉豆蔻酰基-2-油酰基磷脂酰胆碱MOPC，1-棕榈酰基-2-油酰基磷脂酰胆碱POPC，1-油酰-2-棕榈酰甘油-3-磷酸胆碱OPPC，1-油酰-2-硬脂酰甘油-3-磷酸胆碱OSPC，1,2-二月桂酰磷脂酰 胆碱DLPC，1,2-二花生四烯酰基磷脂酰胆碱DAPC，1-棕榈酰-2-肉豆蔻酰基-甘油-3-磷酸胆碱PMPC，1-肉豆蔻酰基-2-硬脂酰甘油-3-磷酸胆碱MSPC，1-肉豆蔻酰基-2-棕榈酰甘油-3-磷酸胆碱MPPC，1-棕榈酰-2花生四烯酰-甘油-3-磷酸胆碱PAPC，1-硬脂酰-2-花生四烯酰甘油-3-磷酸胆碱SAPC，1-油酰-2-肉豆蔻酰基甘油-3-磷酸胆碱OMPC，1-棕榈酰基-2-硬脂酰甘油-3-磷酸胆碱HSPC，二芥酰基甘油-3-磷酸胆碱DEPC，1,2-二月桂酰磷脂酰乙醇胺,二肉豆蔻酰基磷脂酰乙醇胺DMPE，二棕榈酰基磷脂酰乙醇胺DPPE，二硬脂酰基磷脂酰乙醇胺DSPE，二油酰基磷脂酰乙醇胺DOPE，二芥酰基磷脂酰乙醇胺DEPE，1-棕榈酰基-2-油酰基基磷脂酰乙醇胺DLPE，1-棕榈酰基-2-油酰基基磷脂酰乙醇胺POPE，二硬脂酰基磷脂酰乙醇胺-聚乙二醇DSPE-PEG2000(聚乙二醇分子量400-3000)，二硬脂酰磷脂酰聚乙二醇单甲醚(聚乙二醇分子量400-3000)或胆固醇中的一种或多种。助剂还含有一种靶向基团叶酸、半乳糖、抗体、生物素或多肽。
从体外抗肿瘤活性筛选来看，本发明化合物表现良好的抗肿瘤活性。试验表明本发明化合物的体内毒性，小于喜树碱、拓扑替康。因此可作为抗肿瘤药物用于动物，优选用于哺乳动物，特别是人。
从体外抗肿瘤活性筛选来看，本发明化合物脂质体纳米颗粒表现良好的抗肿瘤活性。试验表明本发明化合物的体内毒性远小于喜树碱、拓扑替康。因此可作为抗肿瘤药物用于动物，优选用于哺乳动物，特别是人。
本发明的喜树碱类磷脂化合物脂质体纳米颗粒的制备方法，是由本发明化合物喜树碱类磷脂化合物或本发明化合物与助剂的混合物，通过薄膜分散法、逆相蒸发法、冷冻干燥法、超声波分散法、喷雾干燥法、膜挤压法、或高压均质法等方法制备。
本发明利用2分子的喜树碱或喜树碱类似物作为疏水尾和磷脂亲水头通过共价键相连，制备喜树碱类磷脂化合物，溶解性显著优于喜树碱或喜树碱类似物；本发明将喜树碱类磷脂化合物制备成纳米颗粒，具有脂质体的特性，具有可形成液体制剂、固体制剂、半固体制剂、灭菌制剂和无菌制剂的特性，用于肿瘤治疗。这种喜树碱类磷脂化合物纳米颗粒不仅是一种前药，也是喜树碱类药物全新的药物释放载体，具有靶向功能；本发明的喜树碱类磷脂化合物及其脂质体纳米颗粒也是一种全新的喜树碱类药物的前药，也是药物载体，在体内经酯酶水解发挥药效，，能延长药物释放半衰期，并具有较低的毒副作用。
以下为制备过程中使用的部分试剂代号：
DMAP 4-二甲氨基吡啶
CDI N,N'-羰基二咪唑
DMSO 二甲亚砜
GPC 磷酰胆碱甘油
DBU 1,5-二氮杂二环[5.4.0]十一-5-烯
EDCI N,N'-硫代羰基二咪唑
HOBT 1-羟基苯并三唑
下述通过实施例进一步说明本发明，但本发明不限于以下实施例。
实施例1：
喜树碱-20-磷脂化合物的合成(合成路线见图1)
取喜树碱2g，丁二酸酐1.5g，DMAP 0.2g，三乙胺0.2g，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物喜树碱-20-丁二酸单酯。
取中间产物丁二酸喜树碱单酯1.5g，加入CDI 1g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 1g和DBU 0.75g室温反应，所得反应液通过柱层析纯化，得产物喜树碱-20-磷脂化合物1.2g。1H NMR(500MHz,CD3OD:CDCl31:1):δ8.03-7.59(10H,m),6.25(2H,s),4.71-4.55(7H,m),4.21(2H,t),3.86-3.55(8H,s),3.46(9H,s),2.67-2.41(8H,t),1.98(4H,m),1.12(6H,s)。[M+H]+m/z，1119.34。
实施例2：
喜树碱-20-乙酸酯-10-磷脂化合物的合成(合成路线见图2)
10-羟基喜树碱2g，乙酸2g，EDCI 2g,HOBT 1g，加入二氯甲烷或三氯甲烷为反应溶剂，40℃反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得10-羟基喜树碱-20-乙酸酯。
10-羟基喜树碱-20-乙酸酯1.6g，丁二酸酐3g，DMAP 0.5g，三乙胺0.5g，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物喜树碱-20-乙酸酯-10-丁二酸单酯。
取中间产物喜树碱-20-乙酸酯-10-丁二酸单酯1.2g，加入CDI 1g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 1g和DBU 1g室温反应，所得反应液通过柱层析纯化，得产物喜树碱-20-乙酸酯-10-磷脂化合物0.8g。1H NMR(500MHz,CD3OD:CDCl31:1):δ7.93-7.44(8H,m),6.23(2H,s),4.69-4.43(7H,m),3.61(2H,t),3.43(4H,s),3.22(9H,s),3.09(4H,s),2.68-2.52(8H,m),2.14(6H,s),1.65(4H,m),1.05(6H,t)。[M+H]+m/z,1235.35。
实施例3
7-乙基-喜树碱-20-乙酸酯-10-磷脂化合物的合成
7-乙基-10-羟基喜树碱2g，乙酸3g，EDCI 2g,HOBT 1g，加入二氯甲烷或三氯甲烷为反应溶剂，40℃反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得7-乙基-10-羟基喜树碱-20-乙酸酯。
7-乙基-10-羟基喜树碱-20-乙酸酯1.6g，丁二酸酐3g，DMAP 0.5g，三乙胺3mol，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物7-乙基-喜树碱-20-乙酸酯-10-丁二酸单酯。
取中间产物7-乙基喜树碱-20-乙酸酯-10-丁二酸单酯1.3g，加入CDI 1g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 2g和DBU 1g室温反应，所得反应液通过柱层析纯化，得产物7-乙基-喜树碱-20-乙酸酯-10-磷脂化合物0.9g。将7-乙基-喜树碱-20-乙酸酯-10-磷脂化合物溶于氯化钠水溶液中，冻干，得到含有抗衡离子钠离子和氯离子的7-乙基-10-羟基喜树碱磷脂化合物盐。
实施例4
伊立替康(CPT11，联哌喜树碱)-20-磷脂化合物的合成(合成路线见图3)
联哌喜树碱2g，丁二酸酐2g，DMAP 0.5g，三乙胺2g，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物联哌喜树碱-20-丁二酸单酯。
取中间产物联哌喜树碱-20-丁二酸单酯1.3g，加入CDI 1g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 1g和DBU 1g室温反应，所得反应液通过柱层析纯化，得产物联哌喜树碱-20-磷脂化合物0.8g。1H NMR(500MHz,CD3OD:CDCl31:1):δ8.14-7.39(6H,m),6.41(2H,s),4.73-4.51(7H,m),3.72(2H,t),3.49(4H,s),3.22(9H,s),3.04-2.94(8H,s),2.68-2.52(12H,m),2.14(6H,s),1.75(4H,m),1.46-1.32(24H,m),1.15-1.08(6H,tt)。[M+H]+m/z，1595.67。
伊立替康(CPT11，联哌喜树碱)-20-磷脂化合物溶于0.1M乳酸钙水溶液中，冻干得到含有抗衡离子钙离子和乳酸根离子的伊立替康(CPT11，联哌喜树碱)-20-磷脂化合物固体粉末。
实施例5：
拓扑立康-10-磷脂化合物的合成
9-二甲基氨基-10-羟基喜树碱1g，乙酸2g，EDCI 2g,HOBT 1g，加入二氯甲烷或三氯甲烷为反应溶剂，40℃反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得9-二甲基氨基-10-羟基喜树碱-20-乙酸酯。
9-二甲基氨基-10-羟基喜树碱-20-乙酸酯0.8g，邻苯二甲酸酐1g，DMAP 0.5g，三乙胺1g，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物9-二甲基氨基喜树碱-20-乙酸酯-10-邻苯二甲酸单酯。
取中间产物9-二甲基氨基喜树碱-20-乙酸酯-10-邻苯二甲酸单酯0.5g，加入CDI 0.5g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 0.4g和DBU 0.5g室温反应，所得反应液通过柱层析纯化，得产物拓扑立康-10-磷脂化合物0.3g。
实施例6：
喜树碱-7-亚甲基磷脂化合物的合成(合成路线见图4)
在配有温度计和搅拌的100mL三口圆底烧瓶中加入15mL甲醇和12.5mL水，在冰浴冷却下加入6.5mL浓 硫酸，然后加入0.5g(1.43mmol)喜树碱和0.4g(1.43mmol)硫酸亚铁七水合物，混合物搅拌5分钟后，控制温度在5C以下，缓慢滴加0.24mL(0.21mmol)30％的过氧化氢，溶液颜色逐渐变深，反应物在室温搅拌10小时后，倒入200mL水中，水相放置过夜，过滤，固体水洗，干燥得到黄色固体0.5g，粗品用DMF-乙酸乙酯重结晶得到0.3g 7-羟甲基喜树碱，产率：55％，mp 274-276℃。1HNMR(CDCl3，300MHz)：δ8.17(t，2H，Ar-H)，7.84(t，1H，Ar-H)，7.69(t，1H，Ar-H)，7.33(s，1H，Ar-H)，6.49(s，1H，20OH)，5.79(m，1H，7OH)，5.41d(d，4H，H17，7-CH2)，5.26(d，2H，H5)，1.86(m，2H，18-CH2)，0.87(t，3H，19-CH3)。
取7-羟甲基喜树碱0.3g，丁二酸酐2g，DMAP 0.1g，三乙胺1g，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物丁二酸单酯基亚甲基-7-喜树碱0.25g。
取中间产物丁二酸单酯基亚甲基喜树碱0.25g，加入CDI 0.3g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 0.3g和DBU 0.2g室温反应，所得反应液通过柱层析纯化，得产物喜树碱-7-亚甲基磷脂化合物0.26g。1H NMR(500MHz,CD3OD:CDCl31:1):δ8.13-7.61(8H,m),6.47(2H,s),5.26(4H,s),4.68-4.39(7H,m),3.79(2H,t),3.55(4H,s),3.42(9H,s),3.12(4H,s),2.95-2.72(8H,tt),1.94(4H,m),0.98(6H,t)。[M+H]+m/z，1179.36。
实施例7：
9-硝基喜树碱-20-磷脂化合物的合成(合成路线见图5)
取己二酸2g，加入CDI 4g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入9-硝基喜树碱4g，DBU 2g；室温反应2h；加入GPC 1.5g，DBU 2g，室温反应6h；再次加入9-硝基喜树碱1g，DBU 0.5g，室温反应6h。所得反应液通过柱层析纯化，得产物9-硝基喜树碱-20-磷脂化合物3g。1H NMR(500MHz,CD3OD:CDCl31:1):δ8.22-6.95(10H,m),4.80(4H,s),4.63(4H,s),4.47-4.25(7H,m),3.81(2H,t),3.32(9H,s),2.54(8H,tt),2.03(4H,m),1.56(8H,m),1.04(6H,t)。[M+H]+m/z，1165.11。
9-硝基喜树碱-20-磷脂化合物溶于生理盐水中，冻干得到含有抗衡离子钠离子和氯离子的9-硝基喜树碱-20-磷脂化合物固体粉末。
实施例8：
六环喜树碱(Lurtotecan)-20-磷脂化合物的合成(合成路线见图6)
取辛二酸2g，加入CDI 3.5g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入六环喜树碱(Lurtotecan)4g，DBU 2g，室温反应2h；加入GPC 1.8g，DBU 1.5g，室温反应8h；再次加入9-硝基喜树碱1g，DBU 0.5g，室温反应4h。所得反应液通过柱层析纯化，得产物六环喜树碱(Lurtotecan)-20-磷脂化合物0.8g。1H NMR(500MHz,CD3OD:CDCl31:1):δ7.56-6.98(6H,m),4.85(4H,s),4.66(2H,t),4.42(8H,tt),4.31-4.16(5H,m),3.98(4H,s),3.65(6H,m),3.29(9H,s),2.56(16H,m),2.31(6H,s),2.01(8H,m),1.82(4H,m),1.68(8H,m),1.33(8H,m),1.01(6H,t)。[M+H]+m/z，1571.35。
六环喜树碱(Lurtotecan)-20-磷脂化合物溶于0.1M磷酸钠水溶液中，冻干得到含有抗衡离子硫酸根和钠离子的六环喜树碱(Lurtotecan)-20-磷脂化合物固体粉末。
实施例9：
二氟喜树碱(BN80915)-20-磷脂化合物的合成(合成路线见图7)
取己二酸2g，加入CDI 3g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入二氟喜树碱(BN80915)4g，DBU 2g，室温反应2h；加入GPC 2g，DBU 2g，室温反应4h；再次加入二氟喜树碱(BN80915)0.8g，DBU 0.5g，室温反应4h。所得反应液通过柱层析纯化，得产物二氟喜树碱(BN80915)-20-磷脂化合物2.5g。1H NMR(500MHz,CD3OD:CDCl31:1):δ7.76-6.94(8H,m),4.91(4H,s),4.65-4.26(7H,m),4.07(4H,m),3.75(2H,t),3.31(9H,s),2.37(8H,t),2.07(4H,m),1.67(8H,m),1.02(6H,t)。[M+H]+m/z，1246.36。
二氟喜树碱(BN80915)-20-磷脂化合物溶于0.1M的醋酸水溶液中，冻干得到含有乙酸根抗衡离子的二氟喜树碱(BN80915)-20-磷脂化合物固体粉末。
实施例10：
CKD-602-20-磷脂化合物的合成(合成路线见图8)
取癸二酸2g，加入CDI 3g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入CKD-6024g，DBU 3g，室温反应2h；加入GPC 2g，DBU 1g，室温反应12h；再次加入CKD-6020.6g，DBU 0.4g，室温反应4h。所得反应液通过柱层析纯化，得产物CKD-602-20-磷脂化合物3g。1H NMR(500MHz,CD3OD:CDCl31:1):δ8.10-7.03(10H,m),4.93(4H,s),4.71-4.43(7H,m),4.23(4H,s),3.75(2H,t),3.38(9H,s),3.04(2H,m),2.89(4H,t),2.61(4H,t),2.25(8H,tt),1.99(4H,m),1.72(8H,m),1.34(16H,m),1.05(18H,m)。[M+H]+m/z，1458.71。
实施例11：
9-硝基喜树碱-20-磷脂酰乙醇胺化合物的合成(合成路线见图9)
取3-(4-甲氧基苄氧基)丙烷-1,2-二醇1g，丁二酸酐2.5g，DMAP 0.1g，三乙胺1.5g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加入CDI 2g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入9-硝基喜树碱2g，DBU 1g，室温反应8h，旋蒸干溶剂；加入稀醋酸，室温反应2h，除去溶剂；加入三氯氧磷1g，三乙胺0.8g，加入二氯甲烷、三氯甲烷或THF为反应溶剂，10℃反应24h，旋蒸干溶剂；N-叔丁氧羰基-氨基乙醇1g，三乙胺0.3g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，10℃反应24h，旋蒸干溶剂；加入异丙醇，醋酸水溶液，加热反应24h。所得反应液通过柱层析纯化，得产物9-硝基喜树碱-20-磷脂酰乙醇胺化合物1.4g。
1H NMR(500MHz,CD3OD:CDCl31:1):δ8.22-6.95(10H,m),4.80(4H,s),4.63(4H,s),4.47-4.25(7H,m),3.81(2H,t),3.32(9H,s),2.54(8H,tt),2.03(4H,m),1.56(2H,m)。[M+H]+m/z，1181.02。
实施例12：
9-硝基喜树碱-20-磷脂酰乙醇胺-N-聚乙二醇化合物的合成(合成路线见图10)
取mPEG20002g，CDI 0.2g，二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入实施例11中的9-硝基喜树碱-20-磷脂酰乙醇胺化合物1g，DBU 1g，加热反应24h。所得反应液通过柱层析纯化，得产物9-硝基喜树碱-20-磷脂酰乙醇胺-N-聚乙二醇化合物1.8g。
实施例13：
喜树碱-20-磷脂酰丝氨酸化合物的合成(合成路线见图11)
取喜树碱0.5g，己二酸酐2.5g，DMAP 0.1g，三乙胺1.5g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，加热回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加入CDI 2g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h；加入3-(4-甲氧基苄氧基)丙烷-1,2-二醇0.6g，DBU 1g，室温反应8h，旋蒸干溶剂；加入稀醋酸，室温反应2h，除去溶剂；加入化合物A和B，过氧化叔丁醇为反应溶剂，加热反应；钯碳催化加氢还原。所得反应液通过柱层析纯化，得产物喜树碱-20-磷脂酰丝氨酸化合物0.4g。
1H NMR(500MHz,CD3OD:CDCl31:1):δ8.10-7.24(12H,m),4.93(4H,s),4.74(4H,s),4.56-4.14(8H,m),2.69(8H,tt),2.14(4H,m),1.78(8H,m),1.21(6H,t)。
[M+H]+m/z，1177.67。
实施例14：
喜树碱-20-磷脂酰甘油化合物的合成(合成路线见图12)
取喜树碱-20-己二酸单酯0.5g，CDI 1g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温反应2h，加入3-(4-甲氧基苄氧基)丙烷-1,2-二醇0.6g，DBU 1g，室温反应8h，旋蒸干溶剂；加入稀醋酸，室温反应2h，除去溶剂；加入三氯氧磷1g，三乙胺0.8g，加入二氯甲烷、三氯甲烷或THF为反应溶剂，10℃反应24h，旋蒸干溶剂；加入甘油缩丙酮1g，三乙胺0.3g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，10℃反应24h，旋蒸干溶剂；加入异丙醇，醋酸水溶液，加热反应24h。所得反应液通过柱层析纯化，得产物喜树碱-20-磷脂酰甘油化合物1.1g。
[M+H]+m/z，1164.23。
实施例15：
9-硝基喜树碱-20-邻苯二甲酸酯-磷脂酰胆碱化合物的合成(合成路线见图13)
9-硝基喜树碱2g，邻苯二甲酸酐2g，DMAP 0.5g，三乙胺2g，加入二氯甲烷或三氯甲烷为反应溶剂，加入回流反应，反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼；加甲醇溶解，4℃放置12h，过滤，取滤饼，干燥，得中间产物9-硝基喜树碱-20-邻苯二甲酸单酯。
取9-硝基喜树碱-20-邻苯二甲酸单酯1.2g，CDI 1g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，室温 反应2h，加入3-(4-甲氧基苄氧基)丙烷-1,2-二醇0.6g，DBU 1g，室温反应8h，旋蒸干溶剂；加入稀醋酸，室温反应2h，除去溶剂；加入三氯氧磷1g，三乙胺0.8g，加入二氯甲烷、三氯甲烷或THF为反应溶剂，10℃反应24h，旋蒸干溶剂；加入甘油缩丙酮1g，三乙胺0.3g，加入二氯甲烷、三氯甲烷或DMSO为反应溶剂，10℃反应24h，旋蒸干溶剂；加入异丙醇，醋酸水溶液，加热反应24h。所得反应液通过柱层析纯化，得产物9-硝基喜树碱-20-邻苯二甲酸酯-磷脂酰胆碱化合物1g。
[M+H]+m/z，1305.31。
实施例16：
9-氨基喜树碱-20-磷脂化合物的合成(合成路线见图14)
取Boc保护的9-氨基喜树碱0.5g，丁二酸酐2.5g，DMAP 0.1g，三乙胺1.1g，加入二氯甲烷或三氯甲烷为反应溶剂，加热回流反应12h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼，干燥，得中间产物丁二酸单酯基氨基喜树碱0.4g。
取中间产物丁二酸单酯基氨基喜树碱0.3g，加入CDI 0.4g，二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 0.2g和DBU 0.25g室温反应24h，干燥溶剂；加入三氟乙酸/二氯甲烷溶液，室温反应2h。所得反应液通过柱层析纯化，得产物9-氨基喜树碱磷脂化合物0.2g。
1H NMR(500MHz,CD3OD:CDCl31:1):δ7.91-6.83(10H,m),4.82(4H,s),4.60(4H,s),4.49-4.18(7H,m),3.72(2H,t),3.26(9H,s),2.54(8H,tt),1.83(4H,m),1.02(6H,t)。
[M+H]+m/z，1149.16。
实施例17：
DX-8951f磷脂化合物的合成(合成路线见图15)
取Boc保护的DX-8951f 0.4g，丁二酸酐3g，DMAP 0.1g，三乙胺1.5g，二氯甲烷或三氯甲烷为反应溶剂，加热回流反应24h；稀盐酸洗涤三遍，过滤稀盐酸层，取滤饼，干燥，得中间产物丁二酸单酯基DX-8951f 0.27g。
取中间产物丁二酸单酯基DX-8951f 0.27g，加入CDI 0.35g，加入二氯甲烷，三氯甲烷或者DMSO作为反应溶剂，加入GPC 0.35g和DBU 0.25g室温反应12h，干燥溶剂；加入三氟乙酸/二氯甲烷溶液，室温反应2h。所得反应液通过柱层析纯化，得产物DX-8951f磷脂化合物0.19g。
1H NMR(500MHz,CD3OD:CDCl31:1):δ7.48(2H,s),6.83(2H,s),4.97(4H,s),4.65(2H,t),4.48-4.21(7H,m),3.88(2H,t),3.41(13H,m),2.89(4H,t),2.56(8H,tt),2.21(6H,s),1.86(4H,m),0.97(6H,t)。
[M+H]+m/z，1294.66。
实施例18：
喜树碱-20-磷脂化合物脂质体的制备
取实施例1的喜树碱-20-磷脂化合物1mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH＝7.4)60℃起膜，200nm滤膜过滤，得喜树碱磷脂化合物脂质体纳米颗粒溶液。粒径分析结果如下图16，平均粒径35.40nm。透射电镜测量纳米颗粒的形态如图17。
实施例19：
喜树碱-20-乙酸酯-10-磷脂化合物脂质体的制备
由实施例2得到的喜树碱-20-乙酸酯-10-磷脂化合物1mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH＝7.4)60℃起膜，200nm滤膜过滤，得喜树碱-20-乙酸酯-10-磷脂化合物脂质体纳米颗粒溶液。粒径分析结果显示平均粒径166nm。将喜树碱-20-乙酸酯-10-磷脂化合物脂质体纳米颗粒溶液冻干，得到粉状纳米颗粒。
实施例20：
7-乙基-喜树碱-10-磷脂化合物脂质体的制备
由实施例3得到的7-乙基-喜树碱-10-磷脂化合物1mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH＝7.4)60℃起膜，200nm滤膜过滤，得7-乙基-喜树碱-10-磷脂化合物脂质体纳米颗粒溶液。粒径分析结果显示，平均粒径152nm。
实施例21：
伊立替康-20-磷脂化合物脂质体的制备
由实施例4得到的伊立替康-20-磷脂化合物1mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH＝7.4)60℃起膜，得伊立替康-20-磷脂化合物脂质体纳米颗粒溶液。粒径分析结果显示，平均粒径228nm。
实施例22：
拓扑立康-10-磷脂化合物脂质体的制备
由实施例5得到的拓扑立康-10-磷脂化合物1mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH＝7.4)60℃起膜，得拓扑立康-10-磷脂化合物脂质体纳米颗粒溶液。粒径分析结果显示平均粒径290nm(图18)。
实施例23：
喜树碱-20-磷脂化合物脂质体B的制备
取喜树碱-20-磷脂化合物0.5mmol，二硬脂酰磷脂酰胆碱DSPC 0.5mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH＝7.4)60℃起膜，得喜树碱磷脂化合物脂质体纳米颗粒溶液。粒径分析显示平均粒径380nm。
实施例24：
喜树碱-20-磷脂化合物脂质体C的制备
取喜树碱-20-磷脂化合物0.5mmol，二硬脂酰磷脂酰胆碱DSPC 0.5mmol，二硬脂酰基磷脂酰乙醇胺-聚乙二 醇DSPE-PEG-叶酸(聚乙二醇分子量400-3000)0.1mmol，加入三氯甲烷20ml，60℃旋蒸干溶剂；加入20ml PBS(pH 7.4)60℃起膜，得喜树碱磷脂化合物脂质体纳米颗粒溶液。粒径分析显示，平均粒径620nm。冷冻干燥喜树碱-20-磷脂化合物脂质体纳米颗粒溶液，得到粉末状纳米颗粒。
药理实验
实验例25：
MTT法人癌细胞杀伤试验
药品及试剂：小牛血清为南京生兴生物技术有限公司产品；DMSO分析纯；RPMI1640为GIBCO产品。
仪器：BIORAD 680型酶标仪。
收集生长良好的肿瘤细胞，用含10％小牛血清的RPMI1640培养基配制成1×10-4/mL细胞悬液，于96孔培养板内接种，每空100μL(含1000个肿瘤细胞)，置37℃，5％CO2温箱内培养24小时后加药，实验设空白对照及溶剂对照，受试样品设4个浓度，每浓度3个平行孔，置37℃，5％CO2温箱内培养4天。弃去培养液，每孔加入MTT溶液(0.4mg/mL，RPMI1640配制)100μL，37℃孵育4小时。弃去上清液，每孔加入MTT溶液150μL，溶解颗粒，轻度振荡后，用550型酶标仪在检测波长540nm，参考波长450nm下测定OD值。
结果计算：以药物的不同浓度及对细胞的抑制率作图可得到剂量反应曲线，从中求出半数抑制浓度(IC50)。
本发明衍生物对人肿瘤细胞株的抗肿瘤活性结果见表1.从体外抗肿瘤活性筛选来看，本发明化合物的半数抑制浓度均小于伊立替康，说明其活性优于伊立替康。本发明化合物的半数抑制浓度均小于喜树碱，大约是喜树碱的一半，可能是因为喜树碱类磷脂化合物含有两个喜树碱类分子结构单元。
表1.本发明化合物对人肿瘤细胞株的抗肿瘤活性结果
Figure PCTCN2015089181-appb-000015
MCF-7：人乳腺癌细胞；Hep G2：人肝癌细胞；A2780：人卵巢癌细胞；HCT-8：人结肠癌细胞；A549：人肺腺癌细胞；EC-304：人血管内皮正常细胞；ND：未试验。
实验例26
喜树碱类磷脂化合物在小鼠体内毒性试验
动物：ICR小鼠，雄性，18-22g，购自维通利华实验动物技术有限责任公司。
本发明化合物喜树碱-20-磷脂化合物、10-羟基喜树碱-10-磷脂化合物、7-亚甲基喜树碱磷脂化合物、7-乙基-10-羟基喜树碱-10-磷脂化合物的体内毒性试验的结果表明，其最大耐受剂量大于100mg/kg，表明其毒性远小于喜树碱、拓扑替康。
表2喜树碱衍生物在小鼠体内毒性试验结果
化合物 最大耐受剂量(MTD，mg/kg)
喜树碱 12
拓扑替康 ＜15
喜树碱-20-磷脂化合物 ＞100
喜树碱-20-乙酸酯-10-磷脂化合物 ＞100
喜树碱-7-亚甲基磷脂化合物 ＞100
7-乙基-喜树碱-20-乙酸酯-10-磷脂化合物 ＞100
上述实施例仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和等同替换，这些对本发明权利要求进行改进和等同替换后的技术方案，均落入本发明的保护范围。

Claims (7)

  1. 一种喜树碱类磷脂化合物，其特征在于，该磷脂化合物为下列通式(1)的化合物或所述通式(1)的化合物与抗衡离子所形成的在药学上可接受的盐：
    Figure PCTCN2015089181-appb-100001
    式(1)中，R1和R2为间隔臂，是亚烃基，所述亚烃基定义为碳原子数为1～20的直链或支链的亚烷烃基/亚烯烃基或碳原子数为6～10的芳烃基；L代表2-氨基-2-羧基乙基、2-氨基乙基、2-三甲基胺基乙基阳离子或2、3-二羟基丙基；
    Rx和Ry是结构为下列式(2)的喜树碱类化合物(20S)20位取代基、式(3)的喜树碱类化合物(20S)10位取代基或式(4)的喜树碱类化合物(20S)7位亚甲基取代基：
    Figure PCTCN2015089181-appb-100002
    式(2)和(3)和(4)中，R7、R9、R10、R11和R12均为以下任一种基团：氢、卤素、羟基、C1-C6的直链或支链的烷基/烷氧基、氰基、硝基、氨基、C1-C6的直链或支链的卤代烷基/卤代烷氧基、C1-C6的直链或支链烷基的三取代硅烷基、C1-C6的直链或支链烯烃基、甲醛基、C1-C6的直链或支链烷基酯基、C1-C6的直链或支链烷基羰基、C1-C6的直链或支链的羟基烷基、C1-C6的直链或支链烷氧基亚甲基、苯氧基亚甲基、苯甲酯基亚甲基、二甲胺基甲基、二甲基胺基乙基、具有哌嗪结构
    Figure PCTCN2015089181-appb-100003
    的取代基、具有结构
    Figure PCTCN2015089181-appb-100004
    的取代基、具有联哌羧基结构
    Figure PCTCN2015089181-appb-100005
    的取代基和-CH2NR13R14，所述-CH2NR13R14中，R13和R14为C1-C6的直链烷基、C1-C6的支链烷基、或者R11和R10形成结构
    Figure PCTCN2015089181-appb-100006
    的取代基；
    式(3)和(4)中R代表C1-C18的直链/支链烷基或C6-C10的芳烃基。
    式(2)和(3)和(4)中，20位碳原子均为S构型。
  2. 根据权利要求1所述的喜树碱类磷脂化合物，其特征在于，所述抗衡离子是阳离子质子、钠离子、钾离子、钙离子、铁离子、镁离子、铵离子、锌离子、阴离子氯离子、硫酸根离子、硫酸根离子、硝酸根离子、羧酸根离子、碳酸根离子、溴离子、磷酸根离子、甲酸根、乙酸根、柠檬酸更、乳酸根、富马酸根、酒石酸根、葡萄糖酸根离子的任一种或任两种的组合。
  3. 一种药物组合物，其特征在于，该组合物包括权利要求1或2所述的喜树碱类磷脂化合物或所述的喜树碱类磷脂化合物与药效学上可接受的载体。
  4. 根据权利要求3的药物组合物，其特征在于，所述的药物组合物是液体制剂、固体制剂、半固体制剂、胶囊剂、颗粒剂、凝胶剂、注射剂、缓释制剂或控释制剂。
  5. 根据权利要求3的药物组合物，其特征在于，所述的药物组合物为粒径10-1000纳米的脂质体纳米颗粒，该药物组合物中还包括助剂。
  6. 根据权利要求5所述的药物组合物，其特征在于，所述的助剂是磷脂。
  7. 一种将权利要求1或2所述喜树碱类磷脂化合物在制备抗肿瘤药物中的应用其特征在于，该方法将所述喜树碱类磷脂化合物或其在药学上可接受的盐，与药效学上可接受的载体制备成药剂。
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