CN113332305B - Lipid composition of cytidine compound - Google Patents

Lipid composition of cytidine compound Download PDF

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CN113332305B
CN113332305B CN202110618034.7A CN202110618034A CN113332305B CN 113332305 B CN113332305 B CN 113332305B CN 202110618034 A CN202110618034 A CN 202110618034A CN 113332305 B CN113332305 B CN 113332305B
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lipid composition
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cytarabine
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CN113332305A (en
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夏桂民
刘明亮
王晓葳
冯文凯
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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    • C07H19/09—Pyrimidine radicals with arabinosyl as the saccharide radical

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Abstract

The invention relates to cytidine compounds and anti-tumor application thereof. In particular to a group of novel cytidine compounds with antitumor activity, a preparation method thereof and application in the antitumor aspect. The compound of the invention has chemical structures shown in formula (I) and formula (II) in the specification. The compound prepared by the invention has excellent biological effect on treating mammal tumor or ophthalmia. The tumor is selected from: leukemias (e.g., acute leukemias such as acute myelocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia), malignant lymphomas, lung cancer, cancer of the digestive tract, head and neck cancer; for example, the ophthalmia is selected from: viral keratitis, epidemic conjunctivitis. The lipid composition prepared by the method of the invention has excellent pharmaceutical properties.

Description

Lipid composition of cytidine compound
Technical Field
The invention belongs to the field of medical chemistry, relates to a method for improving the antitumor performance of cytidine drugs, and particularly relates to a group of cytarabine prodrugs with excellent antitumor activity, a preparation method thereof, and applications thereof in the antitumor aspect. Furthermore, the invention also relates to a lipid composition of the cytidine compound and a preparation method of the lipid composition. The lipid composition of the present invention exhibits excellent technical effects.
Background
Cytarabine (Ara-C), also known as cytosine arabinoside, is chemically 1-beta-D-arabinofuranosyl-4-amino-2 (1H) -pyrimidinone having CAS number 147-94-4 and molecular formula C 9 H 13 N 3 O 5 Molecular weight 243.22, its chemical structure is shown in formula (VI) below:
Figure BDA0003098478630000011
cytarabine was first synthesized in 1959 by a pharmacologist such as Walwick R, at berkeley division, university of california. The FDA in the united states approved cytarabine for marketing in 6 months of 1969. The chemical structure of this drug, originally sold under the name Cytosar-U by Upjohn, is a nucleoside in which cytosine is bound to arabinose, hence the name "cytarabine".
Cytarabine is a pyrimidine antimetabolite which mainly acts on the S proliferation phase of cells and interferes with the proliferation of cells by inhibiting the synthesis of cellular DNA. Cytarabine must be activated before entering human body to act, namely, cytarabine needs to be firstly converted into nucleotide 5-phosphate (Ara-CMP) under the catalysis of deoxycytidine kinase, Ara-CMp can react with proper nucleotide kinase to form nucleotide diphosphate and nucleotide triphosphate (Ara-CDP and Ara-CTP), the former can strongly inhibit the synthesis of DNA polymerase, and the latter can inhibit the conversion of cytidine diphosphate into deoxycytidine diphosphate, thereby inhibiting the polymerization and synthesis of cell DNA. The product is a cell cycle specific medicine, is most sensitive to the action of cells in S-phase proliferation stage, and has weak action on inhibiting RNA and protein synthesis.
Cytosine deaminase, which is widely present in solid tumors (but at low levels in leukemia cells), deaminates cytarabine to convert it to the inactive metabolite, uridine (Ara-U) (Shimma N, et al, Bioorganic & Medicinal Chemistry 8(2000)1697-1706), so this product (cytarabine hydrochloride by intravenous administration) is mainly used clinically in the induction remission stage or maintenance consolidation stage of acute lymphocytic and non-lymphocytic leukemia and in the acute phase of chronic myelocytic leukemia. Unfortunately, there are still many patients who cannot be relieved or relapse after relief, and the drug resistance factors account for a large proportion. The common adverse reactions of this herb include bone marrow depression such as leukopenia, thrombocytopenia and megaloblastic anemia, nausea and vomiting are also common. In addition, oral ulcers, thrombophlebitis and impaired liver function may occur. Although cytarabine is still one of the first-line drugs for clinically treating acute myeloid leukemia at present, the clinical application of the cytarabine is strictly limited due to the continuous aggravation of drug resistance and toxic and side effects.
The prior art still expects new methods and expects to have some or some more excellent effects on treating tumors, for example, the antitumor drug cytosine type compound such as cytarabine prodrug with more excellent performance and the composition thereof are expected to be applied to clinic.
Disclosure of Invention
The object of the present invention is to provide a novel method which is expected to have some or some more excellent effects for treating tumors, for example, to provide an antitumor agent having more excellent properties for clinical use. The present inventors have surprisingly found that compounds having the structure of the present invention exhibit one or more excellent effects as antitumor agents, and also found that the preparation of the active compounds of the present invention into lipid compositions exhibits excellent technical effects. The present invention has been completed based on such findings.
To this end, the present invention provides, in a first aspect, compounds of the following formula (I) (which may also be referred to herein as cytarabine carbamate compounds or cytarabine carbamate) and compounds of the formula (II) (which may also be referred to herein as cytarabine triester compounds),
Figure BDA0003098478630000021
or a pharmaceutically acceptable salt, solvate or solvate thereof,
wherein:
R 1 、R 2 、R 3 independently represent C 6-22 Straight or branched (e.g. C) 8-22 Straight or branched, e.g. C 10-22 Linear or branched) saturated alkyl or unsaturated alkenyl, in which 1 or 2 CH groups in the carbon chain are present 2 Optionally substituted by O;
x represents O, NH or is absent.
According to the inventionA compound of the first aspect wherein R is 1 、R 2 、R 3 Selected from: n-decyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, n-docosyl, n-hexadecyloxypropyl, n-octadecyl, n-9-ene) n-decyl, (11-ene) n-dodecyl, (11-ene) n-dodecylethyl.
A compound according to the first aspect of the invention or any other aspect of the invention, wherein the pharmaceutically acceptable salt is a salt with an inorganic acid or with an organic acid.
A compound according to the first aspect of the invention or as referred to in any of the other aspects of the invention, wherein the inorganic acid is selected from: hydrochloric acid, sulfuric acid, phosphoric acid. A particularly preferred pharmaceutically acceptable salt is the hydrochloride salt.
A compound according to the first aspect of the invention or as referred to in any other aspect of the invention wherein the organic acid is selected from: acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, amino acids (e.g. alanine, aspartic acid, lysine), sulfonic acids (e.g. methanesulfonic acid, p-toluenesulfonic acid).
The compounds according to the first aspect of the invention may also exist in the form of solvates (e.g. hydrates), and thus such solvates (e.g. hydrates) are also included in the compounds of the invention.
A compound according to the first aspect of the invention, which is compound 1 to compound 17 selected from:
compound 1: n is a radical of 4 -n-tetradecyloxycarbonyl cytarabine,
compound 2: n is a radical of 4 -n-hexyloxycarbonyl cytarabine,
compound 3: n is a radical of 4 -n-decyloxycarbonyl cytarabine,
compound 4: n is a radical of 4 -n-eicosyloxycarbonyl cytarabine,
compound 5: n is a radical of 4 -n-docosanyloxycarbonyl cytarabine,
compound 6:N 4 - (9-decenyloxycarbonyl) cytarabine,
compound 7: n is a radical of 4 - (13-tetradecenyloxycarbonyl) cytarabine,
compound 8: n is a radical of 4 - (21-docosadienyloxycarbonyl) cytarabine,
compound 9: 2 ', 3 ', 5 ' -tri-O-n-tetradecanoyl cytarabine,
compound 10: 2 ', 3 ', 5 ' -tri-O-n-undecylcytarabine,
compound 11: 2 ', 3', 5 '-tri-O- (21' -n-docosenyl acyl) cytarabine,
compound 12: 2 ', 3 ', 5 ' -tri-O- (n-hexyloxyacyl) cytarabine,
compound 13: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoyloxyacyl) cytarabine,
compound 14: 2 ', 3', 5 '-tri-O- (21' -n-docosadienyloxyformyl) cytarabine,
compound 15: 2 ', 3 ', 5 ' -tri-O- (n-decylaminoyl) cytarabine,
compound 16: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoylamino acyl) cytarabine,
compound 17: 2 ', 3 ', 5 ' -tri-O- (21 "-n-eicosadienoyl) cytarabine.
Further, the second aspect of the present invention provides a process for preparing a compound of formula (I) and a compound of formula (ii), which are carried out according to the following reaction scheme one and reaction scheme two, respectively:
the reaction scheme I:
Figure BDA0003098478630000041
in scheme one, R 1 As defined in any one of the embodiments of the first aspect of the invention.
Reaction scheme two:
Figure BDA0003098478630000042
in scheme two, R 1 、R 2 、R 3 And X is as defined in any one of the embodiments of the first aspect of the invention.
A process according to the second aspect of the invention, wherein the preparation of the compound of formula (I) comprises the steps of:
1) dissolving the compound of formula (III) in a non-polar solvent (such as dichloromethane, chloroform, tetrahydrofuran, dioxane), and reacting with 1-2 times of the equivalent of the compound of formula (IV) in the presence of 1.5-3 times of the equivalent of an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine) at 0-40 ℃ for 3-10 hours with stirring to obtain the compound of formula (V);
2) dissolving the compound of formula (V) in a protic solvent (e.g., water, alcohol or an alcohol-water mixed solvent), adding 0.1 to 2 equivalents of an inorganic base (e.g., 0.1 to 0.5 equivalent; e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate), at 0 deg.C to room temperature for 0.5 to 3 hours with stirring to provide the compound of formula (I).
The process according to the second aspect of the present invention, wherein the preparation of the compound of formula (II) comprises the steps of:
1) dissolving a compound of formula (VI) in a non-polar solvent selected from: dichloromethane, trichloromethane, tetrahydrofuran, dioxane, in the presence of an organic base selected from the group consisting of: triethylamine, pyridine, 4-dimethylaminopyridine and 1-6 times of equivalent weight of Boc 2 Stirring and reacting O at room temperature to 50 ℃ for 3-15 hours to obtain an amino-protected compound of formula (VII);
2) dissolving a compound of formula (VII) in a non-polar solvent selected from: dichloromethane, trichloromethane, tetrahydrofuran, dioxane, in the presence of an organic base selected from the group consisting of: triethylamine, pyridine and 4-dimethylamino pyridine react with 1-4 times of equivalent of a compound of formula (IV), a compound of formula (VIII) or a compound of formula (IX) for 4-15 hours under stirring at 0-room temperature to obtain a compound of formula (X);
3) dissolving a compound of formula (X) in a non-polar solvent selected from: adding 1-2 times of equivalent of acid selected from the following into dichloromethane, trichloromethane, tetrahydrofuran and dioxane: and (3) reacting trifluoroacetic acid and hydrochloric acid at 0-room temperature for 0.5-5 hours under stirring to obtain the compound of the formula (II).
Further, in a third aspect, the present invention provides the use of a compound according to any one of the first aspect of the present invention or a compound prepared by the process according to any one of the second aspect of the present invention in the manufacture of a medicament for the treatment of a tumour or ophthalmia in a mammal.
The use according to the third aspect of the invention, wherein the tumour is selected from the group consisting of: leukemia (e.g., acute leukemia such as acute myelocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia), malignant lymphoma, lung cancer, cancer of the digestive tract, and head and neck cancer.
The use according to the third aspect of the invention, wherein the ophthalmia is selected from: viral keratitis, epidemic conjunctivitis.
The use according to the third aspect of the invention, wherein the compound elicits less adverse effects of thrombocytopenia in a mammal than cytarabine, e.g. a statistically significant difference with a p-value < 0.05.
Further, in a fourth aspect of the present invention, there is provided a method for reducing adverse effects of cytarabine on thrombocytopenia in a mammal, which comprises the step of preparing a compound of formula (I) or a compound of formula (II) as described in any one of the first aspect of the present invention from cytarabine. In one embodiment of the invention, the mammal is a human.
Further, the fifth aspect of the present invention provides a lipid composition comprising:
API (a compound of formula (I) or a compound of formula (II) according to any one of the first aspect of the invention): 100 parts by weight of a water-soluble polymer,
phospholipid: 100 to 2000 parts by weight (for example, 200 to 1500 parts by weight),
pegylated phospholipids: 30 to 300 parts by weight (for example, 40 to 200 parts by weight),
cholesterol: 20 to 200 parts by weight (e.g., 30 to 150 parts by weight), and
and (3) an excipient.
The lipid composition according to the fifth aspect of the invention is a composition in a liquid state (e.g. is a lipid suspension), wherein the excipient is an aqueous vehicle. For example, it is selected from: water, 0.8-1% sodium chloride solution (e.g., 0.9% sodium chloride solution), 2-10% glucose solution (e.g., 5% glucose solution). For example, the amount of the aqueous solvent is such that the concentration of the compound of formula (I) or the compound of formula (II) in the liquid composition is 0.2-20 mg/ml, such as 0.25-15 mg/ml, such as 0.25-10 mg/ml, such as 0.25-5 mg/ml.
The lipid composition according to the fifth aspect of the invention, which is a composition in a solid state (e.g. is a freeze-dried composition), wherein the excipient is a freeze-dried excipient. For example, the lyophilized excipient is selected from: mannitol, sorbitol, lactose, glycine, dextran, sucrose, glucose, and the like. For example, the weight ratio of the compound of formula (I) or the compound of formula (II) to the lyophilization excipient is 1: 20-200, for example, in a weight ratio of 1: 20-150, for example, in a weight ratio of 1: 20 to 100.
The lipid composition according to the fifth aspect of the present invention, wherein the phospholipid is selected from the group consisting of: egg yolk lecithin, hydrogenated egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoyl phosphatidylcholine (i.e., DMPC), dimyristoyl phosphatidylglycerol (i.e., DMPG), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine, dioleoylphosphatidylcholine, dilauroyl phosphatidylcholine, and combinations thereof.
The lipid composition according to the fifth aspect of the present invention, wherein the pegylated phospholipid (may be simply referred to as pegylated phospholipid) is a phospholipid modified with a molecular weight of 1000 to 10000 daltons, such as pegylated distearoylphosphatidylethanolamine, which may be expressed as distearoylphosphatidylethanolamine-polyethylene glycol (may be abbreviated as PEG-DSPE or DSPE-PEG). For example, the pegylated phospholipid is selected from: distearoylphosphatidylethanolamine-polyethylene glycol 1000 (abbreviated as PEG1000-DSPE, and the others may be similarly described), distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 3350, distearoylphosphatidylethanolamine-polyethylene glycol 4000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 6000, distearoylphosphatidylethanolamine-polyethylene glycol 8000, distearoylphosphatidylethanolamine-polyethylene glycol 10000.
The lipid composition according to the fifth aspect of the present invention is prepared by a process for preparing liposomes. The preparation of liposomes is well known in the art, such as, but not limited to: film dispersion method, extrusion preparation method, French pressure method, reverse phase evaporation method, chemical gradient method (for example, pH gradient method, ammonium sulfate gradient method).
The lipid composition according to the fifth aspect of the present invention is prepared by a thin film dispersion method (a classical liposome preparation method) comprising the steps of:
(21) dissolving phospholipids, pegylated phospholipids, cholesterol and active drug in an organic solvent (e.g., dichloromethane, chloroform, methanol, and the like, and combinations thereof, e.g., in an amount of 2-4 times the complete solubility);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (for example, at 40-60 ℃, at a vacuum degree of 200-250 mbar, at a rotation speed of 200-300 rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding an aqueous solvent into a container, hydrating at 40-80 deg.C (e.g. 50-70 deg.C) for 0.1-5 hr (e.g. 0.1-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 15-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), and collecting lipid composition in liquid form of lipid suspension; or
(23b) Adding an excipient solution dissolved in water in advance into a container, hydrating at 40-80 deg.C (e.g. 50-70 deg.C) for 0.1-5 hr (e.g. 0.1-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 15-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), packaging into glass bottles, and freeze-drying in a freeze-dryer to remove water to obtain solid lipid composition.
The lipid composition according to the fourth aspect of the present invention, wherein in the step (23b), the excipient concentration in the excipient solution previously dissolved with water is 3 to 20%, such as 3 to 15%, such as 3 to 10%.
Further, the sixth aspect of the present invention provides a method for preparing the lipid composition according to any one of the fifth aspect of the present invention, which is carried out by a method selected from the group consisting of: film dispersion method, extrusion preparation method, French pressure method, reverse phase evaporation method, chemical gradient method (for example, pH gradient method, ammonium sulfate gradient method).
The method according to the sixth aspect of the present invention, wherein the thin film dispersion method comprises the steps of:
(21) dissolving the phospholipid, pegylated phospholipid, cholesterol and active agent in an organic solvent (e.g., dichloromethane, chloroform, methanol, and the like, and combinations thereof);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (for example, at 40-60 ℃, at a vacuum degree of 200-250 mbar, at a rotation speed of 200-300 rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding an aqueous solvent into a container, hydrating at 40-80 deg.C (e.g. 50-70 deg.C) for 0.1-5 hr (e.g. 0.1-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 15-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), and collecting lipid composition in liquid form of lipid suspension; or
(23b) Adding an excipient solution dissolved in water in advance into a container, hydrating at 40-80 deg.C (e.g. 50-70 deg.C) for 0.1-5 hr (e.g. 0.1-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 15-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), packaging into glass bottles, and freeze-drying in a freeze-dryer to remove water to obtain solid lipid composition.
The method according to the sixth aspect of the present invention, wherein in the step (23b), the excipient concentration in the excipient solution previously dissolved with water is 3 to 20%, such as 3 to 15%, such as 3 to 10%.
Further, the seventh aspect of the present invention provides the use of the lipid composition according to any one of the fifth aspect of the present invention or the lipid composition prepared by the method according to any one of the sixth aspect of the present invention in the preparation of a medicament for treating tumors or ophthalmia in a mammal.
The use according to the seventh aspect of the invention, wherein the tumour is selected from the group consisting of: leukemia (e.g., acute leukemia such as acute myelocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia), malignant lymphoma, lung cancer, cancer of the digestive tract, and head and neck cancer.
Use according to a seventh aspect of the invention, wherein the ophthalmia is selected from: viral keratitis, epidemic conjunctivitis.
The lipid composition according to any aspect of the present invention is a liquid or solid composition, which is diluted with water or dissolved to form a liquid medicine having a concentration of the compound of formula (I) of 0.2mg/ml or less, wherein the liquid medicine has an average particle size of less than 200nm (e.g., an average particle size of 20 to 200nm, e.g., an average particle size of 30 to 200nm, e.g., an average particle size of 40 to 200nm, e.g., an average particle size of 50 to 200nm, e.g., an average particle size of 30 to 180nm, e.g., an average particle size of 30 to 150nm), less than 5% of particles having a particle size of less than 10nm (e.g., less than 5% of particles having a particle size of less than 15 nm), and less than 5% of particles having a particle size of more than 500nm (e.g., less than 5% of particles having a particle size of more than 400nm, and less than 5% of particles having a particle size of more than 300 nm), as measured by a nanometer particle sizer. This term may be referred to as particle size and particle size distribution.
In any aspect of the present invention, the pharmaceutical composition prepared in liquid form or further prepared in the form of a freeze-dried powder injection may be prepared in a manner to control the preparation process to make the composition into a sterile preparation for use in a sterile manner. The process is easy to control, for example, the control mode is that each raw and auxiliary material is sterilized and then prepared into a sterile preparation by whole-process sterile operation; it may also be a post-controlled manner, i.e. the composition in liquid form as prepared is sterilized by filtration through, for example but not limited to, a microfiltration membrane. Thus, according to any aspect of the invention, the pharmaceutical composition prepared in liquid form or further prepared in the form of a lyophilized powder for injection is a sterile formulation.
Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
In the following specific examples section, pharmaceutical compositions in liquid form or formulations in the form of lyophilized compositions are provided, as not otherwise mentioned, in amounts of the respective materials per 100mg or 100 parts by weight of the compound of formula (I) in the composition prepared; in the actual preparation, it is dosed in an amount to prepare a pharmaceutical composition comprising 10g of a compound of formula (I). When the pH value of the liquid medicine needs to be adjusted during the preparation of the composition, a 2M hydrochloric acid solution or a 2M sodium hydroxide solution is used. In the following examples, the content of organic solvent in the freeze-dried powder obtained by freeze-drying the composition was determined to be below the detection limit.
Examples of preparation of Compounds
Example 1、N 4 -n-tetradecyloxycarbonyl cytarabine
2 ', 3 ', 5 ' -tri-O-benzoylcytarabine (III, 555mg,1.0mmol) was dissolved in dichloromethane (15ml) and triethylamine (417. mu.l, 3.0mmol) and tetradecyl chloroformate (IV) were added successively / 333mg,1.2 mmol). Stirring for 8-15 hours at room temperature, and finishing the reaction. And (3) post-treatment: the reaction system was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was used directly in the next reaction. 2 ', 3 ', 5 ' -tri-O-benzoyl cytarabine can be readily prepared by methods well known in the art, for example, from cytarabine as a starting material.
The product V obtained in the previous step / Dissolved in methanol (10mL), and a sodium hydroxide solution (0.5mL,1M) was added thereto, followed by stirring at room temperature for 1 hour to complete the reaction. And (3) post-treatment: the reaction mixture was concentrated, and the residue was separated and purified by column chromatography (dichloromethane: methanol 25: 1 to 10: 1) to give a pure white solid (I) / ) For the title compound, HPLC purity>98.6 percent. The content or purity of the target substance in various materials (including raw material medicines and compositions) is determined by an HPLC method under the following conditions: inertsil ODS-SP column (5 μm, 150 mm. times.4.6 mm), purified with methanol: water (80:20) is used as a mobile phase for isocratic elution, the flow rate is 1.0mL/min, the detection wavelength is 241nm, the column temperature is 30 ℃, and the sample injection amount is 20 mu L.
1 H NMR(500MHz,DMSO)δ9.08(s,1H),8.03(d,J＝7.5Hz,1H),7.01(d,J＝7.6Hz,1H),6.04(d,J＝3.9Hz,1H),5.48(d,J＝5.3Hz,2H),5.07(d,J＝5.1Hz,1H),4.09(t,J＝6.6Hz,2H),4.04(d,J＝5.9Hz,1H),3.92(d,J＝3.4Hz,1H),3.82(d,J＝3.6Hz,1H),3.61(t,J＝5.6Hz,2H),1.59(t,J＝7.1Hz,2H),1.24(d,J＝6.8Hz,22H),0.85(t,J＝6.8Hz,3H).MS-ESI(m/z):483.3(M+H) + .
Example 2、N 4 -n-hexyloxycarbonylCytarabine
Referring to example 1, compound III was substituted with n-hexyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 98.3%.
1 H NMR(500MHz,DMSO)δ9.13(s,1H),7.77(d,J＝7.7Hz,1H),6.91(d,J＝7.7Hz,1H),5.98(d,J＝6.0Hz,1H),5.16(d,J＝4.4Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.9,3.1Hz,1H),3.72–3.60(m,2H),1.72–1.63(m,1H),1.43(dp,J＝8.1,6.9Hz,2H),1.38–1.24(m,6H),0.89(t,J＝6.8Hz,3H).MS-ESI(m/z):371.2(M+H) + .
Example 3、N 4 -n-decyloxycarbonyl cytarabine
Referring to example 1, substitution of compound III with n-decyl chloroformate gave, after removal of the Bz-protecting group, the title compound as a white solid with HPLC purity > 98.7%.
1 H NMR(500MHz,DMSO)δ9.11(s,1H),7.90(d,J＝7.7Hz,1H),6.78(d,J＝7.7Hz,1H),6.19(d,J＝6.0Hz,1H),5.03(d,J＝4.4Hz,1H),4.84(d,J＝4.7Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.72–3.60(m,2H),1.71–1.62(m,2H),1.44–1.34(m,2H),1.34–1.22(m,6H),1.26(s,6H),0.95–0.84(m,3H).MS-ESI(m/z):427.2(M+H) + .
Example 4、N 4 -n-eicosyloxycarbonyl cytarabine
Referring to example 1, compound iii was substituted with n-eicosyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 98.1%.
1 H NMR(500MHz,DMSO)δ7.90(d,J＝7.7Hz,1H),6.71(d,J＝7.7Hz,1H),6.21(d,J＝6.0Hz,1H),5.19(d,J＝4.4Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.02(td,J＝6.8,4.5Hz,1H),3.89(dt,J＝6.8,3.0Hz,1H),3.72–3.60(m,2H),1.67(tt,J＝7.7,6.4Hz,2H),1.44(dt,J＝8.1,7.0Hz,2H),1.37–1.25(m,32H),0.93–0.85(m,3H).MS-ESI(m/z):567.4(M+H) + .
Example 5、N 4 -n-docosanyloxycarbonyl cytarabine
Referring to example 1, compound iii was substituted with n-docosyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 97.7%.
1 H NMR(500MHz,DMSO)δ8.88(s,1H),7.94(d,J＝7.7Hz,1H),6.68(d,J＝7.7Hz,1H),6.14(d,J＝6.0Hz,1H),5.03(d,J＝4.4Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.01(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.72–3.60(m,2H),1.67(tt,J＝7.7,6.4Hz,2H),1.44–1.34(m,2H),1.33–1.22(m,36H),0.93–0.85(m,3H).MS-ESI(m/z):595.4(M+H) + .
Example 6、N 4 - (9-decenyloxycarbonyl) cytarabine
Referring to example 1, compound III was substituted with 9-decenyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 98.3%.
1 H NMR(500MHz,DMSO)δ8.96(s,1H),7.89(d,J＝7.7Hz,1H),6.62(d,J＝7.7Hz,1H),6.12(d,J＝6.0Hz,1H),5.80(tt,J＝10.2,6.8Hz,1H),5.08(ddt,J＝10.3,2.1,1.0Hz,1H),5.00(d,J＝4.4Hz,1H),4.97(ddt,J＝10.3,2.1,0.9Hz,1H),4.84(d,J＝4.8Hz,1H),4.69(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.66(ddd,J＝4.4,3.1,1.1Hz,2H),2.23(tdt,J＝7.8,6.7,1.0Hz,2H),1.77–1.65(m,1H),1.39(dq,J＝8.1,6.7Hz,2H),1.34–1.22(m,8H).MS-ESI(m/z):425.2(M+H) + .
Example 7、N 4 - (13-tetradecenyloxycarbonyl) cytarabine
Referring to example 1, compound III was substituted with (13-tetradecenyl) chloroformate and the Bz-protecting group was removed to give the title compound as a white solid with HPLC purity > 98.6%.
1 H NMR(500MHz,DMSO)δ8.77(s,1H),7.90(d,J＝7.7Hz,1H),6.79(d,J＝7.7Hz,1H),6.03(d,J＝6.0Hz,1H),5.80(tt,J＝10.2,6.8Hz,1H),5.28(ddt,J＝10.3,2.1,1.0Hz,1H),5.00(d,J＝4.4Hz,1H),4.97(ddt,J＝10.2,2.2,0.9Hz,1H),4.84(d,J＝4.8Hz,1H),4.73(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.11(td,J＝6.8,4.5Hz,1H),3.93(dt,J＝6.8,3.0Hz,1H),3.58(ddd,J＝4.4,3.1,1.1Hz,2H),2.03(tdt,J＝7.8,6.7,1.0Hz,2H),1.71–1.62(m,2H),1.39(dq,J＝8.1,7.1Hz,2H),1.34–1.22(m,16H).MS-ESI(m/z):481.3(M+H) + .
Example 8、N 4 - (21-docosadienyloxycarbonyl) cytarabine
Referring to example 1, substitution of compound III with 21-dodecenyl chloroformate (21-docodecenyl ester) gave the title compound as a white solid after removal of the Bz-protecting group, with an HPLC purity of > 97.3%.
1 H NMR(500MHz,DMSO)δ9.04(s,1H),7.33(d,J＝7.7Hz,1H),6.82(d,J＝7.7Hz,1H),6.00(d,J＝6.0Hz,1H),5.79(tt,J＝10.2,6.8Hz,1H),5.08(ddt,J＝10.3,2.1,1.0Hz,1H),5.00(d,J＝4.4Hz,1H),4.97(ddt,J＝10.3,2.2,1.0Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.66(ddd,J＝4.3,3.1,1.1Hz,2H),2.08–1.99(m,2H),1.67(tt,J＝7.7,6.4Hz,2H),1.44–1.34(m,2H),1.34–1.22(m,32H).MS-ESI(m/z):593.4(M+H) + .
Example 92 ', 3 ', 5 ' -tri-O-n-tetradecanoyl cytarabine
Cytarabine (VI, 244mg,1.0mmol) was dissolved in N, N-dimethylformamide (15ml), and Boc was added at 0 deg.C 2 O (437mg,2.0mmol) was added to the solution, stirred for 10 minutes, heated to 60 ℃ and stirred for 6-8 hours. And (3) post-treatment: the reaction solution was washed with water, and extracted with ethyl acetate (10 ml). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Separating and purifying the residue by column chromatography to obtain a compound VII / . Compound VII / Dissolved in dichloromethane solutionTo (10ml) was added triethylamine (417. mu.l, 3mmol) and tetradecanoyl chloride (VIII) / 890mg, 3.6mmol) to the reaction solution, and after stirring at room temperature for 3 to 6 hours, the reaction was completed. And (3) post-treatment: the reaction solution was concentrated to obtain a residue (X) / ) Directly used for the next reaction.
Product X described above / Dissolving in dichloromethane solution (10ml), adding trifluoroacetic acid (2ml) to remove protecting group to obtain white solid compound II / For the title compound, HPLC purity>97.6％。
1 H NMR(500MHz,CDCl 3 )δ7.58(t,J＝5.6Hz,1H),6.33(d,J＝3.9Hz,1H),5.71(dd,J＝7.6,4.6Hz,1H),5.48(d,J＝3.8Hz,1H),5.01(s,1H),4.44(dd,J＝11.8,7.2Hz,1H),4.34(dd,J＝11.8,4.0Hz,1H),4.15(dt,J＝7.1,3.6Hz,1H),2.40–2.31(m,4H),2.27–2.14(m,2H),1.63(q,J＝6.9Hz,4H),1.54–1.46(m,2H),1.36–1.18(m,61H),0.87(t,J＝6.8Hz,9H).MS-ESI(m/z):915.7(M+H) + .
Example 102 ', 3 ', 5 ' -tri-O-n-undecylcytarabine
Referring to example 9, the substitution reaction of compound VII with n-undecanoyl chloride, deprotection of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.1%.
1 H NMR(500MHz,CDCl 3 )δ7.88(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.17(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.43(dd,J＝6.8,4.2Hz,1H),5.25–5.21(m,1H),4.33(dt,J＝6.3,3.9Hz,1H),4.23(dd,J＝11.9,4.0Hz,1H),4.15(dd,J＝11.8,3.9Hz,1H),2.35–2.26(m,6H),1.60–1.49(m,6H),1.34–1.19(m,42H),0.91–0.85(m,9H).MS-ESI(m/z):747.5(M+H) + .
Example 112 ', 3', 5 '-tri-O- (21' -n-docosadienoyl) cytarabine
Referring to example 9, the compound VII was substituted with 21 "-n-docosenylformyl chloride to remove the Bz protecting group to give the title compound as a white solid with HPLC purity > 98.5%.
1 H NMR(500MHz,CDCl 3 )δ7.86(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.17(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.80(tt,J＝10.0,6.9Hz,3H),5.43(dd,J＝6.8,4.2Hz,1H),5.26–5.21(m,1H),5.12–5.04(m,3H),5.01–4.93(m,3H),4.33(dt,J＝6.4,4.0Hz,1H),4.19(d,J＝3.9Hz,2H),2.35–2.26(m,6H),2.08–1.99(m,6H),1.56(dddd,J＝34.0,16.4,8.6,7.3Hz,6H),1.27(ddd,J＝5.8,3.4,2.0Hz,102H).MS-ESI(m/z):1246.1(M+H) + .
Example 122 ', 3 ', 5 ' -tri-O- (n-hexyloxyacyl) cytarabine
Referring to example 9, substitution of compound VII with n-hexyl chloroformate and removal of Bz protecting group gave the title compound as a white solid with HPLC purity > 97.3%.
1 H NMR(500MHz,CDCl 3 )δ7.72(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.18(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.47(dd,J＝6.7,4.1Hz,1H),5.37(t,J＝6.5Hz,1H),4.79(dt,J＝6.1,4.0Hz,1H),4.38(dd,J＝3.9,2.1Hz,2H),4.22–4.13(m,6H),1.71(tt,J＝7.3,6.2Hz,6H),1.44–1.24(m,18H),0.89(t,J＝6.7Hz,9H).MS-ESI(m/z):627.3(M+H) + .
Example 132 ', 3 ', 5 ' -tri-O- (n-tetradecanoyloxy) cytarabine
Referring to example 9, the substitution reaction of compound VII with n-tetradecyl chloroformate and removal of Bz protecting group gave the title compound as a white solid with HPLC purity > 98.6%.
1 H NMR(500MHz,CDCl 3 )δ7.80(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.18(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.47(dd,J＝6.7,4.1Hz,1H),5.37(t,J＝6.5Hz,1H),4.79(dt,J＝6.1,4.0Hz,1H),4.42–4.34(m,2H),4.17(td,J＝6.3,1.8Hz,6H),1.72(tt,J＝7.7,6.2Hz,6H),1.39(p,J＝7.3Hz,6H),1.34–1.21(m,60H),0.93–0.85(m,9H).MS-ESI(m/z):963.7(M+H) + .
Example 142 ', 3', 5 '-tri-O- (21' -n-docosadienyloxyformyl) cytarabine
Referring to example 9, substitution reaction of compound VII with chloroformic acid (21-docodecenyl ester) and elimination of Bz protecting group gave the title compound as a white solid with HPLC purity > 98.4%.
1 H NMR(500MHz,CDCl 3 )δ7.76(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.18(d,J＝4.2Hz,1H),6.08(d,J＝7.3Hz,1H),5.80(tt,J＝10.3,6.9Hz,3H),5.47(dd,J＝6.7,4.1Hz,1H),5.42–5.32(m,1H),5.12–5.04(m,3H),5.01–4.93(m,3H),4.79(dt,J＝6.1,3.9Hz,1H),4.43–4.34(m,2H),4.17(td,J＝6.1,1.7Hz,6H),2.03(dtd,J＝8.0,6.8,1.1Hz,6H),1.72(tt,J＝7.6,6.2Hz,6H),1.39(p,J＝7.3Hz,6H),1.38–1.21(m,96H).MS-ESI(m/z):1294.0(M+H) + .
Example 152 ', 3 ', 5 ' -tri-O- (n-decylaminoyl) cytarabine
Referring to example 9, the substitution reaction of compound VII with n-decyl isocyanate, deprotection of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.2%.
1 H NMR(500MHz,CDCl 3 )δ7.68(d,J＝7.5Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.14(d,J＝3.8Hz,1H),6.08(d,J＝7.3Hz,1H),5.41(dd,J＝6.8,3.8Hz,1H),5.27(dd,J＝6.8,5.9Hz,1H),5.16–5.06(m,2H),4.39–4.30(m,2H),3.15(q,J＝5.5Hz,2H),3.03(dq,J＝13.9,5.5Hz,1H),2.90–2.79(m,2H),1.53–1.41(m,6H),1.33–1.24(m,42H),0.93–0.85(m,9H).MS-ESI(m/z):792.6(M+H) + .
Example 162 ', 3 ', 5 ' -tri-O- (n-tetradecanoylamino acyl) cytarabine
Referring to example 9, the substitution reaction of compound VII with n-tetradecyl isocyanate followed by the removal of the Bz protecting group gave the title compound as a white solid with HPLC purity > 97.9%.
1 H NMR(500MHz,CDCl 3 )δ7.85(d,J＝7.5Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.14(d,J＝3.8Hz,1H),6.08(d,J＝7.3Hz,1H),5.43–5.40(m,1H),5.27(dd,J＝6.8,5.9Hz,1H),5.16–5.06(m,2H),4.36(ddd,J＝5.7,4.5,3.5Hz,1H),4.33–4.31(m,2H),3.15(q,J＝5.5Hz,2H),3.03(dq,J＝13.9,5.5Hz,1H),2.89–2.81(m,2H),1.53–1.41(m,6H),1.34–1.23(m,66H),0.93–0.85(m,9H).MS-ESI(m/z):960.8(M+H) + .
Example 172 ', 3', 5 '-tri-O- (21' -n-eicosadienoyl) cytarabine
Referring to example 9, substitution of compound VII with (21-docodecenyl isocyanate) and removal of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.4%.
1 H NMR(500MHz,CDCl 3 )δ7.77(d,J＝7.5Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.14(d,J＝3.8Hz,1H),6.08(d,J＝7.3Hz,1H),5.80(tt,J＝10.3,6.9Hz,3H),5.41(dd,J＝6.8,3.8Hz,1H),5.28–5.25(m,1H),5.16–5.04(m,6H),4.97(dt,J＝13.7,3.1Hz,3H),4.39–4.28(m,3H),3.15(q,J＝5.5Hz,2H),3.03(dq,J＝13.9,5.5Hz,1H),2.90–2.80(m,2H),2.03(td,J＝7.9,6.8Hz,6H),1.53–1.41(m,6H),1.35–1.23(m,102H).MS-ESI(m/z):1291.1(M+H) + .
The yields in the steps from compound III to compound V in examples 1 to 8 above are 57 to 64% (e.g., 61.6% in example 1), and the yields in the steps from compound VII to compound X in examples 9 to 17 are 52 to 58% (e.g., 56.3% in example 9). In a supplementary example of the present invention (which may be referred to as example 18 in the present invention), with reference to examples 1 to 8, respectively, except that sodium pyruvate was further added together with compound III in the step from compound III to compound V to obtain the title compound; with reference to examples 9 to 17, respectively, except that 0.1 molar equivalent of sodium pyruvate with respect to compound VII was further added along with compound VII in the step from compound VII to compound X to obtain the title compound; it was found that the yield of the step from compound III to compound V in this additional example was 77 to 81% (e.g., 78.4% in the additional example of reference example 1), and the yield of the step from compound VII to compound X in this additional example was 71 to 77% (e.g., 74.7% in the additional example of reference example 9). It has surprisingly been found that in the process according to the invention for preparing the compounds, the addition of 0.1 molar equivalents of sodium pyruvate together with compound III in the step from compound III to compound V or 0.1 molar equivalents of sodium pyruvate together with compound VII in the step from compound VII to compound X significantly increases the reaction yield. Thus, according to any aspect of the present invention, wherein in the preparation of the compound of formula (I) or the compound of formula (II), 0.1 molar equivalent of sodium pyruvate is further added with compound III in the preparation step from compound III to compound V, and 0.1 molar equivalent of sodium pyruvate is further added with compound VII in the preparation step from compound VII to compound X.
Second, biological test example section of Compounds
Test example 1 Effect of Compounds on platelets
Clinical use of cytarabine is known to cause thrombocytopenic adverse reactions, which in turn affects the blood coagulation function. This experiment examined the in vivo performance of the drug by measuring the effect on the platelets and clotting time of animals after administration of the compounds of the invention. All data are expressed as mean ± SD, and comparisons between groups were performed using the t-test.
1. Material
White Kunming mice, each half of male and female, with the weight of 18-22 g, are purchased from the breeding house; cytarabine hydrochloride for injection (H20084073); the sterilized aqueous solution of the products of the embodiments 1-17 of the invention is prepared on site; inverted microscope XDS-900 (Zeikang).
2. Grouping
Test mice were taken and randomly grouped into 10 mice each, including: a normal control group, b cytarabine group, c compound group (including c1 group using the compound of example 1, c2 group using the compound of example 2, and c17 group using the compound of example 17 for …).
3. Composition medicine
a normal control group is injected with 0.9 percent sodium chloride injection with the same volume as that of the cytarabine group b by intraperitoneal injection every day; b, the cytarabine group is administrated with 0.1mmol/kg/d of cytarabine by intraperitoneal injection every day; c, the corresponding compound is administered by intraperitoneal injection every day at a rate of 0.1 mmol/kg/d; each group of medicines is prepared into a solution by using 0.9 percent sodium chloride injection as a solvent according to the concentration that the volume of a liquid medicine injected into the abdominal cavity every time is 0.1 to 0.2ml per animal. The above dose was administered daily for 7 days, and the general condition, activity, infection, bleeding, food intake, etc. of the animals were observed daily.
4. The reference (Chenqi, methodology of pharmacological research in Chinese medicine, Beijing: people's health Press, 1994: 484-: blood was drawn from the orbital capillary, dropped onto a slide glass, and the drop diameter was about 5mm, immediately timed with a stopwatch, the day before the administration (0d) and 6h after the last administration (7d), respectively. The clean No. 4 injection needle is slightly shifted inwards from the edge of the blood drop to observe whether blood filaments are picked up or not, and the blood coagulation time is determined from the beginning of blood sampling to the end of the blood filaments picking up.
As a result: the pre-dose clotting time(s) was summarized in data for all groups of animals as 118.24 ± 26.47; after 7 days of administration, the coagulation time(s) of the b cytarabine group was 282.24 ± 43.72 ×, the coagulation time(s) of the C1 group was 176.63 ± 38.46 × #, the mean coagulation time(s) of the C2 to C17 groups was 161 to 195, showed a significant difference of × # p <0.01 compared to 0 days, and showed a significant difference of # # p <0.01 compared to 7 days of the b cytarabine group. Wherein # is p <0.01 compared to day 0 results and # is p <0.01 compared to day 7 b cytarabine group results.
5. Reference (national institute of health, national operating rules for clinical laboratory, Nanjing: university Press 1997: 22-23) method for blood cell counts: blood was collected one day before administration (day 0) and one day after administration (day 8), and peripheral blood platelets (Bpc) were counted manually using a microscope, according to "visual counting" of "platelet count" of "national clinical laboratory practice". Results are expressed as Bpc change rates: bpc change rate (average of Bpc on day 0-average of Bpc on day 8)/average of Bpc on day 0 × 100%.
As a result: bpc numbers (x 1000/. mu.L) were collected at day 0 before dosing as 847.16 ± 138.34 for all groups of animals; 7 days after administration, the number of Bpc in the b cytarabine group (x 1000/. mu.L) was 402.73 ± 86.54 #, the number of Bpc in the C1 group (x 1000/. mu.L) was 683.48 ± 116.58 #, and the number of Bpc in the C2-C17 group (x 1000/. mu.L) averaged values ranged from # #orin the range 653-698, wherein p is <0.01 compared with the 0-day results, p is <0.05 compared with the 0-day results, and # is p <0.01 compared with the 7-day b cytarabine group results; the change rate of Bpc on day 8 for each group, b cytarabine group 52.46%, C1 group 19.32%, C2-C17 group 17.6-22.9%.
According to the results of this test, it was unexpectedly found that the compound of the present invention is significantly weaker than cytarabine in causing platelet lowering at the equivalent dose, and thus it can be expected that the compound of the present invention will cause significantly less adverse effects in causing platelet lowering. Although the test examples were conducted using mice, it is obvious to those skilled in the art that the results of such animal models can be extrapolated to the results of other mammals such as humans.
Test example 2 pharmacodynamic study of Compound
The influence of the compound on the cell biological characteristics of the acute myeloid leukemia SCID model mouse is examined by referring to the method of Corhong literature (Corhong, et al, Epimedium extract on the cell biological characteristics of the acute myeloid leukemia SCID model mouse, Chinese medicine emergency 2018 (03): 389-393). The HL-60 cell line, namely, the human promyelocytic leukemia cell is obtained from a 36-year-old female suffering from acute promyelocytic leukemia at the national cancer institute, mainly a neutrophil promyelocytic cell, and is a cell line used for laboratory research on how certain blood cells are formed, particularly for the research on the anti-tumor activity of acute myeloid leukemia drugs. HL-60 cells were purchased from Punuisal, Inc., and the remaining materials were commercially available.
Cell culture was performed according to the method of Kyoho. The test mice were randomly grouped into 10 mice each, including: a normal control group, b cytarabine group, c compound group (including c1 group using the compound of example 1, c2 group using the compound of example 2, and c17 group using the compound of example 17 for …), and d model group (no administration after successful molding). And (4) molding by referring to the method of the Corohong literature and reaching the standard of nodulation, wherein the normal control group is not molded, and the other groups are molded.
Administration: fine injection of HL-60The normal control group a and the model group d are injected with 0.9 percent sodium chloride injection with the same volume as that of the cytarabine group b by intraperitoneal injection every day at the last 1d of the 3 rd week after the cell suspension; b Cytarabine group is administered with 0.4mmol/m Cytarabine per day by intraperitoneal injection 2 D; c group was administered the corresponding compound 0.4mmol/m by intraperitoneal injection every day 2 D; each group of medicines is prepared into a solution by using 0.9 percent sodium chloride injection as a solvent according to the concentration that the volume of a liquid medicine injected into the abdominal cavity every time is 0.1 to 0.2ml per animal. The above dose was administered daily for 7 days, and the general condition, activity, infection, bleeding, food intake, etc. of the animals were observed daily.
The sample collection and detection are carried out according to the method of the Kyoho literature, tail vein blood is taken from the animal 48 hours after the last administration, and the number of HL-60 cells (%) is counted by an artificial counting method, and the results are as follows: the results of the experiments showed that the cell numbers of HL-60 in the a normal control group were 0 (%), the cell numbers of HL-60 in the b cytarabine group were 6.62 + -0.87 (%), the cell numbers of HL-60 in the C1 group were 5.12 + -0.63 (%), the average values of the cell numbers of HL-60 in the C2-C17 groups were 4.3-6.4, and the cell numbers of HL-60 in the d model group were 19.73 + -2.04, where p is less than 0.05 compared with the results in the d model group.
The above results indicate that the compounds of the present invention are slightly superior to cytarabine in inhibiting HL-60 cells but with no statistically significant difference.
Third, preparation example section of composition
Example 31 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a water-soluble polymer,
phospholipid (DPPG): 300 parts by weight of a solvent, and a solvent,
pegylated phospholipid (DSPE-PEG) 2000 ): 75 parts by weight of a water-soluble polymer,
cholesterol: 100 parts by weight, and
excipient (aqueous solvent: 5% glucose solution to make liquid composition, the addition amount is to make the final concentration of active drug be 2mg/ml, or lyophilized excipient: mannitol to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 25).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, pegylated phospholipid, cholesterol and active drug in an organic solvent (dichloromethane: methanol 4: 1, added in an amount of 3 times the amount of completely dissolved);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (45 ℃, vacuum degree of 200mbar, rotation speed of 250rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding aqueous solvent into container, hydrolyzing at 55 deg.C for 0.25 hr, performing ultrasonic treatment for 20min, and filtering for sterilization (using 0.22 μ M polyethersulfone microporous filter membrane) to obtain lipid composition in the form of liquid lipid suspension; or
(23b) Adding excipient solution (3.5% concentration) dissolved in water in advance, hydrating at 55 deg.C for 0.25 hr, performing ultrasonic treatment for 15min, filtering for sterilization (such as 0.22 μ M polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 31, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 32 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a water-soluble polymer,
phospholipid (DPPC): 240 parts by weight of a non-woven fabric,
pegylated phospholipid (DSPE-PEG) 2000 ): 60 parts by weight of a water-soluble polymer,
cholesterol: 36 parts by weight, and
excipient (aqueous solvent: 5% glucose solution to make liquid composition, the addition amount is to make the final concentration of active drug be 0.5mg/ml, or lyophilized excipient: mannitol to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 20).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 3 times of the complete dissolution degree);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (45 ℃, vacuum degree of 200mbar, rotation speed of 250rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding aqueous solvent into container, hydrolyzing at 55 deg.C for 0.33 hr, performing ultrasonic treatment for 30min, and filtering for sterilization (using 0.22 μ M polyethersulfone microporous filter membrane) to obtain lipid composition in the form of liquid lipid suspension; or
(23b) Adding excipient solution (3.5% concentration) dissolved in water in advance, hydrating at 55 deg.C for 0.33 hr, performing ultrasonic treatment for 30min, filtering for sterilization (such as 0.22 μ M polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 32, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 33 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a water-soluble polymer,
phospholipid (DPPC): 500 parts by weight of a water-soluble polymer,
pegylated phospholipid (DSPE-PEG) 2000 ): 120 parts by weight of a solvent and a solvent,
cholesterol: 65 parts by weight, and
excipient (aqueous solvent: 5% glucose solution to make liquid composition, the addition amount is to make the final concentration of active drug 1mg/ml, or lyophilized excipient: mannitol to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 35).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 3 times of the complete dissolution degree);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (50 ℃, vacuum degree of 220mbar, rotation speed of 250rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding aqueous solvent into container, hydrating at 70 deg.C for 1 hr, performing ultrasonic treatment for 15min, filtering for sterilization (using 0.22 μ M polyethersulfone microporous membrane), and collecting lipid composition in form of liquid lipid suspension; or
(23b) Adding excipient solution (3.5% concentration) dissolved in water in advance, hydrating at 70 deg.C for 1 hr, performing ultrasonic treatment for 15min, filtering for sterilization (using 0.22 μ M polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 33, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 34 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a water-soluble polymer,
phospholipid (DMPG): 200 parts by weight of a solvent, and a solvent,
PEGylated Phospholipids (PEG 1000-DSPE): 250 parts by weight of a non-woven fabric,
cholesterol: 80 parts by weight, and
excipient (aqueous solvent: 0.9% sodium chloride solution to make liquid composition, the addition amount is to make the final concentration of active drug be 3mg/ml, or lyophilized excipient: glycine to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 30).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (chloroform, 2 times of the amount of the phospholipid dissolved completely);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (60 ℃, vacuum degree of 200mbar, rotation speed of 250rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of the lipid composition:
(23a) adding aqueous solvent into container, hydrating at 60 deg.C for 1.5 hr, performing ultrasonic treatment for 20min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or
(23b) Adding excipient solution (15% concentration) dissolved in water in advance, hydrating at 70 deg.C for 2.5 hr, performing ultrasonic treatment for 45min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 34, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 35 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a water-soluble polymer,
phospholipids (egg yolk lecithin): 1500 parts by weight of a reaction product of (B),
pegylated phospholipid (DSPE-PEG) 4000 ): 60 parts by weight of a water-soluble polymer,
cholesterol: 120 parts by weight, and
excipient (aqueous solvent: water to make liquid composition, the addition amount is to make the final concentration of active drug be 0.5mg/ml, or lyophilized excipient: dextran, to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 50).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane with 4 times of the amount of completely dissolved);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (40 ℃, vacuum degree of 250mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of the lipid composition:
(23a) adding aqueous solvent into the container, hydrating at 70 deg.C for 2.5 hr, performing ultrasonic treatment for 45min, and filtering for sterilization (using 220nm polyethersulfone microporous filter membrane) to obtain lipid composition in the form of liquid lipid suspension; or
(23b) Adding excipient solution (3% concentration) dissolved in water in advance, hydrating at 60 deg.C for 1.5 hr), performing ultrasonic treatment for 20min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 35, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 36 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a solvent, based on the total weight of the composition,
phospholipid (soybean lecithin): 120 parts by weight of a solvent and a solvent,
pegylated phospholipid (DSPE-PEG) 6000 ): 300 parts by weight of a solvent, and a solvent,
cholesterol: 1450 parts by weight, and
excipient (aqueous solvent: 5% glucose solution to make liquid composition, the addition amount is to make the final concentration of active drug 10mg/ml, or lyophilized excipient: lactose to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 20).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 2 times of the complete dissolution degree);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (45 ℃, the vacuum degree is 230mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding aqueous solvent into container, hydrating at 40 deg.C for 2 hr, performing ultrasonic treatment for 35min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or
(23b) Adding excipient solution (6% concentration) dissolved in water in advance, hydrating at 80 deg.C for 5 hr, performing ultrasonic treatment for 35min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 36, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 37 preparation of lipid composition
The formula is as follows:
active drug: 100 parts by weight of a water-soluble polymer,
phospholipid (dipalmitoylphosphatidylcholine): 1800 parts by weight of a reaction solution,
pegylated phospholipid (DSPE-PEG) 3350 ): 35 parts by weight of a solvent, and a solvent,
cholesterol: 25 parts by weight, and
excipient (aqueous solvent: 0.9% sodium chloride solution to make liquid composition, adding amount is to make active drug final concentration 0.5 mg/ml), or lyophilized excipient: mannitol to make solid composition, active drug and the lyophilized excipient weight ratio is 1: 150).
The preparation method adopts a film dispersion method and comprises the following steps:
(21) dissolving phospholipid, PEGylated phospholipid, cholesterol and active drug in organic solvent (dichloromethane with 2.5 times of the total amount of the phospholipid, cholesterol and active drug);
(22) evaporating the liquid obtained in the previous step on a rotary evaporator (50 ℃, vacuum degree 210mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding aqueous solvent into the container, hydrating at 80 deg.C for 2 hr, performing ultrasonic treatment for 40min, and filtering for sterilization (using 220nm polyethersulfone microporous membrane) to obtain lipid composition in the form of liquid lipid suspension; or
(23b) Adding excipient solution (with concentration of 3.5%) dissolved in water in advance, hydrating at 40 deg.C for 1 hr, performing ultrasonic treatment for 25min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.
In this example 37, cytarabine and 17 compounds obtained in examples 1 to 17 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (18 liquid compositions) and freeze-dried lipid compositions in a solid state (18 solid compositions), respectively.
Example 41 characterization of lipid compositions
The lipid composition in the liquid state and the lipid composition in the solid state obtained in examples 31 to 37 were each diluted with water or dissolved and then diluted so that the concentration of the compound of formula (I) was 0.2mg/ml (the obtained liquid composition itself was not diluted if the concentration was lower than this concentration), the particle size of the fine particles in the drug solution was measured using a malvern Zetasizer Nano ZS Nano-particle size potentiometer, the average particle size was calculated, and the percentage of fine particles having a particle size of less than 10nm and the percentage of fine particles having a particle size of more than 300nm were counted.
As a result:
all the liquid lipid compositions obtained in examples 31 to 37 have less than 5% of particles having a particle size of less than 10nm and less than 5% of particles having a particle size of more than 300nm, for example, the liquid lipid composition obtained in example 31 has 1.5% of particles having a particle size of less than 10nm and 0.2% of particles having a particle size of more than 300nm with respect to Compound 1;
all of the solid lipid compositions obtained in examples 31 to 37 had less than 5% of fine particles having a particle size of less than 10nm and less than 5% of fine particles having a particle size of more than 300nm, for example, the solid lipid composition obtained in example 31 had 1.6% of fine particles having a particle size of less than 10nm and 0.4% of fine particles having a particle size of more than 300nm with respect to Compound 1;
the average particle size of the total liquid lipid composition obtained in examples 31 to 37 is in the range of 74 to 127nm, for example, the average particle size of the particles in the liquid lipid composition obtained for compound 1 in example 31 is 92 nm.
The average particle size of the total solid lipid composition obtained in examples 31 to 37 is in the range of 81 to 118nm, for example, the average particle size of the particles in the liquid lipid composition obtained for compound 1 in example 31 is 102 nm; there was no significant difference in particle size between the various liquid compositions obtained in examples 31-37 and their corresponding solid compositions.
The above particle size measurements were made within 15 days of the preparation of the composition and these results do not reflect the stability in particle size properties of the composition, which corresponds to a 0 month result. When all the liquid lipid compositions obtained in examples 31 to 37 were left at room temperature for 18 months, their particle sizes were measured, and as a result, the average particle size of each sample was increased by 3 to 7% as compared with that of the sample at 0 month, for example, the average particle size of the particles in the liquid lipid composition obtained in example 31 for compound 1 was increased by 4.6% after 18 months. In addition, when all the solid lipid compositions obtained in examples 31 to 37 were left at room temperature for 18 months and then their particle sizes were measured, the average particle size of each sample was increased by 3 to 6% as compared with the 0 month result thereof, for example, the average particle size of the particles in the solid lipid composition obtained in example 31 for compound 1 was increased by 4.3% after 18 months.
Example 42 antitumor Activity of lipid composition
This example was conducted in accordance with the method of test example 2 of the present invention, and it was determined that the corresponding compound was administered at 0.25mmol/m by intraperitoneal injection 2 D liposomes with the compounds of examples 1 to 17 of example 31 as API, determination of the intraperitoneal administration of 0.25mmol/m of the corresponding compound 2 The results of the HL-60 cell counts (%) of the API liposomes with the compounds of examples 1 to 17 of example 32 mean values in the range of 4.5 to 6.8, for example, the compound of example 1 of example 31 mean HL-60 cell counts (%) of 5.36 ± 0.48, and antitumor activity comparable to that of the corresponding drug substance can be obtained at a dose corresponding to 62.5% of the API drug substance.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (17)

1. A lipid composition comprising: 100 parts of a compound of a formula (II), 100-2000 parts of phospholipid, 30-300 parts of polyethylene glycol phospholipid, 20-200 parts of cholesterol and an excipient;
the compound shown in the formula (II) has the following structure:
Figure DEST_PATH_IMAGE002
or a pharmaceutically acceptable salt thereof, wherein:
R 1 、R 2 、R 3 each independently represents C 6-22 Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 CH of the carbon chain is 2 Optionally substituted with O;
x represents O or NH.
2. The lipid composition according to claim 1, wherein the amount of the phospholipid is 200 to 1500 parts by weight.
3. The lipid composition according to claim 1, wherein the amount of the pegylated phospholipid is 40 to 200 parts by weight.
4. The lipid composition according to claim 1, wherein the amount of cholesterol is 30 to 150 parts by weight.
5. A lipid composition according to claim 1, wherein R is 1 、R 2 、R 3 Selected from: n-decyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, n-docosyl, n-hexadecyloxypropyl, n-octadecyl, n-9-ene) n-decyl, (11-ene) n-dodecyl, (11-ene) n-dodecylethyl.
6. The lipid composition according to claim 1, said pharmaceutically acceptable salt being a salt with an inorganic or organic acid selected from the group consisting of: hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, alanine, aspartic acid, lysine, methanesulfonic acid, p-toluenesulfonic acid.
7. A lipid composition according to claim 1, said compound being a compound 12 to a compound 17 selected from the group consisting of:
compound 12: 2 ', 3 ', 5 ' -tri-O- (n-hexyloxyacyl) cytarabine,
compound 13: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoyloxyacyl) cytarabine,
compound 14: 2 ', 3', 5 '-tri-O- (21' -n-docosadienyloxyformyl) cytarabine,
compound 15: 2 ', 3 ', 5 ' -tri-O- (n-decylaminoyl) cytarabine,
compound 16: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoylamino acyl) cytarabine,
compound 17: 2 ', 3 ', 5 ' -tri-O- (21 "-n-eicosadienoyl) cytarabine.
8. The lipid composition according to claim 1, which is a composition in the liquid state, wherein the excipient is an aqueous vehicle selected from the group consisting of: water, 0.8-1% sodium chloride solution and 2-10% glucose solution; the dosage of the aqueous solvent is such that the concentration of the compound of formula (II) in the liquid composition is 0.2-20 mg/ml.
9. The lipid composition according to claim 1, which is a composition in a solid state, wherein the excipient is a lyophilized excipient; the lyophilized excipient is selected from: mannitol, sorbitol, lactose, glycine, dextran, sucrose, and glucose; the weight ratio of compound of formula (II) to lyophilized excipient is 1: 20 to 200 parts.
10. A lipid composition according to claim 1, said phospholipid being selected from the group consisting of: egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, and combinations thereof.
11. The lipid composition according to claim 1, wherein the pegylated phospholipid is a phospholipid modified with polyethylene glycol having a molecular weight of 1000 to 10000 daltons.
12. The lipid composition according to claim 1, said pegylated phospholipid being selected from the group consisting of: distearoylphosphatidylethanolamine-polyethylene glycol 1000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 3350, distearoylphosphatidylethanolamine-polyethylene glycol 4000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 6000, distearoylphosphatidylethanolamine-polyethylene glycol 8000, distearoylphosphatidylethanolamine-polyethylene glycol 10000.
13. The lipid composition according to claim 1, which is prepared by a liposome preparation process selected from the group consisting of: film dispersion method, extrusion preparation method, French pressure method, reverse phase evaporation method, pH gradient method, ammonium sulfate gradient method.
14. The lipid composition according to claim 1, which is prepared by a thin film dispersion method comprising the steps of:
(21) dissolving a phospholipid, a pegylated phospholipid, cholesterol, and an active drug in an organic solvent selected from the group consisting of: dichloromethane, chloroform and methanol, wherein the amount of the organic solvent is 2-4 times of the complete dissolution degree;
(22) evaporating the liquid obtained in the previous step on a rotary evaporator to remove the solvent, so that the residue forms a film on the inner wall of the container;
(23) preparation of lipid composition:
(23a) adding an aqueous solvent into a container, hydrating for 0.1-5 hours at the temperature of 40-80 ℃, then carrying out ultrasonic treatment for 15-60 min, and carrying out filtration sterilization to obtain a lipid composition in the form of a liquid lipid suspension; or
(23b) Adding an excipient solution dissolved in water in advance into a container, hydrating at 40-80 ℃ for 0.1-5 hours, carrying out ultrasonic treatment for 15-60 min, filtering for sterilization, subpackaging into glass bottles, and freeze-drying in a freeze dryer to remove water to obtain the solid lipid composition.
15. The lipid composition according to claim 1, which is a composition in a liquid or solid state, and which, when diluted with water or dissolved to form a liquid medicine having a concentration of the compound of formula (II) of 0.2mg/ml or less, has an average particle size of less than 200nm, less than 5% of particles having a particle size of less than 10nm and less than 5% of particles having a particle size of more than 500nm, as measured by a nano-particle sizer.
16. Use of a lipid composition according to any one of claims 1 to 15 in the manufacture of a medicament for the treatment of leukemia in a mammal.
17. Use according to claim 16, the leukemia being selected from acute myelocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia.
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US5968914A (en) * 1987-10-28 1999-10-19 Pro-Neuron, Inc. Treatment of chemotherapeutic agent and antiviral agent toxicity with acylated pyrimidine nucleosides
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