CN110054659B

CN110054659B - Method for improving antitumor activity of medicine

Info

Publication number: CN110054659B
Application number: CN201910369362.0A
Authority: CN
Inventors: 夏桂民; 刘明亮; 罗芳; 汪阿鹏
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2020-09-11
Anticipated expiration: 2039-05-05
Also published as: CN110054659A

Abstract

The invention relates to a method for improving the antitumor activity of a medicament. In particular, one aspect provides compounds of formula (I) or pharmaceutically acceptable salts thereof, wherein the substituents are as described in the specification. Also provided are methods of preparing the compounds, methods of enhancing the antitumor activity of antitumor drugs by preparing the compounds into lipid compositions, and lipid compositions comprising such compounds. The method of the invention can effectively improve the antitumor activity of the medicine.

Description

Method for improving antitumor activity of medicine

Technical Field

The invention belongs to the field of medicinal chemistry, relates to a method for improving antitumor activity of drugs such as gemcitabine, and particularly relates to a group of gemcitabine esterified compounds with antitumor activity, lipid compositions and preparation methods thereof, and application of the gemcitabine esterified compounds in antitumor aspect.

Background

Gemcitabine, english name gemcitabine, Chinese scholars name: 4-amino-1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -1H-pyrimidin-2-one), chemical name: 2' -deoxy-2 ',2' -difluorocytidine of the formula: c₉H₁₁F₂N₃O₄The novel fluoro nucleoside analogue has the chemical structure shown in the following formula (J):

gemcitabine has no pharmacological activity, is activated by deoxycytidine kinase after entering a human body, and is metabolized into corresponding monophosphate, diphosphate and triphosphate by cytidine deaminase to play a role. Gemcitabine is used in the treatment of various solid tumors (e.g., non-small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, etc.), and its hydrochloride is used clinically, but is currently limited to intravenous administration for the treatment of pancreatic cancer and non-small cell lung cancer. Patient compliance with this therapy is poor and there are several adverse effects, typical of thisThe adverse reactions include ① myelosuppression, anemia, leukopenia and thrombocytopenia in the blood system ② gastrointestinal tract, 2/3 patients with liver transaminase abnormalities, which are mostly mild and non-progressive lesions, 1/3 patients with nausea and vomiting, 20% of whom require medication, ③ kidneys, 1/2 patients with mild proteinuria and hematuria, some cases with renal failure of unknown origin, ④ allergy, skin diagnosis in 25% of patients, itching in 10% of patients, bronchospasm in less than 1% of patients, ⑤ other patients with symptoms similar to influenza, edema/peripheral edema incidence of about 30%, alopecia, lethargy, diarrhea, oral toxicity and constipation incidence of 13%, 10%, 8%, 7% and 6%, respectively, which will seriously affect the benefit/risk ratio of gemcitabine clinical medication_1/2: 8-17min), multiple administrations are required (Gang Wang, et al.j.med.chem.2017, 60,2552; tang Li, et al, DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, 2017, 43: 2016).

The prior art still expects a new method and expects some more excellent effect or effects for treating tumor, for example, a drug with more excellent antitumor activity is expected to be applied clinically.

Disclosure of Invention

The object of the present invention is to provide a novel method and to expect some more excellent effect or effects for treating tumors, for example, to provide a drug having more excellent antitumor activity for clinical use. The present inventors have surprisingly found that compounds having the structure of the present invention exhibit excellent antitumor activity.

To this end, a first aspect of the invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof; wherein:

R₁and R₂Identical or different, each represents C_6-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

A compound according to the first aspect of the invention, wherein R₁And R₂Identical or different, each represents C_8-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

A compound according to the first aspect of the invention, wherein R₁And R₂Identical or different, each represents C₁₀22 linear or branched, saturated alkyl or unsaturated alkenyl radicals, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

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 of the other aspects 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 12 selected from the group consisting of:

compound 1: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-decanoate,

compound 2: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dilaurate,

compound 3: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dimyristate,

compound 4: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dipalmitate,

compound 5: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-trans-oleate,

compound 6: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-hexadecyloxypropyl ether,

compound 7: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-octadecyl oxyethyl ether,

compound 8: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -laurate-5 ' -myristate,

compound 9: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -myristate-5 ' -laurate,

compound 10: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -n-tetradecyloxyethyl ether-5 ' -n-hexadecyloxyethyl ether,

compound 11: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -n-hexadecyloxypropyl ether-5 ' -n-tetradecyloxyethyl ether,

compound 12: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -n-hexadecyloxypropyl ether-5 ' -myristate,

or a pharmaceutically acceptable salt thereof.

Further, the present invention provides in a second aspect a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

According to a second aspect of the invention, R in the compound of formula (I)₁And R₂In the same way, the process is shown in scheme 1.

Scheme 1:

in scheme 1, R is as defined herein for R₁And/or R₂Defined, e.g. for C_6-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

Specifically, the preparation method comprises the following steps:

1) dissolving Compound II (commercially available in China) in a nonpolar solvent (e.g., Dichloromethane (DCM), chloroform, tetrahydrofuran, dioxane) in an appropriate amount of an organic base [ e.g., triethylamine, N-lutidine, pyridine, 4-Dimethylaminopyridine (DMAP)]In the presence of 1-6 times of equivalent of Boc₂Stirring and reacting O at the temperature of between room temperature and 50 ℃ for 3 to 15 hours to obtain a compound of formula (III) protected by amino;

2) dissolving the compound of formula (III) in a protic solvent (e.g., water, alcohol or a mixed solvent of alcohol and water, such as methanol), adding 1-2 times of an equivalent of an inorganic base (e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate), and reacting at 0-room temperature for 3-10 hours with stirring to obtain a compound of formula (IV) with a hydroxyl protecting group removed;

3) dissolving a compound of formula (IV) in a nonpolar solvent [ such as Dichloromethane (DCM), chloroform, tetrahydrofuran and dioxane ], and reacting with a compound of formula (V) (obtained by a domestic commercial method) in an amount of 1-4 times of equivalent in the presence of a proper amount of organic base (such as triethylamine, N-dimethylpyridine, pyridine and 4-Dimethylaminopyridine (DMAP)) at 0-room temperature for 4-15 hours under stirring to obtain a compound of formula (VI);

4) dissolving the compound of formula (VI) in a non-polar solvent (e.g., Dichloromethane (DCM), chloroform, tetrahydrofuran, dioxane), adding 1-2 equivalents of an acid [ e.g., trifluoroacetic acid (TFA), hydrochloric acid]Stirring and reacting for 0.5-5 hours at 0-room temperature to obtain the compound of formula (I'), wherein R is₁And R₂The same compound of formula (I).

According to a second aspect of the invention, R in the compound of formula (I)₁And R₂In a different situation, the process is shown in scheme 2.

Scheme 2:

in scheme 2, R₁And R₂As defined herein.

Specifically, the preparation method comprises the following steps:

1) dissolving the compound of formula (IV) in a dipolar solvent [ e.g. Dimethylformamide (DMF), dimethyl sulfoxide]Reacting with 1-2 times of tert-butyldiphenylchlorosilane (TBDPSCl) in the presence of an appropriate amount of organic base (such as triethylamine, N-dimethylpyridine, pyridine, 4-Dimethylaminopyridine (DMAP)) at 0-room temperature for 8-15 hours while stirring to obtain sugar ring 5^/-a compound of formula (VII) wherein the hydroxyl group at position is selectively protected;

2) reacting a compound of formula (VII) with a compound of formula (V) (which may be abbreviated as R)₁COCl) to produce esterification reaction to obtain a compound shown as a formula (VIII);

3) dissolving the compound of formula (VIII) in a non-polar solvent (e.g., Dichloromethane (DCM), chloroform, Tetrahydrofuran (THF), dioxane), adding 1-2 times equivalent of tetrabutylammonium fluoride (may be abbreviated as TBAF), and reacting with stirring at 0-room temperature for 4-15 hours to obtain a compound of formula (IX) with a protecting group removed;

4) reacting a compound of formula (IX) with a compound of formula (V) (R)₂COCl) to obtain a compound of a formula (X);

5) the compound of formula (X) is subjected to Boc protecting group removal to obtain the compound of formula (I), wherein R₁And R₂Different.

Further, the third aspect of the present invention provides a method for enhancing the antitumor activity of the compound of formula (J) or a pharmaceutically acceptable salt thereof,

the method comprises the following steps:

(1) preparing a compound of formula (J) or a pharmaceutically acceptable salt thereof, as a compound of formula (I) or a pharmaceutically acceptable salt thereof, as described in any one of the first aspect of the invention;

(2) preparing a lipid composition comprising a phospholipid from a compound of formula (I) or a pharmaceutically acceptable salt thereof.

A process according to a third aspect of the invention, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is as described in any embodiment of the first aspect of the invention or as prepared by a process as described in any of the second aspects of the invention.

The process according to the third aspect of the invention, wherein the compound of formula (J) or a pharmaceutically acceptable salt thereof is prepared as described in any one of the second aspects of the invention.

The method according to the third aspect of the invention, wherein the lipid composition comprises:

a compound of formula (I): 100 parts by weight of a water-soluble polymer,

phospholipid: 50 to 1000 parts by weight (for example, 50 to 800 parts by weight),

pegylated phospholipids: 5 to 200 parts by weight (for example, 10 to 100 parts by weight),

cholesterol: 5 to 100 parts by weight (e.g., 10 to 50 parts by weight), and

and (3) an excipient.

The method according to the third aspect of the invention, wherein the lipid composition 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 its pharmaceutically acceptable salt in the liquid composition is 0.2-20 mg/ml, such as 0.25-15 mg/ml, such as 0.5-10 mg/ml.

The method according to the third aspect of the invention, wherein the lipid composition is a composition in a solid state (e.g. is a freeze-dried composition), wherein the excipient is a lyophilised 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 a pharmaceutically acceptable salt thereof to the lyophilized excipient is 100: 100-2000, for example, the weight ratio is 100: 200-1500, for example, the weight ratio is 100: 250 to 1000.

The method according to the third aspect of the present invention, wherein the phospholipid is selected from the group consisting of: egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine (i.e., DMPC), dimyristoylphosphatidylglycerol (i.e., DMPG), dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, and combinations thereof.

The method according to the third 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 method according to the third aspect of the present invention, wherein said lipid composition is prepared by a liposome preparation process. 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 method according to the third aspect of the present invention, wherein the lipid composition is prepared by a thin film dispersion method (a classical liposome preparation method) comprising the steps of:

(21) dissolving phospholipid, pegylated phospholipid, cholesterol and active drug in an organic solvent (e.g., dichloromethane, chloroform, etc.);

(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 at 40-80 deg.C (e.g. 60-70 deg.C) for 1-5 hr (e.g. 1.5-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 20-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), and collecting lipid composition in the form of liquid lipid suspension; or

(23b) Adding an excipient solution dissolved in water in advance into a container, hydrating at 40-80 deg.C (e.g. 60-70 deg.C) for 1-5 hr (e.g. 1.5-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 20-45 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 third 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%, for example, 5 to 15%.

Further, the fourth aspect of the present invention provides a lipid composition comprising:

a compound of formula (I): 100 parts by weight of a water-soluble polymer,

cholesterol: 5 to 100 parts by weight (e.g., 10 to 50 parts by weight), and

and (3) an excipient.

The lipid composition according to the fourth 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 its pharmaceutically acceptable salt in the liquid composition is 0.2-20 mg/ml, such as 0.25-15 mg/ml, such as 0.5-10 mg/ml.

The lipid composition according to the fourth 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 a pharmaceutically acceptable salt thereof to the lyophilized excipient is 100: 100-2000, for example, the weight ratio is 100: 200-1500, for example, the weight ratio is 100: 250 to 1000.

The lipid composition according to the fourth aspect of the present invention, wherein the phospholipid is selected from the group consisting of: egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine (i.e., DMPC), dimyristoylphosphatidylglycerol (i.e., DMPG), dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, and combinations thereof.

The lipid composition according to the fourth 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 fourth 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 fourth aspect of the present invention is prepared by a thin film dispersion method (a classical liposome preparation method) comprising the steps of:

(22) evaporating the liquid obtained in the last step on a rotary evaporator (40-60 ℃ and the vacuum degree of 200-250 mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of 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%, for example, 5 to 15%.

Further, the fifth aspect of the present invention provides a method for preparing a lipid composition comprising:

a compound of formula (I): 100 parts by weight of a water-soluble polymer,

cholesterol: 5 to 100 parts by weight (e.g., 10 to 50 parts by weight), and

an excipient;

the method is a liposome preparation process selected from the following: 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 fifth aspect of the invention, wherein the lipid composition 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 its pharmaceutically acceptable salt in the liquid composition is 0.2-20 mg/ml, such as 0.25-15 mg/ml, such as 0.5-10 mg/ml.

The method according to the fifth aspect of the invention, wherein the lipid composition is a composition in a solid state (e.g. is a freeze-dried composition), wherein the excipient is a lyophilised 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 a pharmaceutically acceptable salt thereof to the lyophilized excipient is 100: 100-2000, for example, the weight ratio is 100: 200-1500, for example, the weight ratio is 100: 250 to 1000.

The method according to the fifth aspect of the invention, comprising the steps of:

(23) preparation of lipid composition:

The method 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, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine (i.e., DMPC), dimyristoylphosphatidylglycerol (i.e., DMPG), dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, and combinations thereof.

The method 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 method according to the fifth 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%, for example, 5 to 15%.

Further, the sixth aspect of 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 a process according to any one of the second aspect of the present invention or a lipid composition according to the fourth aspect of the present invention in the manufacture of a medicament for the treatment of cancer. For example, the cancer is, but not limited to, non-small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer.

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%. This term may be referred to as particle size and particle size distribution.

The lipid composition according to any aspect of the present invention, which is a composition in a liquid or solid state, is diluted with water or dissolved to prepare a liquid medicine having a concentration of the compound of formula (I) of 0.2mg/ml or less, which liquid medicine is measured with a nano-particle sizer and calculates the particle diameters D10, D50, and D90 values of the nanoparticles (also commonly understood as a particle diameter where 10% of the particles are smaller than this value, a particle diameter where 50% of the particles are smaller than this value, or a median particle diameter, and a particle diameter where 90% of the particles are smaller than this value, respectively), the diameter Span value of the nanoparticles of the test article is calculated by the following formula: span ═ (Dv90-Dv10)/Dv 50; the composition has a Span of less than 5, particularly less than 3, more particularly less than 2.5, more particularly less than 2. Smaller Span means narrower particle size distribution of the particles and is more desirable in the art, and it is well known in the art that for injectable nanoparticle formulations, a Span of less than 3 is generally considered acceptable, a Span of less than 2.5 is generally considered satisfactory, and a Span of less than 2 is generally considered highly satisfactory. It has been surprisingly found that the compositions prepared by the process of the present invention have nanoparticles with an average particle size of less than 200nm, Span values of less than 2.5, and that some compositions exhibit substantially unchanged effect of average particle size and Span values after prolonged storage.

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 formulated as 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.

In the present invention, references to% are weight/weight percentages, as not otherwise specified.

Gemcitabine, (+)2' -deoxy-2 '2' -difluorocytosine, is usually used clinically in the form of hydrochloride, and a common dosage form is gemcitabine hydrochloride freeze-dried powder injection. Can be clinically used for treating the following diseases: locally advanced or metastatic non-small cell lung cancer, locally advanced or metastatic pancreatic cancer, gemcitabine and paclitaxel in combination are suitable for the treatment of unresectable, locally recurrent or metastatic breast cancer that recurs after adjuvant/neoadjuvant chemotherapy.

Cellular metabolism and mechanism of action of gemcitabine: gemcitabine (dFdC) is a pyrimidine antimetabolite which is metabolized intracellularly to active nucleoside diphosphate (dFdCDP) and triphosphate (dFdCTP) by the action of nucleoside kinases. dFdCDP and dFdCTP inhibit DNA synthesis by two mechanisms of action, thereby achieving the cytotoxic effects of gemcitabine. First, dFdCDP inhibits the activity of ribonucleotide reductase, so that the production of deoxynucleoside triphosphate (dCTP), which is necessary for DNA synthesis, is inhibited. Second, dFdCTP competes with dCTP for incorporation into the DNA strand (self-enhancing effect). Likewise, small amounts of gemcitabine may also be incorporated into RNA molecules. Thus, a decrease in intracellular dCTP concentration is more favorable for incorporation of dFdCTP into the DNA strand. The DNA polymerase is unable to remove the incorporated gemcitabine and repair the DNA strands that have been formed. When gemcitabine is incorporated into a DNA strand, one nucleotide is added to the extended DNA strand. This added nucleotide can completely inhibit further synthesis of the DNA strand (cryptic strand termination). Gemcitabine induces apoptosis upon incorporation into a DNA strand.

Cytotoxic activity of gemcitabine on cultured cells: gemcitabine has significant cytotoxic activity against various cultured human and murine tumor cells. Its action is cell cycle specific, i.e., gemcitabine acts primarily on cells in the DNA synthesis phase (S-phase), under certain conditions, to prevent cell progression at the G1 phase/S phase junction. In vitro, the cytotoxic effect of gemcitabine depends on concentration and time.

Study of antitumor Activity of Gemcitabine in animal models: the antitumor activity of gemcitabine was found to be related to the mode of administration in studies in animal models of tumors. The daily administration results in high mortality and low antitumor activity in the animals. Gemcitabine has good antitumor activity against a variety of tumors in mice at non-lethal doses when administered once every 3-4 days.

Pharmacokinetic profile of gemcitabine: the pharmacokinetic profile of gemcitabine was evaluated in a total of 353 patients in 7 studies. Wherein 121 female patients and 232 male patients are between 29-79 years old. Of these patients, about 45% are non-small cell lung cancer patients and 35% are pancreatic cancer patients. The dosage range for obtaining the following pharmacokinetic parameters is 500-2,592mg/m²The infusion time is within 0.4-1.2 hours. The peak plasma concentration (obtained within 5 minutes after the end of infusion) was 3.2-45.5. mu.g/ml. According to 1000mg/m²The administration is carried out at a dose of 30min, the plasma concentration of the parent compound can be continuously higher than 5 mug/ml within 30min after the transfusion is finished, and the plasma concentration is also higher than 0.4 mug/ml within 1 hour after the transfusion is finished.

Distribution: the distribution volume of the central chamber is 12.4L/m for female²And 17.5L/m for men²(the inter-individual difference was 91.9%). The distribution volume of the peripheral compartment was 47.4L/m². The volume of the peripheral compartment is independent of gender. Plasma protein binding was negligible. Half-life: the half-life was 42-94 minutes, age and sex related. For the recommended dosing regimen, gemcitabine is completely cleared within 5-11 hours after infusion begins. Gemcitabine does not accumulate when administered once a week.

Gemcitabine is rapidly metabolized by cytidine deaminase in the liver, kidney, blood and other tissues. In cells, gemcitabine is metabolized intracellularly to produce gemcitabine monophosphate, diphosphate, and nucleoside triphosphate (dFdCMP, dFdCDP, and dFdCTP), where the dFdCDP and dFdCTP are active. Metabolites formed within these cells were not detected in either plasma or urine. The major metabolite, 2' -deoxy-2 ',2' -diflubenzuron (dFdU), is inactive and is detectable in both plasma and urine.

Gemcitabine has a systemic clearance of 29.2L/hrm²-92.2L/hr/m²Correlated with gender and age (individual differences 52.2%). Clearance is approximately 25% lower in women than in men. Although clearance is rapid, clearance decreases with age in both men and women. Gemcitabine is recommended to be administered at a dose of 1000mg/m²Intravenous drip for 30 minutes, without reducing the gemcitabine dose due to reduced clearance in men and women. Excretion via urine: less than 10% is excreted as bulk drug. Renal clearance: 2-7L/hr/m². Within one week after administration, 92% -98% of gemcitabine doses were detected, 99% of which were excreted mainly in the form of dFdU through urine and 1% through feces.

The present invention surprisingly found that a unique class of chemical structural modifications of gemcitabine exhibits significantly higher antitumor activity when prepared into lipid compositions.

Drawings

FIG. 1: particle size distribution profile of lipid composition.

FIG. 2: transmission electron microscopy of lipid compositions.

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.

Example 1 preparation of 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-decanoate

An exemplary reaction scheme is as follows:

commercially available 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dibenzoate (compound of formula II, 4.7g, 0.01mol) was dissolved in 50mL dioxane, and triethylamine (4.0g, 0.04mol), N, N-lutidine (1.8g, 0.01mol) and Boc were added sequentially₂O (12.9 g, 0.04 mol). The reaction was carried out at 40 ℃ for 5 hours and the completion of the reaction was monitored by TLC. And (3) post-treatment: the reaction solution is poured into water, extracted by dichloromethane, and the organic phase is concentrated (to obtain a compound III) and then directly subjected to the next reaction. Compound III above was dissolved in 20mL of methanol solution, then NaOH solid (28mg, 0.7mmol) was added, stirred at room temperature for 4 hours and the reaction was monitored by TLC for completion. And (3) post-treatment: it was directly filtered through silica gel, the filtrate was concentrated and subjected to column chromatography (ethyl acetate) to obtain 1.8g of a white solid IV (yield in two steps: 50%).

¹H NMR(300MHz,CD₃OD)8.28(d,J＝7.50Hz,1H),7.32(d,J＝7.5Hz,1H),6.23(t,J＝7.80Hz, 1H),4.22-4.36(m,1H),3.96(brs,2H),3.80-3.84(brs,1H),1.54(s,9H).

The above compound IV (1.8g, 4.9mmol) was dissolved in 20mL of dichloromethane, and triethylamine (1.5g, 14.7 mmol), n-C were added in that order₉H₁₉COCl (2.1g,10.7 mmol). Stir at rt overnight and TLC monitored for reaction completion. And (3) post-treatment: water was added, and the mixture was extracted with dichloromethane (to obtain compound VI) and directly subjected to the next step.

Trifluoroacetic acid (0.7g, 6.0mmol) was added to the above dichloro solution, stirred at room temperature for 4 hours and monitored by TLC for completion of the reaction. And (3) post-treatment: the reaction system was diluted with dichloromethane and washed with aqueous sodium bicarbonate, the organic phase was concentrated and subjected to column chromatography (ethyl acetate) to obtain 2.0g of the title compound V' as a white solid (two-step yield: 71%).

¹H NMR(500MHz,CDCl₃)7.45(d,J＝6.85Hz,1H),6.45(brs,1H),5.91(d,J＝7.30Hz,1H),5.28 (d,J＝11.90Hz,1H),4.42(brs,2H),4.26(d,J＝5.00Hz,1H),2.46(t,J＝7.00Hz,2H),2.40(t,J＝7.35Hz, 2H),1.66-1.71(m,4H),1.31-1.34(m,24H),0.92(t,J＝6.50Hz,6H).MS-ESI(m/z):572.3(M+H)⁺.

Example 2 preparation of 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dilaurate

Preparation method referring to example 1, compound IV was reacted with lauroyl chloride and Boc-protecting group was removed by trifluoroacetic acid to obtain the title compound as a white solid.

¹H NMR(500MHz,CDCl₃)7.55(brs,1H),6.32(brs,1H),6.09(brs,1H),5.29(brs,1H),4.34-4.45 (m,3H),2.37-2.40(m,4H),1.60-1.70(m,4H),1.25-1.29(m,32H),0.92(t,J＝6.50Hz,6H).MS-ESI (m/z):628.3(M+H)⁺.

Example 3 preparation of 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dimyristate

Preparation method referring to example 1, compound IV was reacted with myristoyl chloride and Boc protecting group was removed with trifluoroacetic acid to obtain the title compound as a white solid.

Mp:142-144℃；¹H NMR(500MHz,CDCl₃)7.44(d,J＝6.85Hz,1H),6.44(d,J＝8.85Hz,1H), 5.74(d,J＝7.30Hz,1H),5.22(d,J＝11,90Hz,1H),4.38(brs,2H),4.26(brs,1H),2.41(t,J＝7.35Hz,2H), 2.36(t,J＝7.35Hz,2H),1.60-1.70(m,4H),1.25-1.29(m,40H),0.87(t,J＝6.50Hz,6H).MS-ESI(m/z): 684.4(M+H)⁺.

Example 4 preparation of 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dipalmitate

Preparation method referring to example 1, compound IV was reacted with palmitoyl chloride, and Boc-protecting group was removed with trifluoroacetic acid to obtain the title compound as a white solid.

¹H NMR(500MHz,CDCl₃)7.42(d,J＝6.90Hz,1H),6.43(brs,1H),5.87(d,J＝7.30Hz,1H),5.32 (brs,1H),4.42(brs,2H),4.29(d,J＝5.00Hz,1H),2.46(t,J＝7.35Hz,2H),2.40(t,J＝7.25Hz,2H), 1.65-1.70(m,4H),1.25-1.31(m,48H),0.92(t,J＝6.50Hz,6H).MS-ESI(m/z):740.5(M+H)⁺.

Example 5, 2 '-difluoro-2' -deoxycytidine-3 ', 5' -di-trans-oleate

Preparation method referring to example 1, compound IV was subjected to condensation reaction with elaidic acid, and Boc-protecting group was removed by trifluoroacetic acid to obtain the title compound as a white solid.

¹H NMR(500MHz,CDCl₃)7.42(d,J＝6.90Hz,1H),6.43(brs,1H),5.86(d,J＝7.30Hz,1H), 5.32-5.37(m,5H),4.42(brs,2H),4.29(brs,1H),2.46(t,J＝7.35Hz,2H),2.10-2.43(m,12H),1.65-1.71(m, 2H),1.25-1.31(m,40H),0.91(t,J＝7.20Hz,6H).MS-ESI(m/z):792.5(M+H)⁺.

Example 6 preparation of 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-hexadecyloxypropyl ether

Tetradecyl bromide (21.7mmol) and 1, 3-propanediol (4.9g, 65.1mmol) were dissolved in DMSO (50mL) and KOH (4.8g,86.8mmol) powder was added with vigorous stirring. Stir at rt for 4h and TLC monitored for reaction completion. And (3) post-treatment: the reaction solution was diluted with 100mL of water, acidified with concentrated hydrochloric acid, and extracted with ethyl acetate. The organic phase was concentrated and recrystallized from petroleum ether to yield 3g of a white solid (yield 53%). The above white solid was dissolved in dichloromethane (30mL), triethylamine (5mL) and methanesulfonyl chloride (2.1g, 13.8mmol) were added in this order, and the mixture was stirred at room temperature for 2 hours. And (3) post-treatment: the reaction solution was washed with water, and the organic phase was dried over anhydrous sodium sulfate and concentrated to be directly subjected to the next reaction.

Compound IV (2.0g, 5.7mmol) and the compound obtained in the above step were dissolved in acetonitrile (30mL) and sodium methoxide (0.6g,11.6 mmol) was added, and the mixture was stirred at room temperature for 5 hours. And (4) carrying out post-treatment, pouring the compound into water, and precipitating a solid crude product. The crude product was Boc-removed with trifluoroacetic acid to yield 2.3g of the title compound (53% yield).

¹H NMR(500MHz,CDCl₃)7.32(d,J＝6.00Hz,1H),6.42(d,J＝8.15Hz,1H),5.71(d,J＝7.30Hz, 1H),5.21(brs,1H),4.31(brs,2H),4.15(brs,1H),3.57(t,J＝7.15Hz,8H),3.32(t,J＝6.15Hz,4H), 1.62-1.72(m,4H),1.21-1.28(m,56H),0.89(t,J＝6.50Hz,6H).MS-ESI(m/z):828.4(M+H)⁺.

Example 7 preparation of 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-octadecyl oxyethyl ether

Preparation the title compound was obtained as described in example 6, starting from n-hexadecyl bromide and ethylene glycol.

¹H NMR(500MHz,CDCl₃)7.30(brs,1H),6.42(brs,1H),5.73(d,J＝7.30Hz,1H),5.21(brs,1H), 4.10-4.24(m,3H),3.51-3.56(m,8H),3.31(t,J＝6.15Hz,4H),1.62-1.71(m,4H),1.25-1.28(m,60H),0.87 (t,J＝6.50Hz,6H).MS-ESI(m/z):856.5(M+H)⁺.

Example 8, 2 '-difluoro-2' -deoxycytidine-3 '-laurate-5' -myristate

The preparation method is carried out according to the following reaction route:

compound IV (1.8g, 4.9mmol) was dissolved in DMF (20mL) and N, N-lutidine (0.7g, 5.9 mmol), tert-butyldiphenylchlorosilane (1.4g,5.0mmol) were added sequentially. Stir at rt overnight and TLC monitored for reaction completion. And (3) post-treatment: 2.1g of solid VII (yield 69%) are precipitated by addition to ice-water, dried in vacuo (to give the compound of formula VII) and carried directly to the next step.

¹H NMR(300MHz,CD₃OD)8.27(d,J＝7.50Hz,1H),7.31(d,J＝7.5Hz,1H),7.11-7.25(m,10H), 6.21(brs,1H),4.22-4.36(m,3H),3.80-3.84(brs,1H),1.54(s,9H),1.09(s,9H).

Referring to the procedure of example 1, reaction of Compound VII (2.1g, 3.49mmol) with lauroyl chloride (0.8g, 3.50mmol) gave 2.1g of VIII-1 as a white solid (yield 77%). Compound VIII-1(2.1g, 2.68mmol) was dissolved in THF (20mL) and tetrabutylammonium fluoride (3mL, 1M in THF) was added. Stir at rt for 2h and TLC monitored for reaction completion. And (3) post-treatment: the reaction mixture was added to water, extracted with dichloromethane (to give compound IX, 3' -laurate) and directly subjected to the next step.

The title compound was obtained by reacting the above compound (IX) with myristoyl chloride and Boc removal with trifluoroacetic acid in the same manner as in example 1.

¹H NMR(500MHz,CDCl₃)7.54(brs,1H),6.31(brs,1H),6.09(d,J＝8.00Hz,1H),5.29(brs,1H), 4.31-4.42(m,3H),2.31-2.40(m,4H),1.60-1.72(m,4H),1.21-1.26(m,36H),0.91-0.95(m,6H).MS-ESI (m/z):656.3(M+H)⁺.

Example 9, 2 '-difluoro-2' -deoxycytidine-3 '-myristate-5' -laurate

Referring to the procedure of example 8, compound VII was reacted with myristoyl chloride followed by TBAF deprotection of TBDPS followed by reaction with lauroyl chloride and Boc removal with trifluoroacetic acid to afford the title compound.

¹H NMR(500MHz,CDCl₃)7.51(brs,1H),6.30(d,J＝8.15Hz,1H),6.08(d,J＝8.10Hz,1H),5.28 (d,J＝11.00Hz,1H),4.31-4.41(m,3H),2.32-2.40(m,4H),1.60-1.72(m,4H),1.21-1.27(m,36H), 0.91-0.92(m,6H).MS-ESI(m/z):656.3(M+H)⁺.

Example 10, 2 '-difluoro-2' -deoxycytidine-3 '-n-tetradecyloxyethyl ether-5' -n-hexadecyloxyethyl ether

The title compound was prepared by reacting compound VII with tetradecyloxyethyl methanesulfonate, deprotecting TBDPS by TBAF, then hexadecyloxyethyl methanesulfonate and debiBoc with trifluoroacetic acid according to the procedures of reference examples 1, 6 and 8.

¹H NMR(500MHz,CDCl₃)7.31(brs,1H),6.42(d,J＝8.00Hz,1H),5.73(brs,1H),5.21(brs,1H), 4.10-4.23(m,3H),3.51-3.57(m,8H),3.32(t,J＝6.15Hz,2H),3.28(t,J＝6.00Hz,2H),1.65-1.71(m,4H), 1.23-1.28(m,48H),0.87-0.89(m,6H).MS-ESI(m/z):772.5(M+H)⁺.

Example 11, 2 '-difluoro-2' -deoxycytidine-3 '-n-hexadecyloxypropyl ether-5' -n-tetradecyloxyethyl ether

The title compound was prepared by reaction of compound VII with hexadecyloxypropyl methanesulfonate, deprotection of TBDPS by TBAF, followed by tetradecyloxyethyl methanesulfonate reaction and Boc removal by trifluoroacetic acid, according to the procedures of reference examples 1, 6 and 8.

¹H NMR(500MHz,CDCl₃)7.31(brs,1H),6.41(brs,1H),5.72(d,J＝8.15Hz,1H),5.21(brs,1H), 4.10-4.20(m,3H),3.51-3.55(m,8H),3.28-3.32(m,4H),1.64-1.71(m,4H),1.23-1.28(m,50H),0.87-0.89 (m,6H).MS-ESI(m/z):786.5(M+H)⁺.

Example 12, 2 '-difluoro-2' -deoxycytidine-3 '-n-hexadecyloxypropylether-5' -myristate

The title compound was prepared by reacting compound VII with hexadecyloxypropyl methanesulfonate, TBDPS deprotection by TBAF, followed by myristoyl chloride reaction and Boc removal by trifluoroacetic acid, according to the procedures of reference examples 1, 6 and 8.

¹H NMR(500MHz,CDCl₃)7.31(brs,1H),6.31(d,J＝6.20Hz,1H),5.70(d,J＝7.20Hz,1H),5.21 (brs,1H),4.11(brs,2H),4.00-4.03(m,1H),3.97(t,J＝7.20Hz,4H),3.89-3.92(m,2H),3.28-3.32(m,2H), 1.64-1.71(m,4H),1.23-1.28(m,48H),0.87-0.90(m,6H).MS-ESI(m/z):756.5(M+H)⁺.

Example 13 Gemcitabine monoester Compound

Compound 13: 5' -Gemcitabine laurate according to the methods described in [0046] to [0048] of the specification of CN 102675390A.

Compound 14: 5' -Gemcitabine elaidic acid ester is prepared by the method described in example 1 of WO 98/32762.

Compound 15: 5' -Gemcitabine n-octanoate was prepared by the methods described in [0037] to [0039] of CN 102675390A.

Compound 16: 3' -Gemcitabine elaidic acid ester is prepared by the method described in example 1 of WO 98/32762.

Example 21 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (DMPC): 250 parts by weight of a non-woven fabric,

pegylated phospholipid (DSPE-PEG)₂₀₀₀): 50 parts by weight of a water-soluble polymer,

cholesterol: 25 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 100: 500).

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 220mbar) 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 65 deg.C for 2 hr, performing ultrasonic treatment for 30min, 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 (8% concentration) dissolved in water in advance, hydrating at 65 deg.C for 2 hr, performing ultrasonic treatment for 30min, 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 21, gemcitabine and 16 compounds obtained in examples 1 to 13 of the present invention were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (17 liquid compositions) and freeze-dried lipid compositions in a solid state (17 solid compositions), respectively.

Example 22 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (DMPG): 50 parts by weight of a water-soluble polymer,

PEGylated Phospholipids (PEG 1000-DSPE): 100 parts by weight of a water-soluble polymer,

cholesterol: 50 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 1mg/ml, or lyophilized excipient: glycine to make solid composition, the weight ratio of active drug and lyophilized excipient is 100: 600).

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (chloroform, the amount of the chloroform is 2 times of the total dissolution);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (60 ℃, vacuum degree of 200mbar) 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 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 22, using gemcitabine, compound 2, compound 5, compound 13, and compound 14, respectively, as active drugs, lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions) were prepared, respectively.

Example 23 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipids (egg yolk lecithin): 800 parts by weight of a water-soluble polymer,

pegylated phospholipid (DSPE-PEG)₅₀₀₀): 10 parts by weight of a water-soluble polymer,

cholesterol: 10 parts by weight, and

excipient (aqueous solvent: water to make liquid composition, the adding 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 100: 1000).

(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 ℃, the vacuum degree is 250mbar) 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 2.5 hr, performing ultrasonic treatment for 45min, 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 (5% 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 23, using gemcitabine, compound 2, compound 5, compound 13, and compound 14, respectively, as active drugs, lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions) were prepared, respectively.

Example 24 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (soybean lecithin): 50 parts by weight of a water-soluble polymer,

pegylated phospholipid (DSPE-PEG)₄₀₀₀): 200 parts by weight of a solvent, and a solvent,

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 10mg/ml, or lyophilized excipient: lactose to make solid composition, the weight ratio of active drug and lyophilized excipient is 100: 250).

(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 (10% concentration) dissolved in water in advance, hydrating at 80 deg.C for 5 hr, performing ultrasonic treatment for 35min, filtering for sterilization (such as microporous membrane made of 220nm polyethersulfone), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.

In this example 24, using gemcitabine, compound 2, compound 5, compound 13, and compound 14, respectively, as active drugs, lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions) were prepared, respectively.

Example 25 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (dipalmitoylphosphatidylcholine): 1000 parts by weight of a water-soluble polymer,

pegylated phospholipid (DSPE-PEG)₃₃₅₀): 5 parts by weight of a reaction product of (B),

cholesterol: 5 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 5mg/ml, or lyophilized excipient: mannitol to make solid composition, the weight ratio of active drug and lyophilized excipient is 100: 750).

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 2.5 times of the total dissolution degree);

(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 container, hydrating at 80 deg.C for 2 hr, performing ultrasonic treatment for 40min, 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 (7.5% concentration) 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 25, gemcitabine, compound 2, compound 5, compound 13, and compound 14 were used as active drugs, respectively, to prepare lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions), and freeze-dried lipid compositions in a solid state (5 solid compositions).

Example 31 characterization of lipid compositions

The lipid compositions in all liquid states and the lipid compositions in all solid states obtained in examples 21 to 25 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 diameters of the fine particles in the drug solution were measured using a malvern Zetasizer Nano ZS Nano-particle size potentiometer, the average particle diameter was calculated, and the percentage of fine particles having a particle diameter of less than 10nm and the percentage of fine particles having a particle diameter of more than 300nm were counted.

As a result:

all the liquid lipid compositions obtained in examples 21 to 25 had 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 21 for compound 1 had 1.2% of particles having a particle size of less than 10nm and 0.8% of particles having a particle size of more than 300 nm;

all of the solid lipid compositions obtained in examples 21 to 25 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 21 had 1.2% of fine particles having a particle size of less than 10nm and 0.8% of fine particles having a particle size of more than 300nm with respect to Compound 1;

the average particle size of all the liquid lipid compositions obtained in examples 21 to 25 is in the range of 85 to 130nm, for example, the average particle size of the particles in the liquid lipid composition obtained for compound 1 in example 21 is 97nm, and the particle size distribution diagram and the transmission electron micrograph of the sample are shown in FIG. 1 and FIG. 2, respectively;

the average particle size of the total solid lipid composition obtained in examples 21 to 25 is in the range of 85 to 135nm, for example, the average particle size of the particles in the liquid lipid composition obtained for compound 1 in example 21 is 99 nm; there was no significant difference in particle size between the various liquid compositions obtained in examples 21-25 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 21 to 25 were left to stand 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 32 to 37% 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 21 for compound 1 after 18 months was 131nm, which was increased by 35.1%. In addition, when all the solid lipid compositions obtained in examples 21 to 25 were left to stand at room temperature for 18 months, and then their particle sizes were measured, the average particle size of each sample was increased by 29 to 34% 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 21 for compound 1 was increased by 31.3% after 18 months.

Supplementary test (which may be referred to as example 26 in this application): with reference to the preparation methods of examples 21 to 25 herein, respectively, except that magnesium chloride (in an amount of 2.5% by weight of the active drug) was also simultaneously added while adding the aqueous vehicle or excipient solution to the vessel in step (23a) or (23b), thereby obtaining lipid compositions in the form of lipid suspensions in the liquid state or lipid compositions in the solid state, and the average particle diameters, the percentages of particles smaller than 10nm, and the percentages of particles larger than 300nm of these lipid compositions in the form of lipid suspensions in the liquid state at 0 month and 18 months were determined as described above; as a result, the average particle size of all samples at 0 month and 18 month was less than 5% for particles having a particle size of less than 10nm, less than 5% for particles having a particle size of more than 300nm, and in the range of 90 to 125nm (for example, the average particle size of particles in the liquid lipid composition obtained by adding magnesium chloride to compound 1 in example 21 was 95 nm); the average particle size of all samples increased by-1.7% to 2.3% relative to their 0 month particle size at 18 months (showing no significant change, e.g., the average particle size of the 18 month particles of the liquid lipid composition of example 21 for compound 1 with the addition of magnesium chloride was 96nm, an increase of 1.05%). It has been surprisingly found that the results of this example 26 demonstrate that the addition of small amounts of magnesium chloride to a lipid composition helps to improve the particle size stability of the microparticles in the composition. According to the above results, although the lipid compositions of examples 21 to 25, to which no magnesium chloride was added, had an average particle size increased by 29 to 37% after 18 months of storage at room temperature, the average particle size after such an increase still satisfied the general requirements of pharmaceutical products, and therefore the lipid compositions of examples 21 to 25 and 26 were all satisfactory from the clinical point of view, but it was still expected that better quality pharmaceutical products could be clinically applied. Thus, according to any embodiment of any aspect of the invention, the lipid composition further comprises magnesium chloride; for example in an amount of 1 to 5%, for example 2.5% by weight of the compound of formula (I) or a pharmaceutically acceptable salt thereof; for example the magnesium chloride is added with the excipient. In addition, the solid and liquid lipid compositions of examples 21 to 25 and 26 were left to stand at 2 to 8 ℃ for 24 months, and the average particle size of all samples increased by-2.9 to 5.2% from the particle size at 0 month in 24 months, showing no significant change, for example, the average particle size of 24 month particles of the liquid lipid composition obtained by adding magnesium chloride to compound 1 in example 21 was increased by 1.26%, indicating that the particle size did not change significantly after standing for a long time under the condition commonly used for liposome storage at 2 to 8 ℃ regardless of the addition or non-addition of magnesium chloride.

Example 32 antitumor Activity of lipid composition

1. Cells for assay

Human pancreatic cancer (AsPC-1) cells, human pancreatic ductal carcinoma (su.86.86).

2. Test article

Gemcitabine, 12 gemcitabine esters prepared in examples 1 to 12, 6 gemcitabine esters prepared in example 13, and liquid (or solid) lipid compositions prepared in examples 21 to 25.

3. Experimental methods

Culturing AsPC-1, SU.86.86, etc. tumor cells in vitro at 37 deg.C and 5% CO₂Cultured in a cell culture box under the condition to logarithmic phase. The cells were seeded in a 96-well plate at a density of 5000 cells/well and 100. mu.L/well, and then cultured in a cell incubator for 24 hours.

The toxicity of each test substance on the above tumor cells was determined by MTT method, which comprises setting gemcitabine ester compound and gemcitabine as positive control. Diluting the test object by using a cell culture solution in a multiple ratio to obtain a cell culture medium with gemcitabine concentration of 1-100 mu M. After 24h of cell plating, the original medium in the corresponding wells was replaced with 200. mu.L of each drug-containing medium, 4 duplicate wells were set for each concentration, and blank control wells and zero-adjusted wells were set. After further incubation for 24, 48 and 72 hours, 20. mu.L of MTT solution with the concentration of 5mg/mL is added, culture is continued for 4 hours under the same condition, the culture solution is discarded, 150. mu.L of DMSO is added to each well to dissolve formazan, a plate shaking instrument is used for shaking for 10min, the absorbance value at the wavelength of 490nm is measured in an enzyme labeling instrument, and the cell growth inhibition percentage% and IC are calculated₅₀Value (. mu.M). IC of partial compound₅₀The (. mu.M) values are as follows:

IC for AsPC-1, gemcitabine₅₀(. mu.M) is 1198.0, IC of Compound 1₅₀(. mu.M) is 1640.4, IC of Compound 2₅₀(μ M) 1025.5, IC of Compounds 3 to 8₅₀(mu M) in the range of 922.1-1424.2, IC of compounds 9-12₅₀(μ M) in the range of 932.5-1374.8, IC of Compound 13₅₀(μ M) 958.5, IC of Compounds 14 to 16₅₀(μ M) is in the range of 1042.7-1643.3;

IC for SU.86.86, gemcitabine₅₀(. mu.M) 124.1, IC of Compound 1₅₀(. mu.M) 213.2, IC of Compound 2₅₀(μ M) 173.5, IC of Compounds 3 to 6₅₀(mu.M) in the range of 117.4 to 262.4, IC of compounds 7 to 12₅₀(μ M) in the range of 163.3 to 226.3, IC of Compound 13₅₀(μ M) 184.7, IC of Compounds 14 to 16₅₀(mu M) is in the range of 163.2 to 217.5.

As can be seen from the above, the half inhibitory concentration of the esters of the compounds 1-16 to AsPC-1 is basically equivalent to that of gemcitabine and is basically in the same order of magnitude; the half inhibition concentration of the esters of the compounds 1-16 to SU.86.86 is basically equivalent to that of gemcitabine and is basically in the same order of magnitude; the half inhibitory concentration of the esters of the compounds 1-12 and the half inhibitory concentration of the esters of the compounds 13-16 on the same tumor cell are basically equivalent and are basically in the same order of magnitude.

In the test of a tumor cell, the percentage obtained by dividing the IC50 (. mu.M) value of a liquid lipid composition obtained in examples 21 to 25 by the IC50 (. mu.M) value of the chemical substance and multiplying the result by 100% was defined as the relative inhibition percentage (%) of the liquid lipid composition, the smaller the relative inhibition percentage (%) indicates the smaller the half inhibitory concentration, indicating that the liposome has a stronger tumor cell inhibitory activity against the original compound, for example, the percentage of the compound 1 liquid lipid composition prepared in example 21 to AspC-1 divided by the IC50 value of compound 1 to the tumor cells multiplied by 100%, which is the relative percent inhibition (%) of the compound 1 liquid lipid composition obtained in example 21, the relative inhibition percentage (%) was calculated for the solid lipid composition obtained by freeze-drying in the same manner. The results show that it is possible to display,

in the case of the AspC-1 cells,

(i) the relative inhibition percentages of the product of example 21, gemcitabine liquid and solid lipid compositions were 87.7% and 89.3%, respectively; the relative inhibition percentages of the liquid and solid lipid compositions of compounds 1-12 are both in the range of 0.13-0.28% and there is no significant difference between solid and liquid forms of the same chemical, e.g., the relative inhibition percentages of the liquid and solid lipid compositions of compound 1 are 0.173% and 0.164%, respectively; the relative inhibition percentages of compounds 13-16 for both liquid and solid lipid compositions ranged from 43.2-56.4% and there was no significant difference between solid and liquid forms of the same chemical, e.g., 49.72% and 51.24% for compound 13 liquid and solid lipid compositions, respectively;

(ii) the products of examples 22-25, gemcitabine liquid and solid lipid compositions both had relative percent inhibition in the range of 86.4-90.1%; the relative inhibition percentages of compounds 2,5, liquid and solid lipid compositions were all in the range of 0.15-0.26% and there was no significant difference between solid and liquid forms of the same chemical, e.g. compound 2, liquid and solid lipid compositions in example 22 had relative inhibition percentages of 0.203% and 0.196%, respectively; the relative inhibition percentages for compounds 13, 14, both liquid and solid lipid compositions were in the range of 46.5-54.7% and there was no significant difference between solid and liquid forms of the same chemical, e.g. 48.67% and 47.73% for compound 13, liquid and solid lipid compositions, respectively, in example 22.

For the cells of SU.86.86,

(a) the relative inhibition percentages of the product of example 21, gemcitabine liquid and solid lipid compositions were 93.2% and 91.7%, respectively; the relative inhibition percentages of the liquid and solid lipid compositions of compounds 1-12 are both in the range of 1.53-1.83% and there is no significant difference between solid and liquid forms of the same chemical, e.g., the relative inhibition percentages of the liquid and solid lipid compositions of compound 1 are 1.78% and 1.62%, respectively; the relative inhibition percentages of compounds 13-16 for both liquid and solid lipid compositions ranged from 64.6-71.3% and there was no significant difference between solid and liquid forms of the same chemical, e.g., the relative inhibition percentages for compound 13 liquid and solid lipid compositions were 68.43% and 67.95%, respectively;

(b) the products of examples 22-25, gemcitabine liquid and solid lipid compositions both had relative percent inhibition in the range of 89.6-92.4%; the relative inhibition percentages of compounds 2,5, liquid and solid lipid compositions were all in the range of 1.61-1.74% and there was no significant difference between solid and liquid forms of the same chemical, e.g. compound 2, liquid and solid lipid compositions in example 22 had relative inhibition percentages of 1.72% and 1.69%, respectively; the relative inhibition percentages for compounds 13, 14, both liquid and solid lipid compositions were in the range of 66.3-69.6% and there was no significant difference between solid and liquid forms of the same chemical, e.g. compound 13, liquid and solid lipid compositions in example 22, 67.93% and 67.14%, respectively.

The results show that the IC50 value of gemcitabine cannot be significantly reduced by adopting the lipid composition system, namely the effect of inhibiting tumor cells cannot be significantly improved, the IC50 value of gemcitabine monoesters such as compounds 13 to 16 cannot be significantly reduced by adopting the lipid composition system, namely the effect of inhibiting tumor cells cannot be significantly improved, and the IC50 value of gemcitabine diesters such as compounds 1 to 12 can be significantly reduced by adopting the lipid composition system, namely the effect of inhibiting tumor cells can be significantly improved. In other words, the lipid composition system of the present invention can significantly reduce the IC50 value of gemcitabine diester of the present invention, i.e. can significantly improve the effect of inhibiting tumor cells, but it was surprisingly found that such lipid composition system could not effectively improve the effect of gemcitabine and its monoester material on inhibiting tumor cells, indicating that the lipid composition system of the present invention can significantly improve the antitumor activity of gemcitabine diester compound of the present invention. The lipid composition system can obviously improve the effect of the diester compound on inhibiting the tumor cells, but cannot effectively improve the gemcitabine and the monoester thereof, which cannot be predicted at all in the prior art.

Example 33: pharmacodynamic test for inhibiting mouse transplantation tumor

1. Test materials

Medicine preparation: gemcitabine, compound 2, compound 5, compound 13, compound 14, and the liquid lipid composition prepared from gemcitabine, compound 2, compound 5, compound 13, compound 14 obtained in example 21 were administered in a dose equivalent to 10mg gemcitabine/kg body weight/dose. Clean grade C57BL/6N mice. Tumor species: mouse transplantation tumor Lewis lung cancer, S180 sarcoma and H22 liver cancer.

2. Test method

(1) Mouse Lewis lung carcinoma:

male C57BL/6N mice weighing 18-22 g were divided randomly into 11 groups of 10 mice each, and each group was assigned to a saline control group, five groups of the above five compounds, and five groups of the above five liquid lipid compositions.

Neck dislocation and killing Lewis lung cancer, inoculating C57BL/6N tumor-bearing mouse with 12d growth under the skin, aseptically taking fresh tumor tissue, preparing cell homogenate by tissue grinder, and usingAdjusting the content of living cells to 2-3 × 10 with physiological saline⁷and/mL, subcutaneously inoculated in the right axilla of C57BL/6N mice, 0.1mL each.

Dosage and method of administration: normal saline, and intragastric administration, 0.1mL/10g body weight/day daily, 1 time daily, 11 times total; the various reagents are injected intraperitoneally 1 time and 4 times a day.

The administration was started on day 2 of the inoculation, 24 hours after the last administration, the animals were sacrificed, the body weight and the tumor mass weight were weighed, and the tumor weight inhibition ratio was calculated according to the following formula:

the tumor weight inhibition ratio (%) was (1-tumor weight of test group/tumor weight of saline control group) × 100%.

The three tests were combined and statistically analyzed using SPSS10.0 software, and the experimental data for body weight and tumor weight were expressed as x + -s, and the differences between each administration group and the saline control group were compared by one-way analysis of variance.

(2) Mouse S180 sarcoma

Aseptically taking S180 sarcoma and inoculating abdominal cavity with ascites of mice growing for 8 days, and diluting tumor cell content to 3-4 × 10 with normal saline⁷and/mL, inoculated subcutaneously in the right axilla of experimental mice, 0.1mL each. The administration period 11d, the grouping and administration condition and the statistical method are the same as those of the Lewis lung cancer test of mice.

(3) Mouse H22 liver cancer

Taking H22 liver cancer, inoculating abdominal cavity with mouse ascites of 9 days of growth, and diluting with normal saline until the content of tumor cells is 5-6 × 10⁷NIH mice were inoculated per mL. The administration period 11d, the grouping and administration condition and the statistical method are the same as those of the Lewis lung cancer test of mice.

3. Inhibition of mouse graft tumors

The tumor weight inhibition rate of the five compounds of gemcitabine, compound 2, compound 5, compound 13 and compound 14 on Lewis lung cancer is in the range of 63-68% (for example, gemcitabine is 65.3%), the tumor weight inhibition rate of the liquid lipid composition of gemcitabine, compound 13 and compound 14 on Lewis lung cancer is in the range of 67-69% (for example, gemcitabine lipid composition is 68.7%), and the tumor weight inhibition rate of the liquid lipid composition of compound 2 and compound 5 on Lewis lung cancer is in the range of 96-99% (for example, compound 2 lipid composition is 98.3%); the tumor weight inhibition rate of the S180 sarcoma of the five compounds of gemcitabine, compound 2, compound 5, compound 13 and compound 14 is in the range of 53-57% (for example, gemcitabine is 54.7%), the tumor weight inhibition rate of the S180 sarcoma of the liquid lipid composition of gemcitabine, compound 13 and compound 14 is in the range of 51-58% (for example, gemcitabine lipid composition is 53.4%), and the tumor weight inhibition rate of the S180 sarcoma of the liquid lipid composition of compound 2 and compound 5 is in the range of 93-97% (for example, compound 2 lipid composition is 95.8%); the tumor weight inhibition rate of the H22 liver cancer by the five compounds of gemcitabine, compound 2, compound 5, compound 13 and compound 14 is in the range of 57-61% (for example, 59.2% of gemcitabine), the tumor weight inhibition rate of the H22 liver cancer by the liquid lipid composition of gemcitabine, compound 13 and compound 14 is in the range of 60-64% (for example, 63.2% of gemcitabine lipid composition), and the tumor weight inhibition rate of the H22 liver cancer by the liquid lipid composition of compound 2 and compound 5 is in the range of 97-99% (for example, 98.6% of compound 2 lipid composition).

It was surprisingly found that the liquid lipid compositions of both compound 2 and compound 5 have significantly better tumor-inhibiting effects against a variety of tumors than the other substances, and this superior tumor-inhibiting effect is probably due to the specific chemical substances and specific composition combinations of the present invention.

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

1. A compound of the following formula (I):

or a pharmaceutically acceptable salt thereof; wherein:

2. A compound according to claim 1, wherein R₁And R₂Identical or different, each represents C_8-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

3. A compound according to claim 1, wherein R₁And R₂Identical or different, each represents C_10-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

4. A compound according to claim 1, wherein the pharmaceutically acceptable salt is a salt of the compound of formula (I) with an inorganic acid or with an organic acid.

5. The compound according to claim 4, said mineral acid being selected from the group consisting of: hydrochloric acid, sulfuric acid, phosphoric acid.

6. The compound according to claim 4, said organic acid being selected from the group consisting of: 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. The compound according to claim 1, which is compound 1 to compound 12 selected from the group consisting of:

compound 1: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-n-decanoate,

compound 2: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dilaurate,

compound 3: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dimyristate,

compound 4: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -dipalmitate,

compound 5: 2',2' -difluoro-2 ' -deoxycytidine-3 ', 5' -di-trans-oleate,

compound 8: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -laurate-5 ' -myristate,

compound 9: 2',2' -difluoro-2 ' -deoxycytidine-3 ' -myristate-5 ' -laurate,

or a pharmaceutically acceptable salt thereof.

8. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 7,

r in the compound of formula (I)₁And R₂In the same way, as shown in scheme 1,

scheme 1:

the preparation method comprises the following steps:

1) dissolving compound II 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, N-lutidine, pyridine, 4-dimethylaminopyridine, and 1-6 times equivalent of Boc₂Stirring and reacting O at the temperature of between room temperature and 50 ℃ for 3 to 15 hours to obtain a compound of formula (III) protected by amino;

2) dissolving a compound of formula (III) in a protic solvent selected from: adding 1-2 times of equivalent of inorganic base selected from the following inorganic base into water, methanol or a methanol-water mixed solvent: stirring and reacting sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate for 3-10 hours at 0-room temperature to obtain a compound of formula (IV) with a hydroxyl protecting group removed;

3) dissolving a compound of formula (IV) in a non-polar solvent selected from: dichloromethane (DCM), chloroform, tetrahydrofuran, dioxane, in the presence of an organic base selected from: triethylamine, N-dimethylpyridine, pyridine and 4-dimethylaminopyridine react with 1-4 times of equivalent of the compound of the formula (V) at 0-room temperature for 4-15 hours by stirring to obtain a compound of the formula (VI);

4) dissolving a compound of formula (VI) in a non-polar solvent selected from: dichloromethane (DCM), trichloromethane, tetrahydrofuran and dioxane, and adding 1-2 times of equivalent of acid selected from the following: trifluoroacetic acid and hydrochloric acid, stirring and reacting for 0.5-5 hours at 0-room temperature to obtain the compound of formula (I'), wherein R is₁And R₂The same compound of formula (I).

9. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 7,

r in the compound of formula (I)₁And R₂In a different situation, as shown in scheme 2,

scheme 2:

the preparation method comprises the following steps:

1) dissolving a compound of formula (IV) in a dipolar solvent selected from: dimethylformamide, dimethylsulfoxide, in the presence of an organic base selected from: triethylamine, N-dimethylpyridine, pyridine, 4-dimethylaminopyridine and 1-2 times of equivalent weight of tert-butyldiphenylchlorosilane are stirred and react for 8-15 hours at the temperature of 0-room temperature to obtain a compound of a formula (VII) with sugar ring 5' -hydroxyl being selectively protected;

2) reacting a compound of formula (VII) with R₁Carrying out esterification reaction on the COCl compound to obtain a compound shown in a formula (VIII);

3) dissolving a compound of formula (VIII) in a non-polar solvent selected from the group consisting of: adding 1-2 times of equivalent weight of tetrabutylammonium fluoride into dichloromethane, trichloromethane, tetrahydrofuran and dioxane, and stirring at 0-room temperature for reacting for 4-15 hours to obtain a compound of formula (IX) with a hydroxyl protecting group removed;

4) reacting a compound of formula (IX) with R₂Carrying out esterification reaction on the COCl compound to obtain a compound shown in a formula (X);

10. A method for increasing the anti-tumor activity of a compound of formula (J) or a pharmaceutically acceptable salt thereof for non-diagnostic, therapeutic purposes,

the method comprises the following steps:

(1) preparing a compound of formula (J) or a pharmaceutically acceptable salt thereof, as a compound of formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 7;

(2) preparing a lipid composition comprising a phospholipid from a compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein the content of the first and second substances,

the lipid composition comprises:

a compound of formula (I): 100 parts by weight of a water-soluble polymer,

phospholipid: 50 to 1000 parts by weight of a water-soluble polymer,

pegylated phospholipids: 5 to 200 parts by weight of a water-soluble polymer,

cholesterol: 5 to 100 parts by weight, and

an excipient; the lipid composition is a composition in a 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 that the concentration of the compound shown in the formula (I) or the medicinal salt thereof in the liquid composition is 0.2-20 mg/ml; or

The lipid composition is a freeze-dried composition in the solid state, wherein the excipient is a freeze-dried excipient selected from the group consisting of: mannitol, sorbitol, lactose, glycine, dextran, sucrose, glucose, the weight ratio of the compound of formula (I) or its pharmaceutical salt to the lyophilized excipient is 100: 100 to 2000.

11. The method according to claim 10, wherein said phospholipid is 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.

12. The method according to claim 10, wherein the pegylated phospholipid is a modified phospholipid having a molecular weight of 1000 to 10000 dalton.

13. The method according to claim 12, wherein the pegylated phospholipid is pegylated distearoylphosphatidylethanolamine.

14. The method according to claim 12, 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.

15. The method according to claim 10, wherein said lipid composition is prepared by a process for preparing liposomes selected from the group consisting of: film dispersion method, extrusion preparation method, French pressure method, reverse phase evaporation method, and chemical gradient method.

16. The method according to claim 10, wherein the lipid composition is prepared by a membrane dispersion method comprising the steps of:

(21) dissolving phospholipid, pegylated phospholipid, cholesterol, and active drug in an organic solvent;

(23) preparation of lipid composition:

(23a) adding an aqueous solvent into a container, hydrating for 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 1-5 hours, carrying out ultrasonic treatment for 15-60 min, filtering, sterilizing, subpackaging into glass bottles, and freeze-drying in a freeze dryer to remove water to obtain the solid lipid composition.