CN104211742B

CN104211742B - Phosphoryl N-fatty acyl nucleoside analogues for the treatment of viral hepatitis and liver cancer

Info

Publication number: CN104211742B
Application number: CN201410169585.XA
Authority: CN
Inventors: 金义光; 杜丽娜; 汪珊
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2014-04-25
Filing date: 2014-04-25
Publication date: 2020-06-23
Anticipated expiration: 2034-04-25
Also published as: CN104211742A

Abstract

The invention discloses a phosphoryl N-fatty acyl nucleoside analogue for treating viral hepatitis and liver cancer, which is characterized in that the nucleoside analogue is modified by a cyclophosphamide group and then is connected with fatty chains with different carbon atoms. The phosphoryl N-fatty acyl nucleoside analogue can be conveniently prepared into a nano delivery system and has obvious liver cell targeting and tumor targeting. The nano delivery system comprises liposome, nonionic surfactant vesicles, nanoparticles, nanoemulsion and a self-assembly delivery system. The nucleoside drug is selected from lamivudine, vidarabine, cidofovir, gemcitabine, cytarabine, azacitidine, and fludarabine. After intravenous administration, the nanometer delivery system of the phosphoryl N-fatty acyl nucleoside analogue can exert the effect of treating viral hepatitis and liver cancer in a targeted way.

Description

Phosphoryl N-fatty acyl nucleoside analogues for the treatment of viral hepatitis and liver cancer

Technical Field

The invention relates to the field of chemistry and biomedicine, in particular to a phosphoryl N-fatty acyl nucleoside analogue, which can be used for preparing a nano delivery system, including a liposome, a non-ionic surfactant vesicle, a nanoparticle, a nanoemulsion and a self-assembly delivery system.

Background

Liver tissue cells can be divided into parenchymal liver cells and non-parenchymal cells. Nonparenchymal cells mainly include phagocytic cells such as kupffer cells, and belong to the mononuclear macrophage system (MPS). The malignant tumor of the liver comprises liver parenchymal cell cancer (HCC, liver cancer for short), cholangiocellular carcinoma, intrahepatic angiosarcoma, intrahepatic metastatic tumor and the like, wherein 90% of the malignant tumors of the liver are liver parenchymal cell cancer and are 3 rd common tumors except stomach cancer and esophageal cancer in the world. China is the country with the highest incidence rate of liver cancer, is more than 10 times higher than that of the western countries, and is second to lung cancer at the 2 nd position. The number of liver cancer diseases in China accounts for 55% of the whole world, and the number of deaths accounts for 45% of the number of liver cancer deaths in the world. Among the multiple risk factors that can lead to liver cancer, Hepatitis B Virus (HBV) infection, Hepatitis C Virus (HCV) infection, and the first 3 alcohol positions. HBV or HCV, as a hepatotropic virus, mainly infects liver parenchymal cells, which in turn trigger liver parenchymal cell tumors. Particularly, HBV infection is not effectively controlled, so that the incidence rate of liver cancer in China keeps continuously rising.

Nucleosides (Nucleosides) are important chemical components in organisms, from which nucleotides and nucleic acids are derived as carriers of genetic information. The most important genetic materials, DNA and RNA, are assembled from nucleotides. Nucleosides and nucleotides are also involved in many important reactions in organisms. Nucleosides in nature are formed by combining a purine or pyrimidine base with a five-carbon sugar. The bases comprise adenine, guanine, cytosine, uracil and thymine, the five-carbon sugar is ribose and deoxyribose, and adenosine, guanosine, cytidine and uridine, deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine are respectively formed by the five-carbon sugar.

Nucleoside analogues (Nu) are an important class of chemicals. Its structural features are similar to those of nucleosides. Many nucleoside analogs have been synthesized, and many of them have been found to have pharmacological activities such as antiviral action, cytotoxic action, immunosuppressive action, and the like. The current studies on the activity of nucleoside analogues focus mainly on antiviral and anticancer aspects. Antiviral nucleoside analogs undergo phosphorylation in vivo by kinases from viruses and human cells to form phosphorylated nucleoside analogs that inhibit the virus in a variety of ways. According to different action functional parts, the compounds can be divided into DNA polymerase inhibitors, DNA reductase inhibitors, thymidine kinase inhibitors, RNA enzyme inhibitors, reverse transcriptase inhibitors and the like, and are clinically used for treating infections of herpes viruses, cytomegaloviruses, varicella viruses, influenza viruses, hepatitis viruses, SARS viruses, AIDS viruses (HIV), encephalitis viruses and the like. Because viral replication is accomplished intracellularly, most antiviral drugs that inhibit the viral replication process require entry into the cell to exert their antiviral effect. Nucleoside analogs are generally more water soluble, making intracellular nucleosides less likely to exude, which is determined by the cell's natural ability to sustain life. Meanwhile, external nucleoside analogs are difficult to enter the cell, i.e. the permeability of the cell membrane of many nucleoside analogs is poor. Therefore, many nucleoside analog drugs have poor oral bioavailability and small intracellular concentration, which affect the antiviral effect, and the drug can cause the virus to generate drug resistance when the drug keeps low concentration in the cells for a long time. Anticancer nucleoside analogues are generally antimetabolites which exert a cytotoxic effect by inhibiting nucleic acid synthesis, as they also require intracellular entry to function. Therefore, the anticancer nucleoside analogue also has the problems of low bioavailability, difficult crossing of cell membranes and blood brain barriers and the like.

Many approaches have been devised to address the bioavailability and cellular uptake problems of nucleoside analogs, such as the synthesis of prodrugs and the design of specific drug delivery systems, of which lipid soluble prodrugs and liposomal delivery systems are attractive. Administration of lipid soluble prodrugs presents certain difficulties. Since fat-soluble drugs are insoluble or poorly soluble in water, general formulations such as tablets and injections cannot solve the problems of dispersion and dissolution thereof in an aqueous solution. Drugs generally need to be taken up and enter cells in the form of molecules or aggregates of molecules in a highly dispersed state. The human internal environment is aqueous, the biological membrane is liposoluble, and the drug or drug delivery system can reach the target site of action such as cells by passing through the biological membrane for many times in vivo. If insoluble lipid soluble drugs are not soluble or highly dispersed, the drugs are not delivered efficiently.

Liposomes (Liposomes) are vesicles composed of phospholipid bilayers, are highly dispersed in aqueous solutions, and can be used as carriers of various drugs. The high dispersibility of the compound makes the compound have the effects of targeting, slow release and the like in vivo, has lymphatic tropism when being orally taken, is easier to carry the medicine to pass through a blood brain barrier, and is easy to enter cells through ways of fusion, endocytosis and the like. The phospholipid bilayer membrane of the liposome vesicle separates an internal wrapped water phase from an external water phase, and the bilayer is hydrophobic. The drugs are respectively wrapped in the internal water phase or the membrane according to different physicochemical properties. Typically, the water-soluble drug is in the internal aqueous phase; the fat soluble drug is in the membrane layer. The preparation of liposome is the self-assembly process of phospholipid molecules in water, and the volume ratio of the internal and external water phases cannot be large. These factors determine that the encapsulation rate of most water-soluble drugs is low (< 50%), sometimes low (< 5%), and that the encapsulated drug has the potential to leak into the external aqueous phase. If proper fat-soluble groups (such as fatty chains) exist, fat-soluble drug molecules can be inserted into the phospholipid bilayer, the combination is firmer, and the drug molecules are not easy to drop off, so that the drug encapsulation rate is higher. Therefore, in order to increase the encapsulation rate of some water-soluble drugs in liposomes, a method of preparing lipid-soluble prodrugs with long fatty chains is often adopted. If the liposome is prepared on the basis of preparing the fat-soluble prodrug of the nucleoside analogue, the encapsulation rate can be increased, and the characteristics of high dispersity in liposome aqueous solution and easy penetration of the fat-soluble prodrug into cells are also achieved

Nonionic surfactant vesicles (Niosomes) refer to the self-assembly of certain nonionic surfactants (such as span 60) into vesicular structures in water under certain conditions, resembling liposomes. It can be used as drug carrier, and has some in vivo and in vitro characteristics similar to liposome.

Nanoparticles (nanoparticules) generally refer to nano-scale dispersed solid particles, and have the characteristics of improving the bioavailability of the medicament, enhancing the targeting property and the like when being used as a medicament carrier due to high dispersibility of the Nanoparticles. The Solid Lipid Nanoparticles (SLNs) are formed by adopting lipid materials compatible with human bodies as main auxiliary materials, have the characteristics of common nanoparticles and good biocompatibility, and are researched more in recent years.

The blood vessels in most tumors grow rapidly and irregularly, the internal lymphatic system is cleared slowly, and a tumor penetration enhancement factor exists, so that macromolecules and nanoparticles are easy to be retained in the tumors, and the tumor penetration enhancement and Retention effect is called as "enhanced penetration and Retention effect" (EPR). The nano drug delivery system can be passively targeted to the tumor due to EPR effect.

Hepdi prodrug is a new class of phosphate esters and phosphate ester prodrugs. It is a 1, 3-propyl ester containing a 4-aryl cyclic substituent. The prodrug is sensitive to oxidation-fragmentation reaction catalyzed by cytochrome P450(CYP450), can be specifically oxidized by CYP450 isoenzyme CYP3A4 enzyme to release active nucleotide or nucleoside phosphate, and plays a role in drug action. The CYP3a4 enzyme is mainly expressed in parenchymal liver cells, and other cells express less. Therefore, the Hepdirect prodrug is specifically degraded only in liver parenchymal cells to play a role, so that the damage of the prodrug to normal cells is reduced, and the toxic and side effects are reduced. Has significance in the targeted therapy of hepatitis and liver cancer.

Disclosure of Invention

The invention provides a phosphoryl N-fatty acyl nucleoside analogue which is characterized in that the nucleoside analogue is modified by a cyclophosphamide group and then is connected with fatty chains with different carbon atoms. The inventor unexpectedly finds that the phosphoryl N-fatty acyl nucleoside analogue can conveniently prepare a nano delivery system and has obvious liver cell targeting and tumor targeting. As described above, a drug having a highly dispersed state and a high lipid solubility is easily delivered in vivo, and the biological membrane permeability is good. The invention designs and prepares the phosphoryl N-fatty acyl nucleoside analogue with the functions for treating the viral hepatitis and the liver cancer for the first time, and prepares the nano delivery system thereof.

The phosphoryl N-fatty acyl nucleoside analogue is obtained by combining a cyclophosphylated group, a nucleoside analogue and a long fatty chain by adopting a proper reaction. The phosphoryl N-fatty acyl nucleoside analogue has the structure

Hep-Nu-L

Wherein Hep represents a cyclic phosphorylation group substituted by hydroxyl at 5' position of nucleoside sugar ring or short aliphatic chain hydroxyl, Nu is nucleoside analogue group, L represents aliphatic chain, and carbon number in L is 8-20.

In general, the cyclic phosphorylation group can be bonded to the nucleoside analog before bonding to the long aliphatic chain, although the order can be reversed. The carbon number of the long aliphatic chain is 8-20, and the prototype molecule of the long aliphatic chain is preferably selected from monocarboxylic fatty acid with the carbon number of 8-20, such as n-caprylic acid, n-capric acid, lauric acid (lauric acid), myristic acid (myristic acid), palmitic acid (palmitic acid), stearic acid (stearic acid) and arachidic acid (arachidic acid).

The nucleoside analogue prototype molecule for preparing phosphoryl N-fatty acyl nucleoside analogues in the present invention generally contains a reactive group, such as a free hydroxyl group or an amino group, in terms of molecular structural characteristics, preferably more than one hydroxyl group on the aliphatic chain (or ring) of the non-aromatic ring in the nucleoside analogue molecule, and more preferably more than one hydroxyl group on the aliphatic chain (or ring) of the non-aromatic ring. The nucleoside analogs of the present invention are not required to have a specific action, but are preferably drugs having pharmacological activity in terms of action, more preferably drugs having antiviral or anticancer action. The antiviral drug can be selected from lamivudine, vidarabine, and cidofovir, preferably lamivudine. The antineoplastic agent can be selected from gemcitabine, cytarabine, azacitidine and fludarabine, preferably gemcitabine.

The synthesis procedure for phosphoryl N-fatty acyl nucleoside analogues of the present invention is generally divided into two stages. Firstly, the nucleoside analogue is esterified with the cyclophosphylated group and then connected with the fatty chain by an amide bond to obtain a final product, or the two steps are inverted. In the above acylation reaction, an acid or an acid anhydride may be used for acylation with a hydroxyl group or an amino group; or acylation of acyl chloride with hydroxy or amino; or carboxylic acids are first formed into activated intermediates and then acylated with hydroxy or amino groups; or by acylation of carboxylic acids directly with hydroxy or amino groups. High purity phosphoryl N-fatty acyl nucleoside analogues can be obtained by reference to relevant literature methods and by use of general technical expertise.

The phosphoryl N-fatty acyl nucleoside analogue has fat long chain with strong fat solubility, and can be prepared into a nano delivery system selected from liposome, non-ionic surfactant vesicle, nanoparticle and nano emulsion. The particle diameter of these nanosupport systems is generally less than 1 micron, preferably less than 0.5 micron, more preferably less than 0.2 micron. The preparation method can refer to relevant literature methods and professional techniques.

In general, if the liposome is prepared by a membrane dispersion method, the phosphoryl N-fatty acyl nucleoside analogue and a membrane material such as phospholipid are dissolved in an organic solvent together, the mixture is put into a flask, and the mixture is subjected to reduced pressure rotary evaporation to obtain a layer of membrane, and then water or a proper buffer solution is added for oscillation and ultrasonic treatment until a uniform suspension is formed. If the ultrasound time is prolonged, it is also possible to obtain a nano-scale dispersion system. If the reverse phase evaporation method is adopted for preparing the liposome, the phosphoryl N-fatty acyl nucleoside analogue and membrane materials such as phospholipid and the like can be dissolved in an organic solvent together, water or buffer solution is added, high-speed stirring or ultrasound is carried out to prepare emulsion, then reduced pressure rotary evaporation is carried out to obtain gel-state substances, then water or proper buffer solution is added or not added, and the reduced pressure rotary evaporation is continued until uniform liposome suspension is formed. The liposome suspension can also be prepared into solid powder by selecting a proper formula and carrying out freeze drying or spray drying under proper conditions, so that the stability of the preparation can be ensured, and the liposome suspension can be obtained by adding an aqueous solution and shaking before use. Nonionic surfactant vesicles of phosphoryl N-fatty acyl nucleoside analogues can be obtained using the same technique.

The solid lipid nanoparticles in the nanoparticle preparation are more suitable for the phosphoryl N-fatty acyl nucleoside organisms in the invention. Generally, the phosphoryl N-fatty acyl nucleoside analogue and lipid which is solid at normal temperature, such as phospholipid, fatty acid and glyceride, are heated and melted together, then water or a proper buffer solution is added, and the mixture is circularly emulsified on a high-pressure emulsion homogenizer for a plurality of times under the heating condition to form nano-dispersed emulsion droplets, and the nano-dispersed emulsion droplets are rapidly cooled and solidified to obtain the phosphoryl N-fatty acyl nucleoside analogue solid lipid nanoparticles. The phosphoryl N-fatty acyl nucleoside analogue solid lipid nanoparticle can also be prepared by a microemulsion method. The phosphoryl N-fatty acyl nucleoside analogue nanoparticle suspension can also be frozen and dried or sprayed and dried under proper conditions by selecting a proper formula to form solid powder, so that the stability of the preparation can be ensured, and the aqueous solution is added before use and the suspension is shaken to obtain the nanoparticle suspension.

The preparation of the phosphoryl N-fatty acyl nucleoside analogue nanoemulsion can refer to a common prescription, and generally comprises an emulsifier, a co-emulsifier, a cosolvent, an oil phase and a water phase. Typically, the nanoemulsion can be readily formed upon selection of the appropriate formulation. If a proper formula is selected, the nano-emulsion can generally comprise an emulsifier, a co-emulsifier, a cosolvent and an oil phase, and can also form a self-nano-emulsion system, and the system can be self-dispersed into a nano-emulsion after a proper amount of aqueous solution is added.

In addition to the above-described conveniently available nanosupport system of phosphoryl N-fatty acyl nucleoside analogues, the present inventors have unexpectedly discovered that, because of the specific physicochemical properties, particularly the amphiphilicity, the phosphoryl N-fatty acyl nucleoside analogues of the present invention can self-assemble in aqueous solution, either by themselves or with the addition of suitable amounts of additives, to form nanosupport systems. The phosphoryl N-fatty acyl nucleoside analogue molecule contains a long fatty chain group with strong fat solubility and a nucleoside group with larger polarity, thus having amphipathy, and the physicochemical property is similar to that of phospholipid and certain surfactants. If the molecular structure of the amphiphilic molecule meets certain conditions, the amphiphilic molecule can self-assemble into nano ordered aggregates in water, such as bilayers, vesicles obtained by bending bilayers and long nanoparticles obtained by superposing bilayers. The phosphoryl N-fatty acyl nucleoside analogue has longer long fatty chain group with stronger fat solubility and hydrophilic nucleoside group, is easy to form a bilayer, and further obtains vesicles or long nanoparticles, but a certain amount of additives need to be added under certain conditions. Therefore, the invention designs and prepares the self-assembly delivery system consisting of the phosphoryl N-fatty acyl nucleoside analogue or adding a proper amount of additive for the first time.

The preparation method of the self-assembly delivery system consisting of the phosphoryl N-fatty acyl nucleoside analogue is similar to the preparation method of nano delivery systems such as liposome and the like. The phosphoryl N-fatty acyl nucleoside analogue is usually dissolved in an organic solvent, and if necessary, a suitable additive is added, followed by dispersion. The method comprises a film dispersion method, a reverse phase evaporation method, an injection method, a multiple emulsion method and the like. In some cases, additives are not necessary, and the delivery system is composed entirely of phosphoryl N-fatty acyl nucleoside analogs. In some cases, phosphoryl N-fatty acyl nucleoside analogues alone do not form good nanoparticles, and appropriate additives are added to help form ordered structures. Whether additives are required depends on the physicochemical properties of the phosphoryl N-fatty acyl nucleoside analogues and can generally be deduced by preliminary experiments.

The amount of the additive in the self-assembly delivery system is 0-50%, preferably 0-30%, more preferably 0-15% of the total composition in terms of molecular molar ratio, and the rest is the phosphoryl N-fatty acyl nucleoside analogue. The additive can be selected from phospholipid, polyalcohol ester surfactant, polyoxyethylene surfactant, dicetyl phosphatidic acid, cholesterol and its derivatives, and stearylamine. The phospholipid includes synthetic phospholipid, semisynthetic phospholipid, and natural phospholipid. Wherein the synthetic phospholipids include modified phospholipids such as polyethylene glycol-derivatized phospholipids and phospholipids linked to monoclonal antibodies. The polyol ester surfactant is preferably sorbitan fatty acid ester, such as span 60, span 40, and span 20. The polyoxyethylene surfactant is preferably polyoxyethylene polyoxypropylene block copolymer (also called poloxamer), polysorbate and polyoxyethylene fatty alcohol ether, such as poloxamer P188, tween 80, tween 60, tween 40, tween 20, and brij 35. The cholesterol and its derivatives include cholesterol, fatty acid ester of cholesterol, and cholesterol derivatized with polyethylene glycol.

The nano delivery system containing the phosphoryl N-fatty acyl nucleoside analogue comprises a self-assembly delivery system, if the composition or the surface of the nano delivery system is adsorbed with highly hydrophilic molecules or molecular fragments, because a hydrophilic protective layer can be formed in an in vitro environment or an in vivo environment, the nano delivery system can block the polymerization among particles or block the opsonization in vivo to obtain a long circulation effect. A commonly used hydrophilic molecule or molecular fragment is polyethylene glycol (PEG). In the invention, the nano delivery system of the phosphoryl N-fatty acyl nucleoside analogue with hydrophilic surface, which comprises the self-assembly delivery system, can be prepared by a method of adding phospholipid derived by polyethylene glycol or cholesterol derived by polyethylene glycol.

Drawings

FIG. 1.1- (3-phenyl) -1, 3-propanediol synthetic route

FIG. 2.1- (3-chlorophenyl) -1, 3-propanediol-N-dodecanoyllamivudine phosphate Synthesis route

FIG. 3 shows the pi-a isothermal curve of the monomolecular film of phosphoryl N-fatty acyl gemcitabine. PhosphorylN-octanoylgemcitabine (CPOG), phosphorylN-dodecanoyl gemcitabine (CPDG), phosphorylN-tetradecanoyl gemcitabine (CPTG), phosphorylN-hexadecanoyl gemcitabine (CPHG) and phosphorylN-octadecanoyl gemcitabine (CPODG).

FIG. 4 is a transmission electron micrograph of the self-assembled delivery system of phosphoryl N-fatty acyl gemcitabine. A: a phosphoryl N-octanoyl gemcitabine self-assembled delivery system; b: a phosphoryl N-dodecanoyl gemcitabine self-assembled delivery system; c: a phosphoryl N-tetradecanoyl gemcitabine self-assembled delivery system; d: a phosphoryl N-hexadecanoyl gemcitabine self-assembled delivery system; e: a phosphoryl N-octadecanoyl gemcitabine self-assembled delivery system.

FIG. 5 Effect of different concentrations of phosphoryl N-fatty acyl gemcitabine self-assembled delivery system on HepG2 cell survival. Gemcitabine (GEM), PhosphorylN-octanoylgemcitabine (CPOG), PhosphoNdodecanoylgemcitabine (CPDG), Phosphonntetradecanoylgemcitabine (CPTG), Phosphonnhexadecanoylgemcitabine (CPHG) and Phosphonnoctadecanoylgemcitabine (CPODG)

FIG. 6 shows the rate of change of body weight of tumor-bearing mice after intravenous injection of the phosphoryl N-dodecanoyl gemcitabine (CPDG) self-assembled delivery system

FIG. 7 tissue distribution of PhosphoNdodecaylgemcitabine self-assembled delivery System after intravenous injection in H22 tumor-bearing mice

Detailed Description

EXAMPLE 1 Synthesis of Phosphoryl N-fatty acyl Gemcitabine

The phosphoryl N-fatty acyl gemcitabine has the structural formula:

wherein R is a single-chain aliphatic chain, and R ═ C₇H₁₅-，C₉H₁₉-，C₁₁H₂₃-，C₁₃H₂₇-，C₁₅H₃₁-or C₁₇H₃₅First, 1- (3-chlorophenyl) -1, 3-propanediol was synthesized. Weighing magnesium powder (6.3g, 0.26mol), adding ethanol (27ml) and carbon tetrachloride (3ml), stirring under ice bath condition to obtain white powderA colored solid. 33ml of ethyl acetoacetate (0.26mol), 40ml of ethanol and a mixture of 30ml of diethyl ether were added dropwise. After 0.5h, M-chlorobenzoyl chloride (33ml, 0.26mol) was added dropwise, the mixture was oil-washed at 60 ℃ for 2h, after the reaction solution was cooled, it was poured into 150ml of cold water, and the pH was adjusted to 4 with 3M hydrochloric acid solution. Taking the organic phase, adding 5% (w/v) NaHCO₃Washing with water for three times, drying, and concentrating. To the concentrate was added dropwise an ethanol solution (150ml) of KOH (26.9g, 0.48mol), stirred at room temperature overnight, concentrated and dissolved in 390ml of water. Then adding NH₄Cl (29.7g, 0.556mol) and 60ml 30% ammonia were reacted in an oil bath at 45 ℃ for 2.5 h. Dichloromethane was extracted, dried, and concentrated to give ethyl 1- (3-chlorophenyl) -1-carbonyl propionate as an orange liquid. Purification on silica gel (cyclohexane: ethyl acetate 8: 1, v/v). The purified product (2.26g, 10mol) was dissolved in 32ml Tetrahydrofuran (THF) and sodium borohydride (1.5g, 4mol) was added slowly at 65 ℃ under reflux for 5 min. 32ml of methanol were then added dropwise at 65 ℃ under reflux for 4 h. After cooling, 3M diluted hydrochloric acid solution (30ml) was added, extracted with ethyl acetate, dried, concentrated and purified (petroleum ether: ethyl acetate 4: 1, v/v) to give 1- (3-chlorophenyl) -1, 3-propanediol as a pale yellow oil.

And the second step is to synthesize 1- (3-chlorphenyl) -1, 3-phosphoryl lactone p-nitrobenzene. P-nitrophenol (3.47g, 25mmol), POCl3(19.6ml, 125mmol) and 0.1g NaCl were weighed out, refluxed for 6h, and concentrated. The concentrated product (7.6g, 29.8mmol) was dissolved in 150ml THF, and a mixture of 1- (3-chlorophenyl) -1, 3-propanediol (3.0g, 16.10mmol), triethylamine (6.00ml, 59.6mmol) and THF (80ml) was added dropwise under ice-bath conditions. After 2 hours of reaction, 8.2g of p-nitrophenol (59mmol) and 8.4ml of triethylamine were added to the reaction mixture and the reaction was continued for 24 hours. Concentrated, extracted with water and ethyl acetate, and the organic layer was washed with 0.4M NaOH solution, water and saturated brine, respectively. Drying, concentrating and purifying (ethyl acetate: petroleum ether: 3: 7, v/v) to obtain a yellow white solid.

The phosphoryl N-fatty acyl gemcitabine is selected from the group consisting of phosphoryl N-octanoyl gemcitabine (CPOG), phosphoryl N-dodecanoyl gemcitabine (CPDG), phosphoryl N-tetradecanoyl gemcitabine (CPTG), phosphoryl N-hexadecanoyl gemcitabine (CPHG), and phosphoryl N-octadecanoyl gemcitabine (CPODG). The following synthesis of phosphoryl-tetradecanoyl gemcitabine (CPTG) is an example, and the synthesis steps for the remaining derivatives are similar.

Gemcitabine (1.04g, 4mmol) and a proper amount of N, N-Dimethylformamide (DMF) are weighed and dissolved, and then N is introduced₂t-BuMgCl (6ml, 6mmol) was added dropwise over 5min and reacted for 30 min. 1- (3-chlorophenyl) -1, 3-phosphoryl lactone p-nitrobenzene (1.48g, 4.4mmol) was added dropwise and reacted for 24 h. Concentration and purification (dichloromethane: methanol ═ 18: 1, v/v) afforded 1- (3-chlorophenyl) -1, 3-phospholactone gemcitabine (CPG) as a yellow solid. 0.456g (2mmol) of myristic acid and 8ml of anhydrous pyridine are taken, and a THF solution (8ml) of triphosgene (0.16g and 0.6mol) is dropwise added under the ice-bath condition, ice-bath reaction is carried out for 5min, and reaction is carried out for 2h at room temperature. 1- (3-chlorophenyl) -1, 3-phospholactone gemcitabine (0.96g, 2mmol) in DMF was added dropwise over 2 h. After concentration, the mixture was extracted with water and dichloromethane, and the organic phase was washed three times with 1M HCl solution and water, respectively. Drying, concentration and purification (dichloromethane: ethanol ═ 45: 1, v/v) gave a pale yellow fluffy solid.

The synthesis of phosphoryl N-fatty acyl arabinosyl adenosine, phosphoryl N-fatty acyl arabinosyl cytidine, phosphoryl N-fatty acyl azacitidine, phosphoryl N-fatty acyl fludarabine is similar to phosphoryl N-fatty acyl gemcitabine.

Example 2.1- (3-chlorophenyl) -1, 3-propanediol-N-dodecanoyllamivudine phosphate

The product is a product obtained by firstly reacting lamivudine with 1- (3-chlorophenyl) -1, 3-propanediol to generate phosphate and then amidating with dodecanoic acid, and has a molecular formula of C₂₈H₄₁ClN₃O₇PS, molecular weight 642.14. Firstly, 1- (3-chlorphenyl) -1, 3-propanediol is synthesized by the following specific steps. Magnesium powder (6.3g, 0.26mol) was added to a round bottom flask, ethanol (27ml) and carbon tetrachloride (3ml) were added, and mixed with stirring under ice bath conditions. A solution of ethyl acetoacetate (33ml, 0.26mol) in ethanol (40ml) and diethyl ether (30ml) was added dropwise thereto. Reacting for 0.5h, cooling the mixed solution to 0 ℃, then dropwise adding m-chlorobenzoyl chloride (33ml, 0.26mol), reacting for 2h at 60 ℃, and cooling. The reaction was poured into cold water (150ml) and acidified to pH4 with hydrochloric acid solution. The organic phase was separated with a separatory funnel, 5% sodium bicarbonate solution and water were each precipitated 5 times,drying, to which a solution of potassium hydroxide (26.9g, 0.48mol) in ethanol (150ml) is added dropwise with stirring at room temperature overnight, the solution is concentrated and dissolved in water (390ml), ammonium chloride (29.7g, 0.556mol) and 30% aqueous ammonia 60ml are added, mixed in a 45 ℃ oil bath and stirred for 2.5h, after cooling, it is extracted 3 times with dichloromethane (100ml × 3), the organic phases are combined and dried over sodium sulfate, the solvent is removed by rotary evaporation to give an orange liquid, the obtained product is chromatographed on silica gel with an eluent of cyclohexane to ethyl acetate 8: 1 to give about 5g of the product ethyl 1- (3-phenyl) 1-carbonylpropionate, yield is about 10%, the further purified ethyl 1- (trichlorophenyl) 1-carbonylpropionate (2.26g, 10mmol) is taken up and dissolved in 32ml of thf, sodium borohydride (1.5g, 4mmol) is added, stirred for 5min at 65 ℃ and stirred with constant pressure while refluxing, the reaction is carried out at 65 ℃ for 4h, the reaction is carried out on a reflux, the reaction solution is added to give an aqueous solution, after cooling, after extraction, the eluent is added to give a solution of ethyl 1- (3-phenyl) and concentrated, after addition of ethyl 1- (3-phenyl) is added to give an oil, after concentration, the eluent, the reaction solution, which is added to give a yellow oil, and stirred to give a solution, which is added, and stirred to give a.

Lamivudine (1.37g, 6mmol) was weighed out and dissolved in 30ml DMF and 9ml pyridine was added. DCC (3.7g, 18mmol) and 1- (3-chlorophenyl) -1, 3-propanediol (1.13g, 6mmol) in DMF were added and reacted in an oil bath at 100 ℃ for 15 h. Rotary evaporating at 70 deg.C to remove DMF and pyridine, adding 30ml methanol to extract product, suction filtering with Buchner funnel to remove DCU, rotary evaporating filtrate to remove solvent, and purifying with silica gel chromatography, wherein eluent is dichloromethane and methanol (18: 1). After the solvent was removed by rotary evaporation, an appropriate amount of a mixed solvent (dichloromethane/methanol-10/1) was added to dissolve the resulting solution, and the resulting solution was recrystallized in a refrigerator, followed by suction filtration to obtain a lamivudine phosphate prodrug. Thin layer chromatography showed one spot with a yield of about 60%.

Lauric acid (1.5g, 7.5mmol) is weighed into a flask, 2ml of thionyl chloride is added, the reflux reaction is carried out for 1h under 70 ℃ oil bath, and the thionyl chloride is removed by rotary evaporation at 25 ℃. The resulting acid chloride was slowly added dropwise to a stirred solution of 30ml lamivudine phosphate prodrug (1.15g, 2.5mmol) in pyridine in an ice bath. After the dropwise addition, the ice bath was removed 5min, and the reaction was carried out at room temperature for 2.5 h. Removing most of pyridine by rotary evaporation at 60 ℃, adding 20ml of water,a further 20ml of methylene chloride were added. The organic layer was washed three times with 1mol/L HCl solution and 0.1mol/L citric acid solution to remove the remaining pyridine. After removal of the solvent by rotary evaporation, purification by chromatography on silica gel eluting with dichloromethane/methanol 40: 1 gives about 0.6g of a pale yellow solid in 40% yield. The synthetic route is shown in figure 2. Thin layer chromatography revealed one spot.¹H NMR showed chemical shifts 0.88-2.34(23H, CH)₃(CH₂)₁₀)，4.50(1H，CHCH₂CH₂)，7.24、7.32、7.42、7.56(4H，C₆H₄)，8.0(1H，HNNC)。

The synthesis steps of 1- (3-chlorphenyl) -1, 3-propanediol-N-octa-acyl lamivudine phosphate, 1- (3-chlorphenyl) -1, 3-propanediol-N-deca-acyl lamivudine phosphate, 1- (3-chlorphenyl) -1, 3-propanediol-N-tetradecyl lamivudine phosphate, 1- (3-chlorphenyl) -1, 3-propanediol-N-hexadec-acyl lamivudine phosphate, 1- (3-chlorphenyl) -1, 3-propanediol-N-octadeca-acyl lamivudine phosphate are similar to those of 1- (3-chlorphenyl) -1, 3-propanediol-N-dodeca-acyl lamivudine phosphate, only the different fatty acids were replaced.

EXAMPLE 3 preparation of PhosphorylN-Dodecagemcitabine liposomes

Dissolving phosphoryl N-dodecanoyl gemcitabine (25mg) and soybean phospholipid (0.1g) in a 250ml flask by using 20ml of dichloromethane, carrying out reduced pressure rotary evaporation to obtain a layer of organic fat-soluble membrane, adding 10ml of phosphate buffer solution with the pH of 7.4, oscillating, enabling most of the membrane to fall off, carrying out ultrasonic treatment at 50 ℃ until uniform suspension is obtained, and observing under a microscope, wherein the particle diameter of most of the membrane is less than 1 micron, thus obtaining the phosphoryl N-dodecanoyl gemcitabine liposome.

Example 4 preparation of PhosphorylN-Hexadecyldigmcitabine nonionic surfactant vesicles

Dissolving phosphoryl N-hexadecanoyl gemcitabine (30mg) and span 60(0.08g) in a 250ml flask, dissolving the mixture with 20ml dichloromethane, carrying out reduced pressure rotary evaporation to obtain a layer of organic fat-soluble membrane, adding 10ml of phosphate buffer solution with pH7.4, oscillating, enabling most of the membrane to fall off, carrying out ultrasonic treatment at 50 ℃ until a uniform suspension of the phosphoryl N-hexadecanoyl gemcitabine nonionic surfactant vesicles is obtained, and detecting by a laser scattering particle size analyzer, wherein the average particle size is 205 nm.

EXAMPLE 5 preparation of 1- (3-chlorophenyl) -1, 3-propanediol-N-dodecanoyllamivudine phosphate ester Long-circulating liposomes

1- (3-chlorophenyl) -1, 3-propanediol-N-dodecanoyllamivudine phosphate (50mg), soybean phospholipids (0.1g), PEGylated distearoylphosphatidylethanolamine (PEG-DSPE) (0.02g) were taken in a flask, and diluted with 20ml of chloroform: dissolving isopropyl ether (1: 1, v/v), adding appropriate amount of distilled water, performing ultrasonic treatment to obtain emulsion, performing rotary evaporation under reduced pressure to obtain gel-state substance, adding small amount of water, performing rotary evaporation under reduced pressure to obtain uniform suspension, and observing under microscope to obtain 1- (3-chlorophenyl) -1, 3-propylene glycol-N-lauroyl lamivudine phosphate long-circulating liposome with the diameter of most particles smaller than 1 micrometer.

Example 6 preparation of Phosphoryl N-octadecanoyl gemcitabine solid lipid nanoparticle

Phosphoryl N-octadecanoyl gemcitabine (50mg), glyceryl monostearate (0.7g) and tween 80(0.03g) were heated to 80 ℃ in a beaker, and 80 ℃ water (10ml) containing sodium dodecyl sulfate (10mg) was gradually added thereto, and the temperature was kept constant, so that a transparent liquid was obtained. Then the mixture is injected into water with the temperature of 0 ℃ and stirred at a high speed by a syringe to form transparent liquid. Most of the particles are particles of 100nm or less when observed under an atomic force microscope. The phosphoryl N-octadecanoyl gemcitabine solid lipid nanoparticle suspension can be placed at normal temperature for 10 days without precipitation. The solid lipid nanoparticle suspension is added with a proper protective agent and then is frozen into solid powder, and water is added before the solid lipid nanoparticle suspension is used, so that the phosphoryl N-octadecanoyl gemcitabine solid lipid nanoparticle suspension is obtained.

Example 7 preparation of Phosphoryl N-fatty acyl Gemcitabine self-assembled delivery System

25mg of phosphoryl N-fatty acyl gemcitabine is dissolved in methanol to prepare a solution with the concentration of 5 mg/ml. The solution was slowly injected under the surface of pure water with a micro-syringe in a vortexed state, and repeated several times until no further injection (if any, significant precipitation occurred). The newly prepared self-assembled delivery system was placed in a flask and the organic solvent was removed under reduced pressure at 37 ℃ to obtain the phosphoramide N-fatty acyl gemcitabine self-assembled delivery system. The self-assembly delivery system has good dispersion effect and stable property, and can be placed for more than one month at room temperature. The self-assembled delivery system is further subjected to a water bath and reduced pressure to evaporate a portion of the water and concentrate the drug concentration to a certain value. Particularly, a phosphoryl N-capryloyl gemcitabine self-assembly delivery system, a phosphoryl N-lauroyl gemcitabine self-assembly delivery system, a phosphoryl N-myristoyl gemcitabine self-assembly delivery system, a phosphoryl N-palmitoyl gemcitabine self-assembly delivery system and a phosphoryl N-octadecanoyl gemcitabine self-assembly delivery system are prepared.

Example 8 preparation of PhosphorylN-Dodecagemcitabine Long-circulating self-assembled delivery System

20mg of cholesteryl-polyethylene glycol 1500(CHS-PEG1500) is weighed, tetrahydrofuran is dissolved and the volume is determined to 10ml, thus obtaining 2mg/ml solution. 68mg of phosphoryl N-dodecanoyl gemcitabine was weighed and added to the above solution to dissolve. The solution was slowly injected under the surface of pure water with a micro-syringe in a vortexed state, and repeated several times until no further injection (if any, significant precipitation occurred). And (3) placing the newly prepared self-assembly delivery system in a flask, and removing the organic solvent at 25 ℃ under reduced pressure to obtain the phosphoryl N-fatty acyl gemcitabine long-cycle self-assembly delivery system.

Experimental example 1 Langmuir Membrane Property Studies of Phosphoryl N-fatty acyl Gemcitabine

The phosphoryl N-fatty acyl gemcitabine synthesized in example 1 was dissolved in chloroform/methanol (9: 1, v/v) to a concentration of 1mg/ml, 25. mu.l was slowly dropped onto the Langmuir membrane balance level (25 ℃) by a microsyringe for 15min, then the level was pressed at a rate of 10mm/min by a slide barrier, and a pi-a curve of surface pressure (. pi.) and molecular cross-sectional area (a) was recorded and plotted by computer software (FIG. 3). Several phosphoryl N-fatty acyl gemcitabine compounds have proven to be very amphiphilic.

Experimental example 2 Property study of Phospho N-fatty acyl Gemcitabine self-assembled delivery System

5 mul of the phosphoryl N-fatty acyl gemcitabine self-assembly delivery system prepared in example 7 was dropped on the surface of a carbon-sprayed copper mesh, after 1min, excess liquid was sucked on one side of the copper mesh, and then dyed with 5 mul of 2% phosphotungstic acid (w/v) solution, after 1min, excess liquid was sucked on one side of the copper mesh, air-dried, and the form was observed under a transmission electron microscope (fig. 4).

Respectively adding 1.2ml of the assembled phosphoryl N-fatty acyl gemcitabine self-assembled delivery system into a sample cell, and measuring the particle size of the self-assembled delivery system at the temperature of 25 ℃. The Zeta potential of the self-assembled delivery system was determined similarly. The particle sizes of the phosphoryl N-octanoyl gemcitabine, the phosphoryl N-dodecanoyl gemcitabine, the phosphoryl N-tetradecanoyl gemcitabine, the phosphoryl N-hexadecanoyl gemcitabine and the phosphoryl N-octadecanoyl gemcitabine are respectively 95.6 nm, 125.1 nm, 143.2 nm, 264.7 nm and 303.7 nm; zeta potential is-20.8, -31.0, -25.7, -35.0, -32.6mV respectively. The self-assembly delivery system is stable, and the particle size is not obviously changed after the self-assembly delivery system is placed at room temperature for 1 month.

Experimental example 3 cellular pharmacodynamic evaluation of PhosphorylN-Dodecacylgemcitabine self-assembled delivery System

Gemcitabine phosphate solution, the phosphoryl N-dodecanoyl gemcitabine self-assembly delivery system suspension prepared in example 7, was prepared at concentrations of 1.25, 2.5, 5, 10, 20, 40, 80, 160. mu.g/ml, each sample concentration was in 3 wells in parallel, and the control well was complete medium. HepG2 liver cancer cell at 37 ℃ and 5% CO₂Culturing in incubator, collecting logarithmic phase cells, diluting cell suspension to 0.5 × 10⁴After one/ml, the cells are inoculated into a 96-well culture plate, 100 mu l/well, and placed in an incubator for further culture for 24 hours. After attachment, 100. mu.l/well was added to each well to give a final volume of 200. mu.l/well. After further culturing for 24h, adding 20. mu.l/well of MTT solution, further incubating for 4h, discarding the supernatant, adding 150. mu.L of dimethyl sulfoxide into each well, and shaking for 10min to fully dissolve the crystals. The absorbance of each well was measured at 490nm using an enzyme linked immunosorbent assay and the results were calculated as follows (FIG. 5).

Cell survival (%) - (OD drug-OD blank)/(OD control-OD blank) × 100%

Experimental example 4 PhosphorylN-dodecanoyl gemcitabine self-assembly delivery system animal model antitumor drug efficacy evaluation

Inoculating ascites type H22 mouse liver cancer cell into abdominal cavity of male Kunming mouse, and extracting under aseptic condition 7 days laterAscites of mice, diluted to a concentration of about 2 × 10 with sterile physiological saline⁷Cell suspension of one/ml 0.2ml (containing cells 4 × 10) was inoculated in the left anterior axilla of each mouse⁶Pieces/ml). On day 3 after inoculation, tumor-bearing mice of similar tumor size were selected and randomly divided into seven groups of 6 mice each. A: model control group (equal volume of saline given by group C dose volume); b: a positive control group (GEM injection, 5mg/ml, 40 mg/kg); c: phosphoryl N-dodecanoyl gemcitabine self-assembly delivery system low dose group (intravenous, 26.84mg/kg, 1/4 molar weight for group B); d: PhosphorylN-dodecanoylgemcitabine self-assembled delivery system high dose group (intravenous, 52.68mg/kg, 1/2 molar in group B).

The first dose was taken as the first day, once every three days, three times in total, and the body weight of the mice was measured daily to calculate the rate of change of body weight (fig. 6). On day eight, the tumors were dissected out, weighed wet, and tumor inhibition rate was calculated (table 1).

Percent change in body weight (%) - (mean body weight after treatment-mean body weight before treatment)/mean body weight after treatment × 100%

The tumor inhibition rate (%) was (average tumor weight in control group-average tumor weight in experimental group)/average tumor weight in control group was × 100%

Because the high dose group of the phosphoryl N-dodecanoyl gemcitabine self-assembly delivery system is equivalent to 1/2 molar mass of the gemcitabine group, the antitumor effect of the phosphoryl N-dodecanoyl gemcitabine self-assembly delivery system is obvious.

TABLE 1 tumor inhibition rate of phosphoryl N-dodecanoyl gemcitabine self-assembled delivery system on tumor-bearing Kunming mice

Self-assembled delivery system low dose group: PhosphorylN-dodecanoylgemcitabine self-assembled delivery system (low dose group, 1/4 molar equivalent to gemcitabine group)

Self-assembled delivery system high dose group: PhosphorylN-dodecanoylgemcitabine self-assembled delivery system (high dose group, 1/2 molar equivalent to gemcitabine group)

Experimental example 5 tissue distribution of PhosphorylN-Dodecacylgemcitabine self-assembled delivery System

Establishing tumor-bearing mouse model, injecting liver cancer cell H22 into abdominal cavity of male Kunming mouse, culturing until abdominal cavity is full of ascites, extracting ascites of mouse under aseptic condition, diluting with normal saline to concentration of 2 × 10⁷Cell suspension of one/ml Each mouse was inoculated with 0.2ml of cell suspension (containing cells 4 × 10) in the left anterior axilla⁶Pieces/ml). On day 3 after inoculation, 20 tumor-bearing mice with similar tumor sizes were selected for administration.

A tissue distribution protocol. 20 tumor-bearing mice were divided into 4 groups at random, 5 mice in each group were treated with the phosphoryl N-dodecanoyl gemcitabine self-assembly delivery system prepared in example 7 by tail vein injection, and then the mice were sacrificed at 0.5, 1, 1.5, and 2 hours, dissected for coring, liver, spleen, lung, and kidney, and the above tissues were washed with physiological saline, and then the tissue wet weight was determined. Adding equal amount of ultrapure water, homogenizing, centrifuging at 3000rpm for 10min to obtain supernatant, respectively collecting 10 μ l of supernatant, adding protein precipitant 90 μ l, vortexing for 2min, centrifuging at 3000rpm for 10min, collecting supernatant 20 μ l, and determining concentration of phosphoryl N-lauroyl gemcitabine (CPDG).

PhosphorylN-dodecanoyl gemcitabine, after intravenous injection into H22 tumor-bearing mice with self-assembled delivery system, was distributed mostly in liver and tumor (FIG. 7). This is mainly due to the targeting effect and tumor EPR effect of the self-assembled delivery system of the phosphoryl N-dodecanoyl gemcitabine, which is mainly taken up by liver and tumor. The content of phosphoryl N-dodecanoyl gemcitabine in each tissue of tumor-bearing mice after 0.5h of tail vein injection is shown in Table 2. Wherein the highest content of the liver phosphoryl N-dodecanoyl gemcitabine accounts for 35.79 percent of the dose. The phosphoryl N-dodecanoyl gemcitabine is 21.58% at the tumor site, which proves that the phosphoryl N-dodecanoyl gemcitabine self-assembly delivery system has the targeting effect on tumors.

TABLE 2.0.5% post-phosphorylation of gemcitabine N-dodecanoyl in each tissue of tumor-bearing mice

Liver disease

Heart with heart-shaped

Kidney (Kidney)

Spleen

Lung (lung)

Tumor(s)

Percentage of dose (%)

35.79

0.23

1.35

0.34

21.58

Experimental example 6 Effect evaluation of PhosphorylN-Dodecacylgemcitabine on antitumor drug in animal model with Long-circulating self-assembled delivery System

Injecting mouse-derived hepatoma carcinoma cell H22 into abdominal cavity of male Kunming mouse, culturing until abdominal cavity is full of ascites, extracting ascites from mouse under aseptic condition, centrifuging, collecting supernatant, and diluting with normal saline to obtain 2 × 10⁷Cell suspension of one/ml Each mouse was inoculated with 0.2ml of cell suspension (containing cells 4 × 10) in the left anterior axilla⁶One/m 1). On day 3 post-inoculation, 28 tumor-bearing mice with similar tumor sizes were selected for administration. Mice successfully modeled were selected and randomly divided into four groups of 8 mice each. A: negative control group: physiological saline; b: positive control injection group: gemcitabine 5 mg/ml; c: injection group Low dose Phosphorylgemcitabine Long cycle self-assembled delivery System prepared in example 8 (equivalent to 1/4 molar amounts of Gemcitabine B group); d: injection group high dose of the phosphoryl gemcitabine long-cycle self-assembled delivery system prepared in example 8 (equivalent to 1/2 molar amount of gemcitabine in group B).

The administration doses were respectively: group B was dosed at 30mg/kg, group C was dosed at 19.3mg of gemcitabine N-dodecanoyl/kg for group B (1/4 molar basis), group D was dosed at 38.61mg of gemcitabine N-dodecanoyl/kg for group B (1/2 molar basis), and group A was dosed at group D volumetric basis with an equal volume of saline. The first dose was taken as the first day, once every three days, three times in total, and the mice were weighed daily and tumor volume was measured.

The method for evaluating the drug effect was the same as in example 4. The results of tumor inhibition rate are shown in Table 3

TABLE 3 tumor inhibition rate of phosphoryl N-dodecanoyl gemcitabine long-circulating self-assembled delivery system on tumor-bearing Kunming mice

Self-assembled delivery system low dose group: PhosphorylN-dodecanoyl gemcitabine long cycle self-assembled delivery system (low dose group, corresponding to 1/4 molar amount of gemcitabine group)

Self-assembled delivery system high dose group: PhosphorylN-dodecanoyl gemcitabine long cycle self-assembled delivery system (high dose group, corresponding to 1/2 molar amount of gemcitabine group)

The tumor inhibition rate of the high-dose phosphoryl N-dodecanoyl gemcitabine long-cycle self-assembly delivery system group is 65.10%, and the inhibition effect is better. The inhibition effect of the low-dose phosphoryl N-dodecanoyl gemcitabine long-cycle self-assembly delivery system group (the tumor inhibition rate is 42.97%) is equivalent to that of the positive control group. There were significant differences in statistical analysis between the groups. The result shows that after the long-circulating material is added, the phagocytosis of the long-circulating self-assembly delivery system of the phosphoryl N-dodecanoyl gemcitabine by a reticuloendothelial system is reduced, the amount of the medicine reaching the tumor target tissue is increased, and the medicine effect is better.

Claims

1. A nano delivery system is characterized by comprising a phosphoryl N-fatty acyl nucleoside analogue and being selected from liposome, nonionic surfactant vesicles, nanoparticles, nanoemulsion and self-assembly delivery system, wherein the phosphoryl N-fatty acyl nucleoside analogue is characterized by having a structure of

Hep-Nu-L

Hep represents a cyclic phosphorylation group substituted by hydroxyl at the 5' position of a nucleoside sugar ring or short aliphatic chain hydroxyl, Nu is a nucleoside analogue group, L represents an aliphatic chain, and the carbon number in L is 8-20;

the prototypic molecule for the fatty chain is selected from the group consisting of n-octanoic acid, n-decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid;

the nucleoside analog proto-molecule is gemcitabine.

2. The nano-delivery system of claim 1, wherein the amount of phosphoryl N-fatty acyl nucleoside analogs in the particle composition of the self-assembled delivery system is between 50 and 100% by molecular molar ratio of the total composition, with the balance being additives.

3. The nanotransport system of claim 1, wherein the particles of the self-assembled delivery system are comprised entirely of phosphoryl N-fatty acyl nucleoside analogs.

4. The nanotransport system of claim 1, which is a self-assembled delivery system of phosphonogemcitabine having the formula:

wherein R is C₁₁H₂₃-。

5. The nanotransport system of claim 1, which is a phosphoryl N-dodecanoyl gemcitabine long-cycle self-assembled delivery system, the phosphoryl N-dodecanoyl gemcitabine having the formula:

wherein R is C₁₁H₂₃-。