CN104151284B - Synthesis method and application of methacrylamide orthoester monomer and acid-sensitive amphiphilic block copolymer thereof - Google Patents

Abstract

The invention discloses a synthesis method and application of a novel methacrylamide orthoester monomer and an acid-sensitive amphiphilic block copolymer thereof, belonging to the technical field of polymer carriers and slow-release materials. The structure of the new monomer of methacrylamide orthoester is shown in chemical formula I; the side chain synthesized by the new monomer of methacrylamide orthoester contains two orthoester five-membered rings The structure of the sensitive amphiphilic block copolymer is shown in formula II. The micelles formed by the self-assembly of the acid-sensitive amphiphilic block copolymer containing two orthoester five-membered rings in the side chain can be used to deliver the poorly water-soluble drugs (such as anti-tumor paclitaxel) in vivo. The acid-sensitive amphiphilic block copolymer containing two orthoester five-membered rings in the side chain of the present invention has good acid sensitivity, biocompatibility and biodegradability, and has good application in the field of targeted therapy prospect.

Description

Synthesis method and application of a kind of methacrylamide orthoester monomer and acid-sensitive amphiphilic block copolymer thereof

technical field

The invention relates to a synthesis method and application of a novel methacrylamide orthoester monomer and an acid-sensitive amphiphilic block copolymer thereof, and belongs to the technical field of polymer carriers and slow-release materials.

Background technique

Cancer seriously endangers human life and health, and has been listed as the second killer of human beings. For the treatment of tumors, most of the current anti-tumor drugs have obvious deficiencies: the targeting is not obvious, and the treatment process often causes irreversible damage to the normal tissues of the body, reducing the body's immune ability, thereby delaying treatment. Therefore, choosing an appropriate drug carrier has become the key to improving the efficacy of antitumor drugs.

The use of polymeric micelles to deliver anticancer drugs to improve the tumor targeting properties of drugs and overcome drug resistance of tumor cells has aroused extensive research interest. pH-responsive polymer micelles are mainly prepared by the self-assembly of amphiphilic block copolymers to form micelles. The copolymers contain chemical bonds that are rapidly hydrolyzed under acidic conditions, such as hydrazone bonds, glyoxal bonds, and amide bonds. Ester bonds, etc., wherein polymers with orthoester bonds as sensitive groups can now be obtained through fewer steps and simple reactions (Chinese patent CN103588752 Chinese patent CN101880265A), but these polymers degrade slowly in acidic environments, The slow release of the drug after embedding results in a decrease in drug efficacy.

Contents of the invention

The purpose of the present invention is to provide a new monomer of methacrylamide orthoester and its synthesis method and application of acid-sensitive amphiphilic block copolymer.

To achieve the above object, the present invention adopts the following technical solutions:

The structure of the novel monomer of methacrylamide orthoester synthesized by the present invention is shown in chemical formula I:

The synthetic route of methacrylic acid ortho esters novel monomer (chemical formula I) of the present invention is as follows:

In the formula, 1 is 2,2,2-trifluoro-N-{2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3] Dioxolane-4-methyleneoxy)]-ethyl}-acetamide, 2 is 2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy Base-[1,3]dioxolane-4-methyleneoxy)]ethylamine.

The synthetic method of methacrylamide ortho ester novel monomer (chemical formula I) of the present invention comprises the following steps:

(1) Under the action of a catalyst, 4,4'-dimethyloxy-bis-(2-methoxy-1,3-dioxolane) and N-(2-hydroxyethyl)trifluoro Acetamide reaction, the molar ratio of which is 1:1-1.2, was reacted at 130°C for 6h; the reaction system was dissolved in ethyl acetate, washed with aqueous sodium carbonate solution, and dried to obtain pure compound 2,2,2-trifluoro-N- {2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)]-B Base}-acetamide, directly used in next step reaction;

(2) 2,2,2-trifluoro-N-{2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3]di Oxolane-4-methyleneoxy)]-ethyl}-acetamide was dissolved in an organic solvent, added 1.0-2.0M sodium hydroxide solution, stirred overnight at room temperature, extracted with dichloromethane, and dried to obtain pure compound 2 -[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)]ethylamine;

(3) 2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)] Ethylamine, N-succinimide methacrylate and 4-dimethylaminopyridine were dissolved in an organic solvent with a molar ratio of 1:(1-1.1):1, reacted at room temperature in the dark for 24-48 hours, and evaporated to dryness After removing the organic solvent, add ethyl acetate, wash with aqueous sodium carbonate solution, and separate the crude product by silica gel chromatography to obtain the product N-{2-[2-methoxy-[1,3]dioxolane-4- (2-methoxy-[1,3]dioxolane-4-methyleneoxy)]-ethyl}-2-methacrylamide monomer;

The catalyst described in the step (1) is a mixture of pyridinium p-toluenesulfonic acid or p-toluenesulfonic acid and pyridine, the molar ratio of the two is 1:1.1-2, and the mol ratio of the catalyst to the hydroxyl group is 0.001- 0.01:1;

The organic solvent described in step (2) and step (3) is acetonitrile, THF, chloroform, dichloromethane or dioxane;

The silica gel separation eluent described in step (3) is ethyl acetate.

The structure of the acid-sensitive amphiphilic block copolymer with two orthoester five-membered rings in the side chain synthesized by the new monomer of methacrylamide orthoester (chemical formula I) is shown in chemical formula II:

In the formula, X represents a value of 20-120.

The synthetic route of the acid-sensitive amphiphilic block copolymer (chemical formula II) containing two orthoester five-membered rings in the side chain of the present invention is as follows:

The synthesis method of the acid-sensitive amphiphilic block copolymer containing two orthoester five-membered rings in the side chain of the present invention, the specific steps are as follows: the new monomer of methacrylamide orthoester, macromolecular chain transfer agent , Initiator, accurately weighed and put into a dry glass polymerization tube, the molar ratio of the feed is (20-200): 1: (0.1-0.5), the tube is sealed after 30 minutes of degassing with nitrogen, and then the polymerization tube is placed Polymerize in an oil bath at 50-80°C, react for 2-24 hours, dissolve the reactant in an organic solvent, settle the crude product with glacial ether, collect the precipitate by filtration, and dry it in vacuum at room temperature to obtain the target copolymer.

The structure of the macromolecular chain transfer agent is a trithiopolyethylene glycol compound of chemical formula III, and its molecular weight is 5400.

The degree of polymerization of the acid-sensitive amphiphilic block copolymer containing two orthoester five-membered rings in the side chain is 20-120; the organic solvent is tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, chloroform, N, N-dimethylformamide, one of dioxane, benzene, toluene and p-xylene; the initiator is 2,2-azobisisobutyronitrile.

The acid-sensitive amphiphilic block copolymer (chemical formula II) with two orthoester five-membered rings in the side chain of the present invention self-assembles in aqueous solution to form a micelle structure, and the micelle size is different according to the ratio of hydrophilic and hydrophobic blocks. , and load the drug during the self-assembly process.

A polymer micelle pharmaceutical composition, comprising a kind of acid-sensitive amphiphilic block polymer micelle whose side chain contains two orthoester five-membered rings as shown in chemical formula II, and at least one doped Into the active agent in this micelle; Wherein said active agent is selected from anti-inflammatory drug, cancer chemotherapeutic drug, immunosuppressant, metabolic drug, antiallergic drug, liver disease treatment drug, nervous system treatment drug or circulatory system disease treatment drug.

The method for incorporating the active agent into the amphiphilic block polymer micelle includes stirring, heating, ultrasonic wave, solvent evaporation or infiltration treatment.

The cancer chemotherapy drug is selected from paclitaxel, doxorubicin, cyclosporine or carmustine.

The beneficial effect of the present invention is that the synthesized acid-sensitive amphiphilic block copolymer containing two orthoester five-membered rings in the side chain has good acid sensitivity, biocompatibility and biodegradability, and is reduced in acidic environment. The solution is rapid, and the drug embedded in it can be released quickly, so it has a good application prospect in the field of targeted therapy.

Description of drawings

Fig. 1 is N-{2-[2-methoxy-[1,3]dioxolane-4-(2-methoxyl-[1,3]dioxolane-4- ¹ HNMR and ¹³ CNMR spectra of methyleneoxy)]-ethyl}-2-methacrylamide monomer.

Fig. 2 is the pH-dependent hydrolysis change of the orthoester bond of the block copolymer in Example 5.

Figure 3 is the average size distribution of the block copolymer micelles in Example 7.

FIG. 4 shows the pH-dependent size change of block copolymer micelles in Example 7. FIG.

Fig. 5 is the in vitro cytotoxicity test of the block copolymer micelles in Example 8.

Fig. 6 is the in vitro drug release test of the block copolymer micelles in Example 9.

detailed description

Example 1

N-{2-[2-Methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)]- The synthetic method of ethyl}-2-methacrylamide monomer is realized through the following steps:

(1) 2,2,2-Trifluoro-N-{2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3]dioxo Preparation of pentane-4-methyleneoxy)]-ethyl}-acetamide:

Under the action of pyridine-p-toluenesulfonate, 4,4'-dimethyloxy-bis-(2-methoxy-1,3-dioxolane) and N-(2-hydroxyethyl ) trifluoroacetamide reaction, the molar ratio of which is 1:1-1.2, reacted at 130°C for 6h; the reaction system was dissolved in ethyl acetate, washed with 10% sodium carbonate solution, and dried to obtain pure compound 2,2,2- Trifluoro-N-{2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy )]-ethyl}-acetamide, directly used in the next step reaction. ¹ HNMR (400MHz, CDCl ₃ , δ, ppm): 2.0-2.18 (m, NH ₂ -CH ₂ ) 3.27-3.28 (m, 3H, O-CH ₃ ), 3.43-3.710 (m, 6H, CH—

CH ₂ -CH,N-CH ₂ -CH ₂ ),3.68-4.13(m,4H,O-CH _2- CH),4.25-4.40(m,2H,O-CH-CH ₂ ),5.7-5.83( q,2H,O-CH-O).

(2) 2,2,2-Trifluoro-N-{2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3]dioxo Preparation of pentane-4-methyleneoxy)]-ethyl}-acetamide:

2,2,2-trifluoro-N-{2-methoxy-[1,3]dioxolane-2-[4-(2-methoxy-[1,3]dioxolane -4-methyleneoxy)]-ethyl}-acetamide was dissolved in tetrahydrofuran, added 1.0-2.0M sodium hydroxide solution, stirred at room temperature overnight, extracted with dichloromethane, dried to obtain pure compound 2-[2- Methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)]ethanamine. 1HNMR (400MHz, CDCl ₃ ): 1.48(s, CH ₂ -NH ₂ ), 2.86-2.89(m, NH ₂ -CH ₂ ), 3.32-3.33(m, 3H, CH ₃ O), 3.51-3.71(6H ,CH—CH ₂ -CH,N-CH ₂ -CH ₂ ),3.80-4.18(m,4H,O-CH _2- CH),4.32-4.52(m,2H,O-CH-CH ₂ ),5.74 -5.86 (q,2H,O-CH-O).

(3) N-{2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy )]-ethyl}-2-methacrylamide preparation:

2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)]ethylamine, N-succinimide methacrylate and 4-dimethylaminopyridine are dissolved in acetonitrile, the molar ratio is 1:(1-1.1):1, react at room temperature in the dark for 24-48 hours, evaporate to dryness to remove acetonitrile, Add ethyl acetate, wash with sodium carbonate aqueous solution, and separate the crude product by silica gel chromatography (eluent: ethyl acetate) to obtain the product N-{2-[2-methoxy-[1,3]dioxolane Cyclo-4-(2-methoxy-[1,3]dioxolane-4-methyleneoxy)]-ethyl}-2-methacrylamide monomer. ¹ HNMR(400MHz,CDCl ₃ ,δ,ppm):1.97(s,3H,C-CH ₃ ),3.32-3.33(m,3H,O-CH ₃ ),3.48-3.75(m,8H,CH ₂ - O-CH ₂ ,O-CH ₂ -CH ₂ -NH),3.77-4.2(m,4H,CH-O-CH ₂ -CH),4.28-4.51(m,2H,O-CH-CH ₂ ), 5.34-5.36(m,H,C=CH ₂ ), 5.71-5.76(m,2H,CH-(O)3),5.84-5.86(m,H,C=CH ₂ ),6.30-6.38(d, H,CO-NH). ¹³ CNMR(400MHz,CDCl ₃ ,δ,ppm):18.49,42.27,63.13,65.64,66.02,71.62,72.55,74.05,75.02,115.40,115.89,119.81,139.70,168.49.ESI- _MScalcdfor ( _C12H20O5 ), _347.16 ; found m/z, 370.00 (M+Na ⁺ ).

Example 2

1.00g (2.88mmoL) N-{2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4- Methylene oxide)]-ethyl}-2-methacrylamide, polyethylene glycol macroinitiator 0.52g (0.096mmoL), 2,2-azobisisobutylcyanide 3.15mg (0.0192mmoL) is accurate Weigh it into a dry and clean glass polymerization tube, then add N,N-dimethylformamide (2.5mL), degas the polymerization system with nitrogen for 30 minutes, seal the tube, and put the polymerization reaction tube in an oil bath at 70°C Heating and polymerizing, after 24 hours of reaction, the reaction mixture was dissolved in N,N-dimethylformamide, then added dropwise into glacial ether, the sediment was collected, rinsed with a small amount of glacial ether, and the product was vacuum-dried to constant weight to obtain The target copolymer P1, the properties of the polymer are shown in Table 1.

Example 3

1g (2.88mmoL) N-{2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4- Methyloxy)]-ethyl}-2-methacrylamide, polyethylene glycol macroinitiator 0.26g (0.048mmoL), 2,2-azobisisobutyrocyanide 1.58mg (9.6×10 ^-3 mmoL) was accurately weighed into a dry and clean glass polymerization tube, then N,N-dimethylformamide (2.5mL) was added, the polymerization system was degassed with nitrogen for 30 minutes, and then the tube was sealed, and the polymerization reaction tube was placed at 70°C Heated and polymerized in an oil bath. After 24 hours of reaction, the reaction mixture was dissolved in N,N-dimethylformamide, then added dropwise into glacial ether, the sediment was collected, rinsed with a small amount of glacial ether, and the product was vacuum-dried to The target copolymer P2 was obtained by constant weight, and the properties of the polymer are shown in Table 1.

Example 4

1g (2.88mmoL) N-{2-[2-methoxy-[1,3]dioxolane-4-(2-methoxy-[1,3]dioxolane-4- Methyloxy)]-ethyl}-2-methacrylamide, polyethylene glycol macroinitiator 0.17g (0.032mmoL), 2,2-azobisisobutylcyanide 1.05mg (6.4×10 ^-3 mmoL) was accurately weighed into a dry and clean glass polymerization tube, then N,N-dimethylformamide (2.5mL) was added, the polymerization system was degassed with nitrogen for 30 minutes, and then the tube was sealed, and the polymerization reaction tube was placed at 70°C Heated and polymerized in an oil bath. After 24 hours of reaction, the reaction mixture was dissolved in N,N-dimethylformamide, then added dropwise into glacial ether, the sediment was collected, rinsed with a small amount of glacial ether, and the product was vacuum-dried to The target copolymer P3 was obtained by constant weight, and the properties of the polymer are shown in Table 1.

The property characterization of block copolymer P1-P3 in the embodiment 2,3,4 of table 1

a: measured by GPC; b: calculated by 1HNMR

Example 5

Determination of pH-Dependent Degradation of Polymers:

Copolymer P2 was prepared into a 5 mg/mL micellar solution in deuterated phosphate buffer solution with pH = 7.4, 6, 5, and 4 respectively, and ¹ HNMR was measured within the set time of the nuclear magnetic resonance instrument (ADVANCE400). The orthoester peak at -5.82 is a characteristic peak, and 8.14 and 8.19 are detection peaks after hydrolysis. Observe the hydrolysis rate of orthoester, as shown in Figure 2. It can be seen from the figure that the hydrolysis rate of the corresponding orthoester has a certain relationship with its size change, and the size change depends on the hydrolysis rate of the orthoester.

Example 6

Preparation of block copolymer micelles:

Dissolve 100 mg of block copolymers P1-P3 in 1 mL DMSO (with a small amount of triethylamine), then add 10 mL of 50 mM pH=7.4 phosphate buffer solution dropwise with stirring, stir vigorously for 8 hours after the addition, and transfer the solution to a molecular weight cut-off of 3500 In the dialysis bag, water (with a small amount of triethylamine) was used as the dialysate for dialysis for 48 hours, and then the solution in the bag was transferred to a 50 mL centrifuge tube, and freeze-dried to obtain block copolymer micelles.

Example 7

Polymer micelle size measurement and determination of pH-dependent size change:

The copolymer micelles P1-P3 were prepared into a 2 mg/mL micellar solution in phosphate buffer solution with pH=7.4, 4 respectively, and the solution was passed through a 0.45 micron filter head, and each solution was dissolved in Mal The particle size analyzer (MalvernZetasizerNanoZS) measured the size change of the copolymer micelles, with pH=7.4, the size at 0 hours was the initial size, and the average sizes of P1-P3 were 166, 180, and 192 nm, respectively (Figure 3). It can be seen from Figure 4 that the size of the P2 micelles is close to the size of the polyethylene glycol macroinitiator at pH = 4 and in an acidic environment for 48 hours. It is speculated that the micellar structure is completely disintegrated after the orthoester is degraded. .

Example 8

Cytotoxicity test:

NIH3T3 cells (10,000 cells/well) were inoculated in a 96-well culture plate and cultured at 37° C. and 5% carbon dioxide for 24 hours, and the cell confluency was 60-80%. The copolymer solution was added to the above-mentioned 96-well plate that had been inoculated and cultured with NIH3T3 cells, and the culture was continued for 24 hours. Add 20 microliters of MTT (5mg/mL) to each well, aspirate the medium after 4 hours, add 150 microliters of DMSO to each well, shake for 10 minutes, and measure the A value in a microplate reader (ThermoScientific multifunctional microplate reader) at 570nm . The cell survival rate was calculated according to the following formula: cell survival rate (%)=(A _sample -A _blank )/(A _control -A _blank )×100%. It can be seen from Figure 5 that compared with the control group, the survival rate of NIH3T3 cells is 90% even at a higher concentration of 5 mg/mL, which proves that the copolymer has almost no cytotoxicity.

Example 9

Dissolve 30 mg of the copolymer P1 obtained in Example 2 and 10 mg of the hydrophobic drug model drug Nile Red in 1 mL of dimethyl sulfoxide (with a small amount of triethylamine), stir to dissolve and mix well, then slowly add 10 times the volume of phosphoric acid Salt buffer solution, after the dropwise addition, continue to stir for 8-24 hours, centrifuge the solution, transfer the supernatant to a dialysis bag with a molecular weight cut-off of 3500 and dialyze for 2 days, freeze-dry the solution in the dialysis bag, and obtain the gel loaded with the model drug Nile Red bundle. Take 10 mg of each of the micelles, dissolve them in 5 mL of 50 mM phosphate buffer with pH=7.4 and pH=5, transfer to a dialysis tube with a molecular weight cut-off of 20,000, and dialyze in 300 mL of the corresponding buffer. The amount of Nile Red released from the micelles was measured at predetermined times. It can be seen from Figure 6 that the loaded model drug Nile Red is released faster in a slightly acidic environment at pH=5, and only half of it can be released in 11 hours. At 48 hours, due to the disintegration of the micelle structure, Nile Red Almost completely released.

Claims (9)

1. a Methacrylamide ortho acid esters monomer, is characterized in that, its structure is as shown in chemical formula I:

2. contain two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters by the synthetic side chain of Methacrylamide ortho acid esters monomer claimed in claim 1, it is characterized in that, its structure is as shown in chemical formula II:

In formula, X represents numerical value 20-120.

3. a side chain as claimed in claim 2 is containing the synthetic method of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters, it is characterized in that, concrete steps are as follows: by Methacrylamide ortho acid esters monomer, macromolecular chain transfer agent, initator, accurately weigh and put into dry glass polymerization pipe, its molar ratio is (20-200): 1:(0.1-0.5), logical nitrogen tube sealing after degassed 30 minutes, then polymerization pipe is placed into polymerization in the oil bath of 50-80 DEG C, react after 2-24 hour, by reactants dissolved in organic solvent, ice ether sedimentation for crude product, filter collecting precipitation, under room temperature, vacuum drying obtains target copolymer.

4. side chain according to claim 3 is containing the synthetic method of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters, it is characterized in that, described macromolecular chain transfer agent structure is three sulfo-polyethylene glycols compounds of chemical formula III, and its molecular weight is 5400;

5. side chain according to claim 3, containing the synthetic method of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters, is characterized in that, described side chain is 20-120 containing the degree of polymerization of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters; Described organic solvent is oxolane, methyl alcohol, ethanol, acetonitrile, acetone, chloroform, DMF, the one in dioxane, benzene, toluene, paraxylene; Described initator is 2,2-azodiisobutyronitrile.

6. a side chain as claimed in claim 2 is containing the application of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters, it is characterized in that, described side chain forms micellar structure containing the self assembly in the aqueous solution of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters, cause micella size difference according to close hydrophobic block ratio difference, and in self assembling process carrying medicament.

7. a polymeric micelle medicine composition, is characterized in that, comprise the micella of a kind of side chain as claimed in claim 2 containing two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters, and at least one is incorporated in the activating agent in this micella; Wherein said activating agent is selected from anti-inflammatory agent, cancer chemotherapeutic drug, immunodepressant, metabolic drug, Claritin, hepatosis treating medicine, nervous system medicine or circulation system disease medicine.

8. polymeric micelle medicine composition according to claim 7, is characterized in that, described cancer chemotherapeutic drug is selected from taxol, adriamycin, cyclosporin or BCNU.

9. polymeric micelle medicine composition according to claim 7, it is characterized in that, described activating agent is incorporated into side chain and comprises stirring, heating, ultrasonic wave, solvent evaporation or infiltration processing containing the method in the micella of two pentacyclic acid-sensitive amphipathic segmented copolymers of ortho esters.