Synthesis and application of novel lipid

Abstract

The present invention relates to alpha-tocopherol succinate (alpha-TOS) derivatives having ionizable properties or of the cationic type. The alpha-TOS derivative prepared by the invention can be used for preparing ionizable or cationic liposomes and lipid nano preparations. The compounds have good nano preparation formability and in-vivo anti-tumor activity superior to that of alpha-TOS, and have a synergistic anti-tumor effect when being combined with anti-tumor drugs such as doxorubicin hydrochloride and the like. The lipid nano preparation entrapped with the doxorubicin hydrochloride has high tumor inhibition capacity and does not have obvious toxic reaction. The invention firstly provides a class of relatively safe ionizable lipids and cationic lipids for preparing ionizable nano preparations, and secondly provides a class of new medicines and preparations thereof for treating tumors.

Description

Technical Field

[0001] The invention belongs to the field of new drug synthesis / pharmaceutical preparations, and discloses a preparation method of a cationic, especially ionizable α-tocopherol succinate (α-TOS) derivative, a microparticle preparation prepared based on the compound, and its application in anti-tumor preparations. Background Art

[0002] Cationic lipids play an important role in drug delivery. The positively charged head group of cationic lipids can form electrostatic interactions with negatively charged biomacromolecules (such as DNA and RNA), which helps to encapsulate and compress genetic material to form lipid complexes (lipoplexes), thereby improving its cellular uptake rate. Positive charges also help liposomes interact with negatively charged molecules on the cell membrane, thereby promoting the fusion of liposomes with cell membranes or entering cells through other mechanisms. Therefore, the polar head group of cationic lipids plays a role in the mutual binding of liposomes and DNA, liposome-DNA complexes and cell membranes or other components in cells. The advantages of cationic lipid-mediated transfection include high efficiency, ability to transfect a variety of cell types, repeatability and simplicity. The construction of reasonable prescriptions and preparations can achieve the purpose of eliminating problems such as hemolytic toxicity and exerting the effects of cationic lipids.

[0003] Ionizable cationic lipids are sensitive to pH and can overcome some of the disadvantages of permanent cationic lipids. The special thing about ionizable lipids is that they are electrically neutral or nearly neutral under normal physiological pH conditions. Therefore, lipid nanocarriers (LNPs) composed of ionizable lipids have less toxicity and a longer in vivo circulation cycle. After being taken up by cells, in the acidic environment of organelles such as lysosomes, the ionizable lipids will be protonated and converted to a positively charged state, making it easier to fuse with the lysosomal membrane, allowing the delivered drugs to quickly escape from the lysosomes and enter the cytoplasm to exert their therapeutic effects. The current ionizable cationic lipids cannot fully meet the demand, and there is an urgent need to develop new materials with high safety.

[0004] α-Tocopheryl succinate (α-TOS), also known as vitamin E succinate, is a substitute for vitamin E with high safety, high stability, water solubility and surface activity. The introduction of succinic acid groups also endows α-TOS with anti-tumor effects. α-TOS has anti-cancer activity against a variety of human cancer cells, including breast cancer, lung cancer, cervical cancer, colon cancer, etc., and has a certain effect of reversing drug resistance. In general, α-TOS has high safety and relatively mild anti-tumor effects. However, the research and application of its cationic α-TOS derivatives, especially those with ionizable properties, are not sufficient. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, one of the purposes of the present invention is to provide several novel cationic α-TOS derivatives synthesis methods and their uses. Such compounds are themselves cationic or ionizable, and the ionizability is specifically reflected in that when the pH value is lower than 7, the preparation can have a significant positive charge, so it has the use of preparing cationic and ionizable lipid nanoformulations. Such derivatives also include pharmaceutically acceptable deuterated derivatives or prodrugs thereof.

[0006] The basic structure of the α-tocopherol succinate derivative is:

[0007]

[0008] Wherein X in Formula 1 is an atom or group such as NH, O or S that is conducive to in vivo degradation, and Y is Compounds with CH2 replaced by CH n , (n=1-5) compounds.

[0009] In terms of ionizability and anti-tumor properties, the preferred compounds are those containing of compounds.

[0010] The second object of the present invention is to provide a method for synthesizing and purifying α-tocopherol succinate derivatives. The basic method is: α-TOS is dissolved in an organic solvent such as dichloromethane, and condensation reagents such as HATU (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) / EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) are added in sequence under stirring, and nucleophilic reagents such as DIPEA (N,N-diisopropylethylamine) / DMAP (4-dimethylaminopyridine) are added, and Y-XH is added. The reaction is stirred at low temperature for a period of time, and then stirred continuously at room temperature for 2-24 hours. After the reaction is completed, an organic solvent such as dichloromethane can be added to the reaction mixture in an amount of about several times the volume, and washed with ultrapure water and saturated saline for multiple times, the organic phase is collected, the solvent is removed, and the residue is separated by silica gel column chromatography to obtain the final product. Other suitable methods can also be selected according to the product conditions for purification and separation operations. The basic reaction process is:

[0011]

[0012] The third purpose of the present invention is to provide a preparation of such a compound. Our experiments show that several synthesized compounds have good nanoformulation properties. They can be used to prepare various microparticle drug delivery systems in pharmacy, including but not limited to: the compound self-assembles into nanoparticles (emulsifiers such as F68 can be added); or the compound is used alone as a carrier to encapsulate other drugs to form nanoparticles; or the compound is used together with other carrier materials to form liposomes, emulsions, nanoparticles, lipid nanocarriers (LNP), micelles and other microparticle drug delivery systems; or the compound is used together with various phospholipid materials to encapsulate drugs to prepare liposome drug delivery systems; or the compound is used together with other carrier excipients to encapsulate drugs to prepare emulsions, nanoparticles, LNP, micelles and other microparticle drug delivery systems.

[0013] Taking liposome preparation as an example, the compound synthesized by the present invention can replace components such as cholesterol and various types of phospholipids, and is prepared by a common liposome preparation method. The encapsulation rules of various types of drugs are the same as those of liposomes prepared from cholesterol and phospholipids. Lipid-soluble drugs are mainly encapsulated in the phospholipid layer, while water-soluble drugs are encapsulated in the inner water phase of the liposome. Drugs such as doxorubicin hydrochloride can also obtain higher drug loading and encapsulation efficiency by using active drug loading method.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: compared with α-TOS or cholesterol, the nanoformulations formed by the synthesized derivatives have the advantages of higher encapsulation rate, stronger cell uptake rate, faster intracellular release or higher anti-tumor rate for drugs. The synthesized derivatives have the properties of cationic lipids or ionizable lipids, and have the potential to be applied to biotechnology drugs such as negatively charged RNA and prepared into preparations such as LNP. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 TZ's 1 H-NMR.

[0016] Figure 2 TZ's 13 C-NMR.

[0017] Figure 3 TA's 1 H-NMR.

[0018] Figure 4 TA's 13 C-NMR.

[0019] Figure 5 TY 1 H-NMR.

[0020] Figure 6 TY 13 C-NMR.

[0021] Figure 7 TTS 1 H-NMR.

[0022] Figure 8 TTS 13 C-NMR.

[0023] Fig. 9 MPE 1 H-NMR.

[0024] Fig.10 MPE 13 C-NMR.

[0025] Fig.11 Appearance of self-assembled nanoparticles (A), mPEG-PLGA nanoparticles (B), liposomes (C), and DOX liposomes (D) prepared from three α-TOS derivatives TZ, TA, and TY, respectively.

[0026] Fig.12 Uptake of DOX liposomes in different preparations by MGC803 cells DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] Example 1 Synthesis and purification of TZ

[0029] D-α-tocopherol succinate (531 mg) was dissolved in 10 mL of dichloromethane and placed at 0°C. HATU (380 mg), DIPEA (696 μl) and N-(3-aminopropyl)imidazole (119 μL) were added in sequence under stirring, stirred at 0°C for 1 hour, and the temperature was raised to room temperature and stirred continuously for about 8 hours. Then, dichloromethane was added to the reaction mixture, and the mixture was washed with water and saturated brine respectively, the solvent was removed by rotary evaporation, and compound 1 (TZ) was obtained after separation and purification by silica gel column chromatography with a yield of about 82%.

[0030]

[0031] TZ H NMR spectrum is attached Figure 1 : 1H NMR (400MHz, Chloroform-d) δ7.61(s,1H),7.05(s,1H),6.92(s,1H),6.07(t,J=6.0Hz,1H),3.98(t,J=6.9Hz,2H),3.26(q,J=6.4Hz,2H),3.02–2. 88(m,4H),2.56(td,J=6.7,3.6Hz,4H),2.09–1.92(m,9H),1.76(ddq,J=2 0.0,13.3,6.7Hz,2H),1.61–0.98(m,24H),0.85(dd,J=10.4,5.2Hz,12H).

[0032] The C NMR spectrum of TZ is attached. Figure 2 : 13 C NMR (151 MHz, Chloroform-d): δ172.01 for C35, δ171.90 for C32, δ136.99 for C5, δ128.3 for C2, δ126.63 for C45, δ124.91 for C43, δ123.05 for C46, ​​δ12.96 for C14, δ12.11 for C7, δ11.82 for C13.

[0033] Example 2 Synthesis and Purification of TY

[0034] D-α-tocopherol succinate (531 mg) was dissolved in 10 mL of dichloromethane and placed at 0°C. HATU (380 mg), DIPEA (696 μl) and N-methyl-2-(2-aminoethyl)-pyrrolidine (149 μL) were added in sequence under stirring, stirred at 0°C for 1 hour, and the temperature was raised to room temperature and stirred continuously for about 8 hours. Then, dichloromethane was added to the reaction mixture, and the mixture was washed with water and saturated brine respectively, the solvent was removed by rotary evaporation, and compound 2 (TY) was obtained after separation and purification by silica gel column chromatography with a yield of about 75%.

[0035]

[0036] The C NMR spectrum of TY is attached. Figure 3 : 1H NMR (400MHz, Chloroform-d) δ6.72(s,1H),3.45(dq,J=13.0,6.5Hz,1H),3.24(dtd,J=13.8,7.2,4.1Hz,1H),3.06(ddd,J=9.6,7.3,2.7Hz,1H),2.97(t,J= 6.6Hz,2H),2.57(q,J=6.8Hz,4H),2.40–2.04(m,9H),1.98(d,J=15.9Hz,5H), 1.75(tdd,J=17.4,13.9,7.4Hz,6H),1.65–0.99(m,26H),0.96–0.75(m,12H).

[0037] The C NMR spectrum of TY is attached. Figure 4 : 13 C-NMR (151 MHz, Chloroform-d) δ149.41 for C35, δ140.50 for C32, δ126.68 for C5, δ122.99 for C2, δ12.97 for C14, δ12.11 for C7, δ11.80 for C13.

[0038] Example 3 Synthesis and purification of TA

[0039] D-α-tocopherol succinate (531 mg) was dissolved in 10 mL of chloroform and placed at 0°C. HATU (380 mg), DIPEA (696 μl) and N,N-dimethylethylenediamine (109 μL) were added in sequence under stirring, stirred at 0°C for 1 hour, and the temperature was raised to room temperature and stirred continuously for about 16 hours. Then, dichloromethane was added to the reaction mixture, and the mixture was washed with water and saturated brine respectively, the solvent was removed by rotary evaporation, and compound 3 (TA) was obtained after separation and purification by silica gel column chromatography with a yield of about 80%.

[0040]

[0041] The chemical structure and C NMR spectrum of TA are attached. Figure 5 . 1H-NMR(400MHz,Chloroform-d)δ6.23(s,1H),3.63(t,J=6.5Hz,1H),3.35(q,J=5.6H z,1H),2.97(t,J=6.9Hz,2H),2.70(s,2H),2.59(dt,J=14.0,7.0Hz,2H),2.50(t,J=6 .5Hz,2H),2.42(t,J=5.9Hz,3H),2.24(d,J=6.0Hz,6H),2.18–2.04(m,3H),1.98(d, J=15.7Hz,2H),1.86–1.67(m,3H),1.57–1.02(m,24H),0.85(dd,J=9.5,6.5Hz,12H).

[0042] The C NMR spectrum of TA is attached. Figure 6 . 13 C NMR (151 MHz, Chloroform-d): δ177.36 for C35, δ171.65 for C32, δ171.20 for C5, δ122.99 for C2, δ117.37 for C1, δ12.96 for C14, δ12.11 for C7, δ11.80 for C13.

[0043] Example 4 Synthesis and purification of TTS

[0044] Weigh 530 mg (1.0 mmol) of α-TOS and 620 mg of EDC·HCl (4.0 mmol), dissolve in 35 mL of dichloromethane, stir in an ice bath for 30 min to dissolve, then add 55 μL (1.0 mmol) of 3-(1H-imidazol-1-yl)propanol and 61 mg of DMAP (0.5 mmol), stir for 7 h. Wash the reaction solution with saturated brine three times, remove the organic solvent by rotary evaporation, and separate by silica gel column chromatography to obtain a colorless oily product of TTS with a molecular formula of C39H62N2O5 and a molecular weight of 638.93, with a yield of about 65%.

[0045]

[0046] The C NMR spectrum of TTS is attached. Figure 7 The C NMR spectrum of TTS is attached. Figure 8 Its carbon spectrum meets the requirements of TTS. Compared with the carbon spectrum of TZ, it has an additional carbon peak at 60-70ppm, which is consistent with the difference between the two.

[0047] Example 5 Synthesis and purification of MPE

[0048] Weigh 531 mg (1 mmol) of α-TOS and 764 mg (4 mmol) of EDC·HCl, add 70 ml of anhydrous dichloromethane to dissolve, and stir for 30 min in an ice bath using a magnetic stirrer. Then add 135 μL (1 mmol) of N-methyl-2-(2-hydroxyethyl)pyrrolidine and 61 mg (0.5 mmol) of DMAP in sequence, return to room temperature and stir for 10 h. Wash with water and saturated brine respectively, remove the solvent by rotary evaporation, and separate and purify on a silica gel column to obtain compound MPE.

[0049]

[0050] The chemical structure and C NMR spectrum of MPE are attached. Fig. 9 The C NMR spectrum of MPE is attached. Fig.10 Compared with the carbon spectrum of TY, there is an additional carbon peak at 60-70ppm, which is consistent with the difference between the two.

[0051] Example 6 Preparation of self-assembled nanoparticles

[0052] Self-assembled nanoparticles were prepared by emulsification solvent evaporation method: 10 mg of the above compounds were taken, dissolved in 400 μl of dichloromethane and 100 μl of anhydrous ethanol, 2 ml of 5% F68 was added, and the mixture was stirred at 150 W for 10 min. The appearance of the obtained nanoparticle preparations, including TZ, TY and TA nanoparticles, is shown in the attached figure. Fig.11 (A) The other two preparations have similar appearances, and both are semi-transparent liquids with light blue opalescence, which is consistent with the optical characteristics of nanoparticles.

[0053] Example 7 Preparation of mPEG-PLGA Nanoparticles

[0054] Take 10mg (5:1) of TA, TZ and TY and mPEG-PLGA (50 / 50, 18k) respectively, dissolve in 400μl dichloromethane and 100μl anhydrous ethanol, add 2ml 5% F68, and test (power 150W, 10min). Fig.11 (B).

[0055] Example 8 Preparation of TZ Lip

[0056] Accurately weigh 20 mg of egg yolk phosphatidylcholine (EPC) and 7.5 mg of TZ in an eggplant-shaped bottle, then add 2 mL of dichloromethane and 1 mL of ethanol to completely dissolve the two. Place the eggplant-shaped bottle in a rotary evaporator, heat the water bath to 50°C, evaporate the organic solvent until a uniform and continuous film is formed on the bottle wall, then remove the eggplant-shaped bottle, add 2 mL of ultrapure water, and hydrate it by ultrasound at 40°C to form uniform milky white crude liposomes. Then use an ultrasonic cell crusher (10 min, working 3 s, rest 5 s, power 300 W) to make the liposome particle size uniform, and then obtain TZ Lip.

[0057] Example 9 Preparation of TALip

[0058] Accurately weigh 20 mg EPC and 7.5 mg TA in an eggplant-shaped bottle, then add 2 mL of dichloromethane and 1 mL of ethanol to completely dissolve the two. Place the eggplant-shaped bottle on a rotary evaporator, heat the water bath to 50°C, evaporate the organic solvent until a uniform and continuous film is formed on the bottle wall, then remove the eggplant-shaped bottle, add 2 mL of ultrapure water, and hydrate it by ultrasound at 40°C to form uniform milky white crude liposomes. Then use an ultrasonic cell crusher (10 min, working 3 s, rest 5 s, power 300 W) to make the liposome particle size uniform, and then obtain TA Lip.

[0059] Example 10 Preparation of TY Lip

[0060] 20 mg EPC and 7.5 mg TY were accurately weighed into an eggplant-shaped bottle, and then 2 mL of dichloromethane and 1 mL of ethanol were added to completely dissolve the two. The eggplant-shaped bottle was placed in a rotary evaporator, and after the water bath was heated to 50°C, the organic solvent was evaporated to form a uniform continuous film on the bottle wall. Then the eggplant-shaped bottle was removed, 2 mL of ultrapure water was added, and ultrasonic hydration was performed at 40°C to form uniform milky white crude liposomes. Then, an ultrasonic cell crusher (10 min, working 3 s, resting 5 s, power 300 W) was used to make the liposome particle size uniform, and TY Lip was obtained. The appearance of the preparations of Examples 6-8 is shown in the attached Fig.11 (C).

[0061] Example 11 Preparation of TOS Lip

[0062] Accurately weigh 20 mg EPC and 7.5 mg α-TOS in an eggplant-shaped bottle, then add 2 mL of dichloromethane and 1 mL of ethanol to completely dissolve the two. Place the eggplant-shaped bottle on a rotary evaporator, heat the water bath to 50°C, evaporate the organic solvent until a uniform and continuous film is formed on the bottle wall, then remove the eggplant-shaped bottle, add 2 mL of ultrapure water, and hydrate it by ultrasound at 40°C to form uniform milky white crude liposomes. Then use an ultrasonic cell crusher (10 min, working 3 s, rest 5 s, power 300 W) to make the liposome particle size uniform, and thus obtain TOS Lip.

[0063] Example 12 Preparation of DOX Lip

[0064] The ammonium sulfate gradient method was used to actively encapsulate DOX. 20 mg EPC and 4 mg cholesterol were accurately weighed in an eggplant-shaped bottle, and then 2 mL of dichloromethane and 1 mL of ethanol were added to completely dissolve the two. The eggplant-shaped bottle was placed in a rotary evaporator, and the water bath was heated to 50 ° C, and the organic solvent was evaporated to form a uniform and continuous film on the bottle wall. Then the eggplant-shaped bottle was removed, 2 mL of ammonium sulfate solution (250 mmoL) was added, and it was hydrated by ultrasound at 40 ° C to form uniform milky white crude liposomes. An ultrasonic cell crusher (10 min, working 3 s, resting 5 s, power 300 W) was used to make the liposome particle size uniform. In order to remove the ammonium sulfate solution outside the liposome, the prepared liposome was transferred to a dialysis bag (8000-14000 Da), and the dialysis bag was placed in a beaker filled with 1 L of ultrapure water, stirred at a uniform speed, and the water was changed every 1 h, and the dialysis was performed for 4 h. Finally, 1 mg of DOX was added for 2 h. To remove the unencapsulated DOX, the loaded liposomes were transferred to a dialysis bag (3000 Da). The dialysis bag was placed in a beaker filled with 1 L of ultrapure water and stirred at a constant speed. The water was changed every 1 h and dialyzed for 4 h to obtain DOX Lip.

[0065] Example 13 Preparation of DOX@TZ Lip, DOX@TA Lip, DOX@TY Lip, and DOX@TOS Lip

[0066] The ammonium sulfate gradient method was used to actively encapsulate DOX. First, TZ Lip, TALip, TY Lip and TOS Lip were prepared according to the methods of Examples 8-11, and then the same method as Example 12 was used. After obtaining a uniform and continuous lipid film, the eggplant-shaped bottle was removed, and 2 mL of ammonium sulfate solution (250 mmol) was added respectively. The liposomes were hydrated by ultrasound at 40°C to form uniform milky white crude liposomes. An ultrasonic cell crusher (10 min, working 3 s, rest 5 s, power 300 W) was used to remove the ammonium sulfate solution outside the liposomes. The prepared liposomes were transferred to a dialysis bag (8000-14000 Da), and the dialysis bag was placed in a beaker filled with 1 L of ultrapure water. The mixture was stirred at a uniform speed, the water was changed every 1 h, and the dialysis was performed for 4 h. Finally, 1 mg of DOX was added for 2 hours. To remove the unencapsulated DOX, the loaded liposomes were transferred to a dialysis bag (3000 Da), which was placed in a beaker filled with 1 L of ultrapure water. The dialysis bag was stirred at a constant speed, and the water was changed every 1 hour. The dialysis was performed for 4 hours to obtain DOX@TZ Lip, DOX@TA Lip, DOX@TY Lip, and DOX@TOS Lip. The appearance of the preparation of Example 13 is shown in Fig.11 (D).

[0067] Example 14 Preparation of HADOX@TZ Lip, HADOX@TA Lip, HADOX@TY Lip, and HADOX@TOS Lip

[0068] Take 1 ml of DOX Lip, DOX@TZ Lip, DOX@TA Lip, DOX@TY Lip, and DOX@TOS Lip obtained in Examples 12 and 13 above, respectively, add 100 μl of 2% (W / V) HA aqueous solution thereto while stirring, and stir for 1 hour to obtain HA DOXLip, HATZ@DOX Lip, HATA@DOX Lip, HATY@DOX Lip, and HA TOS@DOX Lip.

[0069] Example 15 Preparation of MPE lipid nanoformulation

[0070] The MPE lipid nanoformulation was prepared by thin layer dispersion method. 8 mg MPE and 16 mg egg yolk lecithin were accurately weighed into an eggplant-shaped bottle, and 2 ml dichloromethane and 1 ml anhydrous ethanol were added. The eggplant-shaped bottle was placed in an ultrasonic cleaner to dissolve the product and phospholipids by ultrasound. After complete dissolution, the eggplant-shaped bottle was connected to a rotary evaporator, and the solvent was evaporated by rotary evaporation in a 40°C constant temperature water bath. After the solvent was dried, a continuous and uniform lipid film was observed at the bottom of the eggplant-shaped bottle. 2 ml of pure water was added, and ultrasonic hydration was performed for 20 min using an ultrasonic cleaner to obtain a crude suspension in the bottle. The suspension was transferred to a 5 ml EP tube, and an ultrasonic cell crusher was used to detect the liquid to make the particle size of the lipid nanoformulation uniform. The detection time was set to 10 min, the opening time to 3 s, the closing time to 5 s, and the power to 300 W. Then a 0.45 μm microporous filter membrane was used to filter the lipid nanoformulation.

[0071] Example 16 Preparation of TTS liposomes

[0072] TTS liposomes were prepared by thin film dispersion method. According to the drug-lipid ratio of 1:4, 8 mg TTS and 32 mg lecithin were accurately weighed into a pear-shaped bottle, 2 mL of dichloromethane and 1 mL of anhydrous ethanol were added to dissolve them, and after ultrasonication, the organic solvent was removed by rotary evaporation to form a uniform film in the pear-shaped bottle. 2 mL of ultrapure water was added, and the obtained suspension was ultrasonicated for 10 min (25 ° C, 65 W) to obtain TTS liposomes with a concentration of 4 mg / mL. Using a laser particle size potential meter, with water as the dispersion medium, the average particle size (z average) was measured to be 76.14 ± 1.13 nm, the polydispersity coefficient was 0.061, and the zeta potential was 0.14 ± 0.27 mV.

[0073] Example 17 Preparation of Paclitaxel TZ Lip

[0074] Accurately weigh 20 mg of egg yolk phosphatidylcholine (EPC), 7.5 mg of TZ, and 1 mg of paclitaxel into an eggplant-shaped bottle, then add 2 mL of dichloromethane and 1 mL of ethanol to completely dissolve the two, place the eggplant-shaped bottle on a rotary evaporator, heat the water bath to 50°C, evaporate the organic solvent until a uniform and continuous film is formed on the bottle wall, then remove the eggplant-shaped bottle, add 2 mL of ultrapure water, and hydrate it by ultrasound at 40°C to form uniform milky white crude liposomes, and then use an ultrasonic cell crusher (10 min, working 3 s, rest 5 s, power 300 W) to make the liposome particle size uniform, and thus obtain paclitaxel TZ Lip.

[0075] Example 18 Preparation of HA DOX Lip, HA TZ Lip, HA TA Lip, HA TY Lip, and HA TOS Lip

[0076] Take 1 ml of TZ Lip, TA Lip, TY Lip, DOX Lip and TOS Lip obtained in Examples 8-12 respectively, add 100 μl of 2% (W / V) hyaluronic acid (HA) aqueous solution thereto while stirring, and stir for 1 hour to obtain HA-modified liposomes, HA TZ Lip, HA TA Lip, HA TY Lip, HA DOX Lip and HA TOS Lip.

[0077] The representative examples mentioned above will be selected below to conduct relevant experiments to illustrate the characteristics and advantages of α-TOS derivatives and their preparations. The grouping, preparation and conclusion are all set and analyzed according to the relevant basic rules of scientific experiments. Therefore, if there are any omissions in the specific details of the experiment, please refer to the basic principles.

[0078] Experimental Example 1 Determination of pKa of α-TOS derivative lipid nanoformulation

[0079] The pKa value of α-TOS liposomes was determined by titration or TNS method. Lipid nanoformulations were prepared with derivatives TZ, TA, TY, TTS, MPE and egg yolk phosphatidylcholine (see relevant Examples 8-10, 15, 16 for specific prescriptions). After calculation and drawing, it can be seen that the apparent pKa of liposomes prepared by 5 kinds of α-TOS derivatives TA, TZ, TY are about 7.2, 6.95, 8.12 (titration method), and the apparent pKa of liposomes prepared by TTS and MPE are 5.63 and 5.54I (TNS method). Since the prescription will affect the determination results, the prescription can be adjusted according to the purpose in the later stage. Therefore, the several compounds synthesized by us have the possibility of becoming ionizable lipids and preparing ionizable nanoformulations.

[0080] The basic method of the TNS method is as follows: prepare a series of buffer solutions with pH values ​​ranging from 3 to 10 (150 mM NaCl, 10 mM boric acid solution, 10 mM phosphoric acid solution, and 10 mM citric acid solution). Dilute the liposomes in each solution. Dissolve TNS in DMSO at a concentration of 300 μM, and mix 2 μL of TNS solution with 100 μL of lipid nanoformulation. Measure the fluorescence value under the conditions of excitation light 325 nm and emission light 435 nm.

[0081] Experimental Example 2 Encapsulation efficiency of DOX by α-TOS derivative lipid nanoformulation

[0082] Ultrafiltration was used to separate the encapsulated and free DOX in the liposomes, and the content of DOX was determined respectively, and the encapsulation rate was calculated. Results The liposomes prepared by replacing cholesterol with α-TOS derivatives had a high encapsulation rate for DOX. The liposomes prepared with the participation of derivatives TZ, TA and TY (Example 13) had encapsulation rates for DOX of 98.89±2.14%, 96.49±1.67% and 94.74±2.32%, respectively. After optimizing the ratio of cholesterol to phospholipids, the encapsulation rate of DOX in the prepared ordinary liposomes was 92.09±1.96%, which was significantly lower than the encapsulation rate of DOX after the three α-TOS derivatives replaced cholesterol. Therefore, the experimental results show that the three α-TOS derivatives can form a stable lipid bilayer with phospholipids, and the active drug loading method can achieve perfect encapsulation of DOX.

[0083] In addition to liposomes, these five derivatives can also be prepared into a variety of nanopreparations, including self-assembled nanoparticles, nanopreparations made in combination with mPEG-PLGA, nanoemulsions, etc. Therefore, several α-TOS derivatives have good drugability and will be widely used in nanopreparations in the future.

[0084] Experimental Example 3 Relationship between zeta potential and pH of α-TOS derivative lipid nanoformulation

[0085] We investigated the zeta potential of six liposomes in PBS buffer solutions with different pH values ​​(Examples 8-11, 15, 16). As a result, TZ Lip, TA Lip, and TTS Lip were all close to electrical neutrality at pH 7.4, and their positive charge increased significantly (>25mV) when the pH decreased (pH=4-5), indicating that the nanoformulations prepared with these three compounds have significant ionizable properties. The zeta potential of TOSLip is negative at 4-7.4, and MPE Lip and TY Lip are positive at 4-7.4 (basically above 20mV), indicating that the latter two derivatives are cationic lipids. TTS and TZ have the same ionizable group, and the pKa value of their nanoformulations and the change of the zeta potential of the nanoformulations with pH all meet the requirements of ionizable lipids. Therefore, it can be inferred that derivatives with similar structures to these two compounds and having both the ionizable group and the TOS group should also have the properties of ionizable lipids. MPE and TY have the same cationic group, and the nanoformulations prepared from them have similar potentials, which are all positive at physiological pH. The above results show that when the α-TOS group is the same, the main factor affecting the ionizability of the compound is the ionic head group. Therefore, it can be inferred that the derivatives of the α-TOS group, if they contain the same cationic head group as in this patent, have similar properties.

[0086] In addition to being used in the preparation of DOX liposomes, the several ionizable lipids we synthesized can also have good application prospects in preparations that require rapid release of drugs in cells to avoid degradation and inactivation of drugs in the acidic environment of lysosomes. For example, biotechnology drugs including various RNAs can be loaded with drugs by adjusting the pH value and other means.

[0087] Experimental Example 4 Tumor cell uptake experiment and small animal imaging experiment

[0088] We used MGC803 cells as model cells to investigate the uptake of HADOX@TOS Lip, HA DOX@TZLip, and HADOX@TALip in Example 14. At the same time, we compared them with the control preparation HADOX@Lip containing DOX. The results were ( Fig.12 ), the red fluorescence intensity of the HADOX@TZ Lip group was the strongest, followed by the HA DOX@TA Lip group. The DOX uptake by the HADOX@TOS Lip group was also significantly higher than that of the HA DOX Lip group. The results of the uptake experiment showed that compared with α-TOS and cholesterol, the addition of TZ and TA could significantly promote the uptake of DOX liposomes by tumor cells. Moreover, we used IR780 as a model drug to prepare the corresponding liposomes, and performed in vivo imaging of small animals after intravenous administration to determine the tissue distribution. The results confirmed that TZ and TA can improve the tumor tissue targeting of liposomes after intravenous administration.

[0089] Experimental Example 5 Hemolysis Experiment

[0090] Example 14 was used as the experimental object, and the experiment was conducted according to the standard operating procedure for hemolysis rate. As a result, the preparations all met the requirement that the 2 mg / ml hemolysis rate was less than 5%, and could meet the dosing requirements of different preparations in the later stage.

[0091] Experimental Example 6 Cytotoxicity Experiment

[0092] H22 cells in the logarithmic growth phase were seeded in 96-well plates at a cell seeding density of approximately 1.0 × 10 5 / well, add drugs after 3 hours of culture, and after co-culture of cells and drugs for 24 hours, add 10μL CCK-8 solution to each well, continue to culture in the incubator for 4 hours, and use a microplate reader to measure the absorbance OD at a wavelength of 490nm to calculate the median lethal dose (IC50). The results show that the IC50 of HADOXLip, HA TOS Lip, HATZ Lip and HATALip are 3.8, 106.9, 237.6 and 1019.5μg / ml, respectively. This result shows that compared with α-TOS, the liposome preparations synthesized from the two derivatives have a weaker inhibitory effect on the activity of H22 in vitro.

[0093] Experimental Example 7 Tumor inhibition experiment in tumor-bearing animals

[0094] The H22 tumor transplantation model was prepared according to conventional methods. The basic operation was to extract the ascites of the mice with ascites tumors containing H22 tumor cells, dilute it appropriately, and inoculate it into the subcutaneous part of the right upper armpit of the mice. Each mouse was inoculated with 200 μl of ascites. When the average tumor volume reached about 100 mm 3 Then they were randomly divided into 10 groups, with 5 mice in each group. Among them, the normal saline group was the negative control group (No. 1), and then 9 groups of HA-modified liposomes were prepared respectively. The prescription of the liposomes can be found in the relevant embodiments. The basic prescription is egg yolk lecithin: cholesterol / α-TOS or its derivatives, and it is based on the principle of single factor investigation. Among them, the ordinary liposome groups are No. 2-6, which are liposomes loaded with DOX, α-TOS, TZ, TA and TY respectively, and groups 7-10 are liposomes co-loaded with DOX and α-TOS, TZ, TA and TY. The dosages of DOX and four α-TOS or their derivatives were 5 mg / kg and 20 mg / kg respectively. Weigh the body weight every three days, measure the tumor volume, and administer intravenously once, for a total of 7 times. Two days after the last time, the mice were killed.

[0095] Results The average tumor inhibition rates of HADOX Lip, HATOS Lip, HATZ Lip, HATA Lip, HA TY Lip, HADOX@TOS Lip, HADOX@TZ Lip, HA DOX@TA Lip, and HA DOX@TY Lip were 51%, 36%, 47%, 43%, 40%, 68%, 84%, 75%, and 72%, respectively. In general, the antitumor effect of the α-TOS derivatives prepared by us is better than that of α-TOS. After being made into liposomes to encapsulate DOX, the antitumor effect of the α-TOS derivatives combined with DOX is also better than that of the α-TOS combined with DOX. Although the cytotoxicity of the α-TOS derivatives prepared by us to tumor cells in vitro is significantly lower than that of α-TOS. However, the tumor inhibition ability of the α-TOS derivatives after in vivo administration is higher, which indicates that it may have other non-cytotoxic pathways such as enhancing immunity to inhibit tumor growth in vivo.

[0096] Therefore, the experimental results of H22 tumor-bearing mice showed that compared with the ordinary liposome preparation of DOX containing cholesterol, the liposome preparations prepared by replacing cholesterol with α-TOS and its derivatives can significantly enhance the anti-tumor activity of DOX. In terms of overall toxicity, the body weight of mice in the ordinary liposome preparation group of DOX was significantly reduced, and the mice intravenously administered with the preparation containing α-TOS and its derivatives showed no abnormal performance, and the body weight was not significantly different from that of the saline group. Therefore, the liposome preparations containing α-TOS and its ionizable derivatives can significantly enhance the anti-tumor effect of DOX while overcoming the toxicity of DOX. Among them, the best effect was achieved by several derivatives synthesized by us.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An ionizable or cationic α-tocopherol succinate (α-TOS) derivative and a pharmaceutically acceptable deuterated derivative or prodrug thereof. The compound has the basic structure of formula I. Wherein X in Formula 1 is NH, O or S, and Y is an ionizable group, including 2. The use of the compound according to claim 1, characterized in that This type of compound has good nano-formulation properties and can be used in the preparation of microparticle-type drug delivery systems such as nanoparticles, liposomes, and lipid nanocarriers (LNPs). The preferred particle size range is 10-500 nm.

3. The use of the compound according to claim 1-2, characterized in that: The compound can be used to prepare a microparticle drug delivery system, including but not limited to: the compound self-assembles into nanoparticles; or the compound is used alone as a carrier to encapsulate other drugs to form nanoparticles; or the compound is used together with other carrier materials to prepare liposomes, emulsions, nanoparticles, lipid nanocarriers (LNP), micelles and other microparticle drug delivery systems; or the compound is used together with various phospholipid materials to encapsulate drugs to prepare liposome drug delivery systems; or the compound is used together with other carrier excipients to encapsulate drugs to prepare emulsions, nanoparticles, LNP, micelles and other microparticle drug delivery systems.

4. The use of the compound according to claims 1-3, characterized in that: The compound has a certain anti-tumor effect and can be used for the preparation of tumor preparations.

5. The use of the compound according to claims 1-4, characterized in that: The compound has a synergistic effect when used in combination with tumor treatment or prevention drugs, including but not limited to doxorubicin hydrochloride.

6. The use of the compound according to claims 1 to 5, characterized in that: The compound can be used to prepare ionizable or cationic nano preparations.

7. The use of the compound according to claims 1 to 6, characterized in that: The nanoformulation containing the compound has the effects of promoting tumor cell uptake and improving tumor tissue targeting after administration.

8. The use of the compound according to claims 1-3, characterized in that It has the potential to have therapeutic abilities related to alpha-tocopheryl succinate or vitamin E.

9. The compound according to claim 1, characterized in that It can be prepared by the following method: Dissolve α-TOS in an organic solvent such as dichloromethane, and add condensation reagents HATU / EDC, nucleophilic reagents DIPEA / DMAP, and Y-XH in sequence under stirring. Stir at low temperature for a period of time at the beginning of the reaction, and then continue to stir at room temperature for 2-24 hours. After the reaction is completed, add a certain volume of dichloromethane or other organic solvents to the reaction mixture, and wash it with ultrapure water and saturated brine for multiple times, collect the organic phase, remove the solvent, and separate and purify the residue by silica gel column chromatography to obtain the final product. The basic reaction process is:

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