Polymer prodrug compound and preparation method thereof, polymer prodrug micelle and preparation method thereof

Abstract

The present invention relates to the technical field of polymer drugs, and in particular, to polymer prodrug compounds and preparation methods thereof, and polymer prodrug micelles and preparation methods thereof. The structural formula of the polymer prodrug compound is as follows: The polymer prodrug compound achieves effective tumor targeting, prolongs the circulation time, increases the accumulation of drugs in tumor cells, achieves precise targeting of drugs to tumor cells, and increases the controllable release of drugs in tumor tissues, avoiding the problem that polymer drugs formed by physical encapsulation are easily leaked during the circulation process in the organism.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer drugs, in particular to polymer prodrug compounds and preparation methods thereof, and polymer prodrug micelles and preparation methods thereof. Background Art

[0002] Cancer has become a disease that seriously endangers the lives and health of people around the world, with high morbidity and mortality rates. Chemotherapy is still the most widely used treatment method, but traditional anti-tumor drugs lack selectivity. While killing cancer cells, they also damage normal tissue cells, causing multiple complications, which limits the clinical application and development of anti-tumor drugs. At present, nanoparticle drug delivery carriers have made great progress in cancer treatment, especially polymer prodrug micelles. These polymer prodrug micelles can passively accumulate in tumor tissues, have high stability and can prolong the circulation time of drugs in the blood. In recent years, the main potential mechanism of cancer treatment based on nanomedicine is nonspecific passive targeted therapy related to the enhanced permeability and retention (EPR) effect. However, more and more studies have shown that although the EPR effect exists in animals such as mice, it plays a less important role in humans due to the heterogeneity of tumors or the lack of fenestrations in tumor vasculature. Specific active targeting of ligand-receptor recognition can show better efficacy than passive targeting in human cancer treatment, but since active targeting is based on the recognition and binding of ligands to tumor cell surface receptors, its targeting effect will be affected by receptor expression. However, the receptors (surface markers) of tumor cells show dynamic changes as the tumor develops. In addition, ligand-receptor binding is a saturable process because the recycling and synthesis of receptors takes time. These factors will affect the drug delivery efficiency of single drugs and nanomedicines, thereby reducing the efficacy of the drug. Therefore, compared with single ligand targeting, which may lead to uncontrollable and fluctuating drug delivery efficiency, the dual ligand strategy can have better cell selectivity and higher cell uptake rate in cancer treatment. At the same time, different effects can be achieved according to different ligand combinations. This is because the combination of two targeting ligands can improve the selectivity and uptake of nanomedicines by specific tumor cells, provide the possibility of targeting different cells involved in tumor development, and even achieve specific targeting of cells and organs at the same time, so as to achieve the precise delivery of anticancer drugs to their intracellular therapeutic active sites, thereby improving their anti-tumor efficacy. In addition, targeting ligands can also be combined with cell penetrating peptides (CPPs) to further enhance the uptake of nanomedicines by specific tumor cells. Therefore, it is very necessary to develop environmentally responsive specific dual-targeted prodrug micelles.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a polymer prodrug compound and a preparation method thereof, and a polymer prodrug micelle and a preparation method thereof. The polymer prodrug compound provided in the embodiment of the present invention achieves effective tumor targeting, prolongs the circulation time, increases the accumulation of drugs in tumor cells, achieves precise targeting of drugs to tumor cells, and increases the controllable release of drugs in tumor tissues, avoiding the problem that polymer drugs formed by physical encapsulation are easily leaked during the circulation process in the organism.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a polymer prodrug compound, the structural formula of which is shown below:

[0007] Wherein, A is selected from any one of a polyethylene glycol group or a derivative group thereof, a composite group or a composite long-chain group containing both an alkenyl group and a carbonyl group, a composite group or a composite long-chain group containing both a carbonyl group and a phenyl group, a composite group or a composite long-chain group containing both an amine group and a carbonyl group, a composite long-chain group containing both a carbonyl group and an ether group, a composite group or a composite long-chain group containing both an amino acid residue and a carbonyl group, a composite group or a composite long-chain group containing both at least one of carbonyl, alkenyl, phenyl, amine, ether and amino acid residue and Su, and a composite group or a composite long-chain group containing only a carbonyl group and an alkylene group;

[0008] B is selected from any one of a sulfur-containing group, a selenium-containing group, an amino acid residue-containing group and an amine-containing group;

[0009] Y is selected from N, O or C;

[0010] U is selected from anti-tumor compounds;

[0011] m is 8-17.

[0012] In a second aspect, the present invention provides a method for preparing the polymer prodrug compound described in the aforementioned embodiment, comprising:

[0013] Reacting folic acid with hyaluronic acid to form intermediate 1;

[0014] The antitumor compound is reacted with a substance containing a B group, and then reacted with a compound containing an A group to form an intermediate 2;

[0015] The intermediate 1 and the intermediate 2 react to form the polymer prodrug compound.

[0016] In a third aspect, the present invention provides a polymer prodrug micelle, which is prepared by the polymer prodrug compound described in the aforementioned embodiment.

[0017] In a fourth aspect, the present invention provides a method for preparing the polymer prodrug micelles described in the aforementioned embodiment, comprising: using the polymer prodrug compound described in the aforementioned embodiment to form the polymer prodrug micelles.

[0018] In a fifth aspect, the present invention provides an anti-tumor agent, which includes the polymer prodrug compound described in the aforementioned embodiment or the polymer prodrug micelle described in the aforementioned embodiment.

[0019] The present invention has the following beneficial effects: the polymer prodrug compound of the embodiment of the present invention is based on hyaluronic acid, with environmental response sensitive bonds and polyethylene glycol chains or amino acid residue chains or pure carbon chains as bridges for hyaluronic acid and anti-tumor compounds, and the other side of the hyaluronic acid is connected to folic acid, which can make up for the tumor targeting ability of single hyaluronic acid or folic acid modified polymer prodrugs and the lack of controllable and target administration efficiency, and through the action and EPR effect of hyaluronic acid and folic acid targeting CD44 or FR receptors on the surface of tumor cells, the cumulative amount of drugs in tumor cells is increased, and the off-target amount of anti-tumor compounds is reduced, so as to achieve the effect of accurately and efficiently killing tumor cells. At the same time, when the polymer prodrug compound forms polymer prodrug micelles, the anti-tumor compound is wrapped inside the micelles without showing toxic side effects, and the anti-tumor compound is actively released under conditions of high glutathione concentration, weak acidity, etc. under the conditions of tumor microenvironment, to achieve the controlled release of drugs and the effect of accurately killing tumor cells. And the polymer prodrug micelle avoids the problem that the polymer drug formed by physical encapsulation is easily leaked during the circulation process in the organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The H-NMR spectrum of compound 8 provided in Example 1 of the present invention;

[0022] Figure 2 The H-NMR spectrum of compound 9 provided in Example 1 of the present invention;

[0023] Figure 3 The H-NMR spectrum of compound 15 provided in Example 2 of the present invention;

[0024] Figure 4 The C-NMR spectrum of compound 15 provided in Example 2 of the present invention;

[0025] Figure 5 The H-NMR spectrum of compound 16 provided in Example 2 of the present invention;

[0026] Figure 6 The H-NMR spectrum of compound 20 provided in Example 3 of the present invention;

[0027] Figure 7 The C-NMR spectrum of compound 20 provided in Example 3 of the present invention;

[0028] Figure 8 The H-NMR spectrum of compound 21 provided in Example 3 of the present invention;

[0029] Fig. 9 The H-NMR spectrum of compound 25 provided in Example 4 of the present invention;

[0030] Fig.10 The C-NMR spectrum of compound 25 provided in Example 4 of the present invention;

[0031] Fig.11 The H-NMR spectrum of compound 26 provided in Example 4 of the present invention;

[0032] Fig.12 UV spectra of the FA-HA-SS-PPT and FA-HA-CC-PPT prodrug compounds provided in Examples 1 and 2 of the present invention;

[0033] Fig.13 The infrared spectra of the FA-HA-SS-PPT and FA-HA-CC-PPT prodrug compounds provided in Examples 1 and 2 of the present invention;

[0034] Fig.14 The particle size diagram of FA-HA-SS-PPT and FA-HA-CC-PPTT prodrug micelles provided in Examples 1 and 2 of the present invention;

[0035] Fig.15 Transmission electron microscopy images of FA-HA-SS-PPT and FA-HA-CC-PPTT prodrug micelles provided in Examples 1 and 2 of the present invention;

[0036] Fig.16 The prodrug micelle drug release curves of FA-HA-SS-PPT and FA-HA-CC-PPT provided in Examples 1 and 2 of the present invention;

[0037] Fig.17 Hemolytic test evaluation of the prodrug micelles of FA-HA-SS-PPT and FA-HA-CC-PPT provided in Examples 1 and 2 of the present invention;

[0038] Fig.18The targeting evaluation of the FA-HA-SS-PPT prodrug micelles provided in Example 1 of the present invention;

[0039] Fig.19 This is the in vitro cytotoxicity inhibition rate curve of the FA-HA-SS-PPT prodrug micelles provided in Example 1 of the present invention;

[0040] Fig. 20 This is a graph showing the in vitro cell proliferation inhibition effect of the FA-HA-SS-PPT prodrug micelles provided in Example 1 of the present invention;

[0041] Fig.21 This is a diagram showing the in vitro cell apoptosis effect of the FA-HA-SS-PPT prodrug micelles provided in Example 1 of the present invention;

[0042] Fig. 22 This is a diagram of the in vivo anti-tumor effect of the FA-HA-SS-PPT prodrug micelles provided in Example 1 of the present invention;

[0043] Fig.23 A particle size diagram for screening the preparation conditions of polymer prodrug micelles provided in an embodiment of the present invention;

[0044] Fig.24 The H-NMR spectrum of compound 29 provided in Example 5 of the present invention;

[0045] Fig.25 The H-NMR spectrum of compound 33 provided in Comparative Example 1 of the present invention;

[0046] Fig.26 The H-NMR spectrum of compound 35 provided in Comparative Example 2 of the present invention;

[0047] Fig. 27 The H-NMR spectrum of compound 7 provided in Example 1 of the present invention;

[0048] Fig.28 The C-NMR spectrum of compound 7 provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] The serious toxic side effects caused by fat-soluble anti-tumor compounds due to their poor water solubility, poor selectivity, low bioavailability and other problems limit the application of fat-soluble anti-tumor compounds in tumor treatment.

[0051] Polymer prodrug (polyprodrug) usually refers to a macromolecular prodrug formed by connecting a drug to a polymer carrier by chemical bonds, or by the drug itself through polymerization reaction. Such polymer prodrugs can usually self-assemble into nanoparticles in aqueous solution, and can use the EPR effect to achieve passive targeting of drugs. At the same time, the chemical bonds connecting the drug to the carrier are usually responsive to tumor microenvironment stimulation and can be broken, such as disulfide bonds and trisulfide bonds that can respond to high levels of glutathione (GSH) in tumor cells, carbonate bonds and hydrazone bonds that can respond to low pH in the tumor microenvironment, so as to achieve active targeted release and activation of drugs in tumor tissues. Compared with the polymer micelles formed by the currently commonly used physical embedding, polymer prodrugs closely connect different drugs and polymer carriers into a whole through chemical response bonds, which gives them tumor microenvironment responsiveness and enhances the stability and controllability of drugs in the body. With the above advantages, polymer prodrugs are used to solve the existing defects of anti-tumor drugs.

[0052] Hyaluronic acid (HA) is a natural macromolecular acidic mucopolysaccharide formed by repeated alternating connections of disaccharide units consisting of D-glucuronic acid and N-acetylglucosamine. It can specifically bind to the CD44 receptor overexpressed on the surface of tumor cells. It has good biocompatibility, biodegradability, receptor binding, and is non-immunogenic and non-toxic. At the same time, there are multiple active groups in its structure, which can be physically or chemically modified to prepare derivatives with different properties. It can be used as a relatively ideal drug delivery carrier and tumor targeting ligand.

[0053] Folic acid (FA), a water-soluble B vitamin, is also known as vitamin B9 or pteroylglutamate. It is a substance necessary for the survival of all living cells, especially some cells that grow and reproduce rapidly, and plays a very important role in the DNA replication process. Since folate receptors are overexpressed on the surface of tumor cells, folic acid can be targeted into tumor cells through folate receptor-mediated endocytosis. Therefore, using folic acid as a targeting ligand can deliver a variety of drug payloads to folate receptor-positive cells, thereby enhancing the cell's uptake of drugs.

[0054] Therefore, an embodiment of the present invention provides a polymer prodrug compound, the structural formula of which is shown below:

[0055] Wherein, A is selected from any one of a polyethylene glycol group or a derivative group thereof, a composite group or a composite long-chain group containing both an alkenyl group and a carbonyl group, a composite group or a composite long-chain group containing both a carbonyl group and a phenyl group, a composite group or a composite long-chain group containing both an amine group and a carbonyl group, a composite long-chain group containing both a carbonyl group and an ether group, a composite group or a composite long-chain group containing both an amino acid residue and a carbonyl group, a composite group or a composite long-chain group containing both a carbonyl group, an alkenyl group, a phenyl group, an amine group, an ether group and an amino acid residue and Su, and a composite group or a composite long-chain group containing only a carbonyl group and an alkylene group; B is selected from any one of a sulfur-containing group, a selenium-containing group, an amino acid residue-containing group and an amine-containing group; Y is selected from N, O or C; U is selected from an antitumor compound; m is 8-17, for example, m is any integer between 8-17 such as 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

[0056] It can be seen that the polymer prodrug compound provided by the embodiment of the present invention is mainly based on hyaluronic acid, and one side is connected with folic acid, which makes up for the tumor targeting ability of single hyaluronic acid or folic acid modified polymer prodrugs and the problem of uncontrollable and insufficient target administration efficiency, and makes the polymer prodrug compound target the action and EPR effect of CD44 or FR receptor on the surface of tumor cells through hyaluronic acid and folic acid, increases the cumulative amount of drugs in tumor cells, reduces the off-target amount of anti-tumor compounds, and achieves the effect of accurately and efficiently killing tumor cells. Using different environmental responsiveness (pH sensitive, enzyme sensitive, glutathione sensitive, etc.) connecting bonds as bridges can control the release of drugs by responding to low pH and high concentrations of glutathione in the tumor microenvironment, thereby achieving efficient drug delivery and enhanced anti-tumor effects. At the same time, the polymer prodrug compound solves the problems of poor water solubility of fat-soluble anti-tumor drugs, large toxic and side effects, and single ligand targeting that may lead to uncontrollable and fluctuating target administration efficiency. It can be seen that polymer prodrugs have the characteristics of specific dual targeting, controlled release of drugs, good biocompatibility, and precise targeting of tumors.

[0057] At the same time, when the polymer prodrug compound self-assembles to form micelles, the lipophilic anti-tumor compound will be encapsulated inside the micelles without showing toxic side effects. Under the conditions of high glutathione concentration and weak acidity in the tumor microenvironment, the anti-tumor compound will be actively released to achieve controlled release of the drug and precise killing of tumor cells. In addition, the micelles formed by this single prodrug compound avoid the problem of easy leakage of polymer drugs formed by physical encapsulation during the circulation process in the body.

[0058] Further, A is selected from -CH2-(OCH2)n-CH2-, -CO-(CH2)n-CO-, -CH=CH-CO-, -CH=CH-(CH2)n-CO-, -CH(OH)-Ph-CO-, -CH(OH)- Ph-(CH2)n-CO-, -CH2-Ph-(CH2)n-CO-, --CH(OCH3)-Ph-CO-, -CH(OCH3)-Ph-(CH2)n-CO-, -CO-NH-CO-, -CO-NH-( CH2)n-CO-, -CH2-CH=CH-CO-, -CH2-CH=CH-(CH2)n-CO-, -CO-(CH2)n-, -(CH2)n-CO-, -CO-O-(CH2)n-CO-, -SO2- Ph-CO-, -SO2-Ph-(CH2)n-CO-, -CO-O-PAB-Cit-Val-CO-(CH2)n-CO-, -CO-O-PAB-Lys-Phe-CO-(CH2)n-CO-, -CO- (CH2)n--Su-, -CH=CH-Su-, -CH=CH-(CH2)n-Su-, -CH(OH)-Ph-Su-, -CH(OH)-Ph-(CH2)n-Su-, -CH2-Ph-(CH2)n- Su-, -CO-NH-Su-, -CO-NH-(CH2)n-Su-, -CH2-CH=CH-Su-, -CH2-CH=CH-(CH2)n-Su-, -CO-(CH2)n-, -(CH2)n-Su-, Any one of -CO-O-(CH2)n-Su-, -SO2-Ph-Su-, -SO2-Ph-(CH2)n-Su-, -CO-O-PAB-Cit-Val-CO-(CH2)n-Su-, -CO-O-PAB-Lys-Phe-CO-(CH2)n-Su-, wherein 1≤n≤14; preferably 1≤n≤7; for example, n is any value between 1-14 such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.

[0059] And (CH2)n in A also contains straight-chain or branched saturated alkyl, unsaturated alkyl, aralkyl and alkylaralkyl, aromatic hydrocarbon, halogen, heteroatom group, heterocyclic substituent replacing one or more hydrogen in (CH2)n, the saturated alkyl includes but is not limited to methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl, hexyl or heptyl; the unsaturated alkyl includes but is not limited to vinyl, 1-propenyl, allyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, or its E, Z isomers, ethynyl, propynyl, 2-butynyl; examples of aralkyl and alkylaralkyl include but are not limited to benzyl, diphenylmethyl, tolylmethyl, triphenylmethyl, Cinnamyl, phenethyl, styryl, phenylbutyl, neophenyl, phenylalkynyl, phenylethynyl; the aromatic group includes but is not limited to phenyl, diphenyl, tolyl, benzyl, 2,4,6-trimethylphenyl, (tert-butyl) phenyl, anthracenyl, indenyl, naphthyl, halogenated aryl, halogenated arylalkyl phenoxy, tolyloxy, xylyloxy, diphenyl, anilino, toluamino, toluenesulfonyl, allylbenzyl or phenyl, furanyl, pyridyl, 2-pyridyl (pyridin-2-yl), indol-1-yl, chloromethylbenzyl or phenyl, trifluoromethylbenzyl or phenyl, hydroxybenzyl or phenyl, methoxybenzyl or phenyl, ethoxybenzyl or phenyl, methoxyethoxybenzyl or phenyl, allyloxybenzyl or phenyl, phenoxybenzyl or phenyl, acetoxybenzyl or phenyl, benzoyloxybenzyl or phenyl, methylthiobenzyl or phenyl, phenylthiobenzyl or phenyl, tolylthiobenzyl or phenyl, methylaminobenzyl or phenyl, dimethylaminobenzyl or phenyl, ethylaminobenzyl or phenyl, diethylaminobenzyl or phenyl, acetylaminobenzyl or phenyl, carboxybenzyl or phenyl, methoxycarbonylbenzyl or phenyl, ethoxycarbonylbenzyl or phenyl, phenoxycarbonylbenzyl or phenyl, chlorophenoxycarbonylbenzyl or phenyl, N-cyclohexylcarbamoyloxybenzyl or phenyl, allyloxycarbonylbenzyl or phenyl, carbamoylbenzyl or phenyl, N-methylcarbamoylbenzyl or phenyl, N,N-dipropylcarbamoylbenzyl or phenyl, N-phenylcarbamoylbenzyl or phenyl The halogen includes fluorine, chlorine, bromine or iodine; the heteroatom includes but is not limited to methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, methylsulfinyl, ethylsulfinyl, isopropylsulfinyl, methylsulfonyl, ethylsulfonyl, isopropylsulfonyl; the heterocycle includes but is not limited to pyridyl, quinolyl, thienyl, furanyl, oxazolyl, tetrazolyl, thiazolyl, imidazolyl, pyrazolyl or indolyl.

[0060] B is selected from any one of -SSS-, -SS-, -S-, -Se-Se-, -Se-, -C=N-NH- and -CO-O-PAB-Cit-Val-CO-(CH2)n--CO-.

[0061] U is selected from existing fat-soluble anti-tumor compounds, for example, including but not limited to any one of podophyllotoxin, hydroxycamptothecin, paclitaxel, vinblastine, norvinblastine, methotrexate and pemetrexed.

[0062] The present embodiment uses podophyllotoxin as the anti-tumor compound, but it is understandable that the polymer prodrug compound of the above structure formed by other anti-tumor compounds is also within the protection scope of the present embodiment.

[0063] Specifically, it is selected from the compounds represented by the following structural formula:

[0064] Among them, the selection of A, B, Y and m refers to the selection of A, B and Y mentioned above.

[0065] Furthermore, the polymer prodrug compound is selected from any one of the compounds represented by the following structural formulas:

[0066]

[0067] wherein n is 1-14, preferably 7-14, for example, n is any value between 1-14, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14. m is 8-17, for example, any value between 8-17, such as 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

[0068] Specifically, FA-HA-CC-PPT can controllably release drugs in response to the low pH in the tumor microenvironment, and FA-HA-SS-PPT can controllably release drugs in response to the low pH and high concentration of glutathione in the tumor microenvironment, thereby achieving efficient drug delivery and enhanced anti-tumor effects.

[0069] In a second aspect, an embodiment of the present invention provides a method for preparing the above-mentioned polymer prodrug compound. The embodiment of the present invention uses hyaluronic acid, folic acid and an anti-tumor compound; for example, podophyllotoxin as raw materials, firstly connects folic acid and hyaluronic acid by chemical bonding to form a FA-HA conjugate with CD44 / FR dual targeting, and then modifies the anti-tumor compound by a tumor environment responsive linker and connects it to FA-HA by an amide bond to obtain an environmentally responsive CD44 / FR dual-targeting podophyllotoxin nanoprodrug compound, specifically comprising: synthesizing with reference to the following synthesis path:

[0070]

[0071] Reacting folic acid with hyaluronic acid to form intermediate 1;

[0072] The antitumor compound is reacted with a substance containing a B group, and then reacted with a compound containing an A group to form an intermediate 2;

[0073] The intermediate 1 and the intermediate 2 react to form the polymer prodrug compound.

[0074] In the preparation method of the polymer prodrug compound of the embodiment of the present invention, the environmental response sensitive bond and the polyethylene glycol chain (i.e., the compound containing the A group) are first bonded to the anti-tumor compound and then bonded to the hyaluronic acid. If the environmental response sensitive bond and the polyethylene glycol chain (i.e., the compound containing the A group) are first bonded to the folic acid-modified hyaluronic acid and then bonded to the anti-tumor drug, the anti-tumor drug cannot be grafted successfully, that is, the polymer prodrug compound cannot be successfully obtained.

[0075] For example, please refer to the following synthesis path diagram:

[0076]

[0077] Wherein Y=C or O, A=S or C, B=-COO- or -COO-CH2-CH2-COO-

[0078] Specifically, the step of forming intermediate 1 includes: mixing folic acid, CDI and DMSO for reaction, and then mixing with hyaluronic acid, DMAP and amide solution for reaction for 24-26 hours; and dialyzing after the reaction. The selection of condensing agent in the preparation process of intermediate 1 provided by the embodiment of the present invention has a great influence on the purity of the product. If DCC and EDCI are used as condensing agents, the reaction rate of folic acid is low, and folic acid solids and by-products will be precipitated. The DCC by-product is not easy to be dialyzed and removed. When CDI is used as a condensing agent, the folic acid reaction can reach about 80%, and there is no folic acid precipitation.

[0079] The step of forming the polymer prodrug compound comprises: mixing the intermediate 1 and the amide solution, and then reacting with the intermediate 2, DMSO, 4-PPy, CDI and DMAP at 40-45° C. for 48-52 hours. The reaction temperature in this step is not easy to be too high. If the reaction temperature is too high, such as 60° C., folic acid and the anti-tumor compound prodrug may be broken, resulting in a decrease in the grafting rate of the anti-tumor compound.

[0080] In a third aspect, an embodiment of the present invention provides a polymer prodrug micelle, which is prepared by using the polymer prodrug compound described above. The polymer prodrug micelle has a small and uniform particle size.

[0081] In a fourth aspect, an embodiment of the present invention provides a method for preparing polymer prodrug micelles, comprising: using the above-mentioned polymer prodrug compound to form the polymer prodrug micelles. The polymer prodrug compound self-assembles in an aqueous solution to form micelles with a small particle size (within 100 nm) and uniform dispersion (PDI is below 0.3), further improving the water solubility and biocompatibility of the anti-tumor drug.

[0082] Specifically, the polymer prodrug compound and water are mixed, then ultrasonicated, and then allowed to stand; preferably, 2.5 mg of the polymer prodrug compound corresponds to 2.2-2.5 ml of water, and the ultrasonication time is 5-6 minutes.

[0083] In the embodiments of the present invention, if ultrasound is not used, the polymer micelles obtained generally have a larger particle size. After ultrasonic crushing, the particle size becomes smaller, but as the power increases and the time increases, the particle size becomes larger again. This may be due to the aggregation of the micelles and the breaking of the micelles. Therefore, the above-mentioned ultrasound time and power are selected to obtain a suitable particle size of the polymer prodrug micelles.

[0084] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0085] Example 1

[0086] The present invention provides a method for preparing polymer prodrug micelles, comprising:

[0087] The polymer prodrug compound (denoted as FA-HA-SS-PPT) was synthesized according to the following synthetic route:

[0088]

[0089] The details are as follows:

[0090] Synthesis of compound 1: Under room temperature, take mercaptoethanol (500mg, 6.40mmol, 1eq) and dithiodipyridine (705mg, 3.20mmol, 0.5eq) and dissolve them in 50ml of anhydrous methanol, and add 1.5ml of acetic acid drop by drop. The solution changes from colorless to yellow and reacts at room temperature for 8h. Spot plate and LC-MS detection show that the reaction is no longer in progress. The methanol is dried, dissolved in dichloromethane and washed with saturated sodium bicarbonate solution, and the organic phases are combined. The organic phase is dried with anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which is purified by silica gel column to obtain a colorless liquid compound 1. HRMS (ESI) m / z caled for C4H 10 O2S2Na[M+Na] + 177.0019, found 177.0013.

[0091] Synthesis of compound 2: Under nitrogen conditions, tert-butyldimethylsilyl chloride (3.51 g, 23.29 mmol, 1.2 eq) was added dropwise to a mixed solution of compound 1 (3.00 g, 19.45 mmol, 1 eq) and imidazole (2.80 g, 41.23 mmol, 2.1 eq) in THF (50 mL), and stirred for 4 hours in an ice bath. Then the reaction was quenched with water, extracted three times with dichloromethane, and the organic phase was collected. The organic phase was dried with anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by silica gel column to obtain the target compound 2 as a colorless liquid. HRMS (ESI) m / z caled for C 10 H 24 O2S2SiNa[M+Na] + :291.0887 found 291.0882.

[0092] Synthesis of compound 3: Under nitrogen conditions, N,N'-diisopropylethylamine (DIPEA) (2.51g, 19.40mmol, 2eq) and compound 3 (2.60g, 9.70mmol, 1eq) were dissolved in a round-bottom flask with dry dichloromethane (80mL). Then, a solution of bis(p-nitrobenzene) carbonate (5.90g, 19.40mmol, 2eq) dissolved in dichloromethane was slowly dripped into the above mixed solution and stirred at room temperature overnight. The reaction was completed by spot plate and LC-MS detection, the solution was washed with aqueous hydrochloric acid solution (0.2mol / L, 20mL), the aqueous layer was extracted three times with dichloromethane (30mL), the organic phases were combined, the solvent was removed under reduced pressure, and the crude product was concentrated. The crude product was purified by silica gel column to obtain the target compound 3 as a light yellow liquid. HRMS (ESI) m / z caled for C 17 H 27 NO6S2SiNa[M+Na] + :456.0949,found 456.0944.

[0093] Synthesis of compound 4: Under nitrogen conditions, 4-dimethylaminopyridine (1.48 g, 12.08 mmol, 2 eq) was dissolved in dichloromethane in a round-bottom flask, and then compound 3 (3.00 g, 6.03 mmol, 1.5 eq) and podophyllotoxin (2.50 g, 5.78 mmol, 1 eq) were slowly added to the round-bottom flask at the same time with a syringe, and stirred overnight at room temperature. The reaction was completed by spot plate and LC-MS detection, and the reaction was quenched with aqueous hydrochloric acid (0.2 mol / L, 20 mL×2) to separate the organic phase. The aqueous layer was extracted twice with dichloromethane (30 mL), and the organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 4 as a white solid. HRMS (ESI) m / z caled for C 33 H 44 O 11 S2SiNa[M+Na] + :713.1994,found 731.1989.

[0094] Synthesis of compound 5: Compound 4 (2.96 g, 4.17 mmol, 1 eq) was dissolved in a round-bottom flask with distilled THF (10 mL), and then Et3N·3HF (3.37 g, 20.88 mmol, 5 eq) was added dropwise to the round-bottom flask under ice bath conditions and stirred at room temperature for 5-6 hours. The reaction was detected by spot plate and LC-MS. After THF was dried, it was dissolved in dichloromethane and washed with water. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 5 as a white solid. HRMS (ESI) m / z caled for C 27 H 30 O 11 S2Na[M+Na] + :617.1130, found 617.1119.

[0095] Synthesis of Compound 6 HOOC-PEG8-NH2-FMOC (200 mg, 0.03 mmol, 1 eq) and compound 5 (200 mg, 0.33 mmol, 1.1 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (115 mg, 0.61 mmol, 2 eq) and DMAP (18 mg, 0.15 mmol, 0.5 eq) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 6 as a white solid. HRMS (ESI) m / z caled for C 61 H 77 NO 22 S2Na[M+Na] + :1262.4279,found 1262.4293.

[0096] Synthesis of compound 7: Compound 6 was dissolved in dichloromethane (10 mL) in a round-bottom flask, and then 10 μL DBU was added and stirred at room temperature for 1-2 hours. The reaction was detected by spot plate and LC-MS. The mixture was extracted with dichloromethane and brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was obtained by concentration. The crude product was purified by silica gel column to obtain the target compound 7 as a colorless liquid. HRMS (ESI) m / z caled for C 46 H 67 NO 20 S2H[M+H] + :1018.3778,found 1018.3776.

[0097] Synthesis of compound 8: Folic acid (FA) (49 mg, 0.112 mmol, 4 eq) and N,N'-carbonyldiimidazole (CDI) were dissolved in a round-bottom flask with dimethyl sulfoxide (DMSO), and the mixture was reacted at room temperature for 1-2 hours. Then, hyaluronic acid (HA) (200 mg, 0.028 mmol, equivalent to 0.486 mmol-COOH groups) and DMAP were dissolved in formamide and added to the round-bottom flask. The mixture was reacted at room temperature for 24-48 hours. After the reaction, the mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times. The mixture was then filtered and freeze-dried to obtain compound 8 as a light yellow solid.

[0098] Synthesis of compound 9: Compound 8 (50 mg, 0.007 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Compound 7, 4-PPy and DMAP were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT) as a light yellow solid.

[0099] Preparation of FA-HA-SS-PPT polymer prodrug micelles:

[0100] Compound 9 (2.5 mg) was dissolved in pure water (2 mL), and was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 5 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles.

[0101] Example 2

[0102] The present invention provides a method for preparing polymer prodrug micelles, comprising:

[0103] The polymer prodrug compound (denoted as FA-HA-CC-PPT) was synthesized according to the following synthetic route:

[0104]

[0105] The details are as follows:

[0106] Synthesis of compound 10: Under nitrogen conditions, tert-butyldimethylsilyl chloride (4.59 g, 30.46 mmol, 1.2 eq) was added dropwise to a mixed solution of 1,6-hexanediol (3.00 g, 25.38 mmol, 1 eq) and imidazole (2.80 g, 41.23 mmol, 2.1 eq) in THF (50 mL), and stirred in an ice bath for 4 hours. The reaction was then quenched with water, extracted three times with dichloromethane, and the organic phase was collected. The organic phase was dried with anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by silica gel column to obtain the target compound 10 as a colorless liquid. HRMS (ESI) m / z caled for C 12 H 28 O2SiNa[M+Na] + :255.1759,found255.1748.

[0107] Synthesis of compound 11: Under nitrogen conditions, N,N'-diisopropylethylamine (DIPEA) (1.34 g, 10.35 mmol, 2 eq) and compound 10 (1.20 g, 5.17 mmol, 1 eq) were dissolved in a round-bottom flask with dry dichloromethane (80 mL). Then, a solution of bis(p-nitrobenzene) carbonate (3.15 g, 10.35 mmol, 2 eq) dissolved in dichloromethane was slowly dripped into the above mixed solution and stirred at room temperature overnight. The reaction was completed by spot plate and LC-MS detection, the solution was washed with aqueous hydrochloric acid solution (0.2 mol / L, 20 mL), the aqueous layer was extracted three times with dichloromethane (30 mL), the organic phases were combined, the solvent was removed under reduced pressure, and the crude product was obtained by concentration. The crude product was purified by silica gel column to obtain the target compound 11 as a light yellow liquid. HRMS (ESI) m / z caled for C 19 H 31 NO6SiNa[M+Na] + :420.1821, found 420.1808.

[0108] Synthesis of compound 12: Under nitrogen conditions, 4-dimethylaminopyridine (448 mg, 3.66 mmol, 2 eq) was dissolved in dichloromethane in a round-bottom flask, and then compound 11 (800 mg, 2.01 mmol, 1.1 eq eq) and podophyllotoxin (760 mg, 1.83 mmol, 1 eq) were slowly added to the round-bottom flask at the same time with a syringe, and stirred overnight at room temperature. The reaction was completed by spot plate and LC-MS detection, and the reaction was quenched with aqueous hydrochloric acid (0.2 mol / L, 20 mL×2) to separate the organic phase. The aqueous layer was extracted twice with dichloromethane (30 mL), and the organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 12 as a white solid. HRMS (ESI) m / z caled for C 35 H 48 O 11 SiNa[M+Na] + :695.2866,found 695.2863.

[0109] Synthesis of compound 13: Compound 12 (2.96 g, 4.17 mmol, 1 eq) was dissolved in a round-bottom flask with distilled THF (10 mL), and then Et3N·3HF (3.37 g, 20.88 mmol, 5 eq) was added dropwise to the round-bottom flask under ice bath conditions and stirred at room temperature for 5-6 hours. The reaction was detected by spot plate and LC-MS. After THF was dried, it was dissolved in dichloromethane and washed with water. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 13 as a white solid. HRMS (ESI) m / z caled for C 29 H 34 O 11 Na[M+Na] + :582.2001,found 581.1982.

[0110] Synthesis of compound 14: HOOC-PEG8-NH2-FMOC (100 mg, 0.15 mmol, 1 eq) and compound 13 (100 mg, 0.17 mmol, 1.1 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (57 mg, 0.30 mmol, 2 eq) and DMAP (10 mg, 0.08 mmol, 0.5 eq) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 14 as a white solid. HRMS (ESI) m / z caled for C 63 H 81 NO 22 Na[M+Na] + :1226.5150,found 1226.5114.

[0111] Synthesis of compound 15: Compound 14 was dissolved in dichloromethane (10 mL) in a round-bottom flask, and then 10 μL DBU was added and stirred at room temperature for 1-2 hours. The reaction was detected by spot plate and LC-MS. The mixture was extracted with dichloromethane and brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was obtained by concentration. The crude product was purified by silica gel column to obtain the target compound 15 as a colorless liquid. HRMS (ESI) m / z caled for C 48 H 71 NO 20 H[M+Na] + :982.4649,found 982.4612.

[0112] Synthesis of compound 16: Compound 8 (50 mg, 0.06 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Compound 15, 4-PPy and DMAP were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 16 (FA-HA-CC-PPT) as a light yellow solid.

[0113] Preparation of FA-HA-CC-PPT polymer prodrug micelles:

[0114] Compound 16 (2.5 mg) was dissolved in pure water (2 mL), and was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 5 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-CC-PPT prodrug micelles.

[0115] Example 3

[0116] The present invention provides a method for preparing polymer prodrug micelles, comprising:

[0117] The polymer prodrug compound (denoted as FA-HA-SSC-PPT) was synthesized according to the following synthetic route:

[0118]

[0119]

[0120] Synthesis of compound 17: Podophyllotoxin (500 mg, 1.2 mmol, 1 eq) and 3,3'-dithiodipropionic acid (1009 mg, 4.8 mmol, 4 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (920 mg, 4.8 mmol, 4 eq) and DMAP (73 mg, 0.6 mmol, 0.5 eq,) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 17 as a white solid. HRMS (ESI) m / z caled for C 30 H 34 O 12 S2Na[M+H] + :593.2001,found 593.1974.

[0121] Synthesis of compound 18: Compound 17 (300 mg, 0.53 mmol, 1 eq) and ethylene glycol (130 mg, 2.11 mmol, 4 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (203 mg, 1.06 mmol, 2 eq) and DMAP (33 mg, 0.27 mmol, 0.5 eq) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 18 as a white solid. HRMS (ESI) m / z caled for C 30 H 34 O 12 S2Na[M+Na] + :637.2260,found 637.2251.

[0122] Synthesis of compound 19: HOOC-PEG8-NH2-FMOC (100 mg, 0.15 mmol, 1 eq) and compound 18 (104 mg, 0.17 mmol, 1.1 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (57 mg, 0.30 mmol, 2 eq) and DMAP (10 mg, 0.08 mmol, 0.5 eq) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 19 as a white solid. HRMS (ESI) m / z caled for C 30 H 34 O 12 S2Na[M+Na]+:1282.5409, found 1282.5408.

[0123] Synthesis of compound 20: Compound 19 was dissolved in dichloromethane (10 mL) in a round-bottom flask, and then 10 μL DBU was added and stirred at room temperature for 1-2 hours. The reaction was detected by spot plate and LC-MS. The mixture was extracted with dichloromethane and brine, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 20 as a colorless liquid. HRMS (ESI) m / z caled for C 30 H 34 O 12 S2H[M+H] + :1038.4912,found 1038.4907.

[0124] Synthesis of compound 21: Compound 8 (50 mg, 0.046 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Compound 20, 4-PPy and DMAP were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 21 (FA-HA-SSC-PPT) as a light yellow solid.

[0125] Preparation of FA-HA-SSC-PPT polymer prodrug micelles:

[0126] Compound 21 (2.5 mg) was dissolved in ultrapure water (2 mL), and was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 5 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SSC-PPT prodrug micelles.

[0127] Example 4

[0128] The present invention provides a method for preparing polymer prodrug micelles, comprising:

[0129] The polymer prodrug compound (denoted as FA-HA-CCC-PPT) was synthesized according to the following synthetic route:

[0130]

[0131] The details are as follows:

[0132] Synthesis of compound 22: Podophyllotoxin (500 mg, 1.2 mmol, 1 eq) and 1,8-octanedioic acid (835 mg, 4.8 mmol, 4 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (920 mg, 4.8 mmol, 4 eq) and DMAP (73 mg, 0.6 mmol, 0.5 eq) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 22 as a white solid. HRMS (ESI) m / z caled for C 28 H3O 11 S2Na[M+Na] + :629.1130, found 629.1110.

[0133] Synthesis of compound 23: Compound 22 (300 mg, 0.47 mmol, 1 eq) and ethylene glycol (292 mg, 4.7 mmol, 10 eq) were dissolved in a round-bottom flask with dichloromethane. After stirring for three minutes, EDCI (180 mg, 0.94 mmol, 2 eq) and DMAP (29 mg, 0.24 mmol, 0.5 eq) were added. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 23 as a white solid. HRMS (ESI) m / z caled for C 30 H 34 O 12 S2Na[M+Na] + :673.1392, found 673.1390.

[0134] Synthesis of compound 24: HOOC-PEG8-NH2-FMOC (100 mg, 0.15 mmol, 1 eq) and compound 23 (108 mg, 0.17 mmol, 1.1 eq) were dissolved in dichloromethane in a round-bottom flask, and EDCI (58 mg, 0.30 mmol, 2 eq) and DMAP (10 mg, 0.08 mmol, 0.5 eq) were added after stirring for three minutes. Stir at room temperature for 4-5 hours. The reaction was completed by spot plate and LC-MS detection, and extracted with dichloromethane and brine, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 24 as a white solid. HRMS (ESI) m / z caled for C 64 H 81 NO 23 S2Na[M+Na]+:1318.4541 found 1318.4502.

[0135] Synthesis of compound 25: Compound 24 was dissolved in dichloromethane (10 mL) in a round-bottom flask, and then 10 μL DBU was added and stirred at room temperature for 1-2 hours. The reaction was detected by spot plate and LC-MS. The mixture was extracted with dichloromethane and brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was obtained by concentration. The crude product was purified by silica gel column to obtain the target compound 25 as a colorless liquid. HRMS (ESI) m / z caled for C 30 H 34 O 12 S2H[M+H] + :1074.4040,found 1074.4051.

[0136] Synthesis of compound 26: Compound 8 (50 mg, 0.046 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Compound 25, 4-PPy and DMAP were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 26 (FA-HA-CCC-PPT) as a light yellow solid.

[0137] Preparation of FA-HA-CCC-PPT polymer prodrug micelles:

[0138] Compound 26 (2.5 mg) was dissolved in ultrapure water (2 mL), and was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 5 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-CCC-PPT prodrug micelles.

[0139] Example 5

[0140] The present invention provides a method for preparing polymer prodrug micelles, comprising:

[0141] The polymer prodrug compound (denoted as FA-HA-C-PPT) was synthesized according to the following synthetic route:

[0142]

[0143] Synthesis of compound 27: Dissolve podophyllotoxin (500 mg, 1.2 mmol, 1 eq) and Fmoc-11-aminoundecanoic acid (361 mg, 4.8 mmol, 1.5 eq) in a round-bottom flask with dichloromethane. After stirring for three minutes, add EDCI (460 mg, 4.8 mmol, 2 eq) and DMAP (73 mg, 0.6 mmol, 0.5 eq). Stir at room temperature for 4-5 hours. The reaction was detected by spot plate and LC-MS. The reaction was extracted with dichloromethane and brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was concentrated. The crude product was purified by silica gel column to obtain the target compound 27 as a white solid. HRMS

[0144] (ESI) m / z caled for C 48 H 53 NO 11 H[M+H] + :819.3619, found 819.3621.

[0145] Synthesis of compound 28: Compound 27 was dissolved in dichloromethane (10 mL) in a round-bottom flask, and then 10 μL DBU was added and stirred at room temperature for 1-2 hours. The reaction was detected by spot plate and LC-MS. The mixture was extracted with dichloromethane and brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was obtained by concentration. The crude product was purified by silica gel column to obtain the target compound 28 as a colorless liquid. HRMS (ESI) m / z caled for C 33 H 43 O9H[M+H] + :598.2971,found598.2974.

[0146] Synthesis of compound 29: Compound 8 (50 mg, 0.046 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Compound 28, 4-PPy and DMAP were dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 29.

[0147] (FA-HA-C-PPT), a light yellow solid.

[0148] The specific synthesis conditions are similar to those in Example 1 and will not be described in detail in this example.

[0149] Characterization

[0150] The compounds and micelles in Examples 1-6 and Comparative Examples 1-2 were characterized. The characterization results are shown in Figure 1-Figure 11 as well as Figure 24-28 .

[0151] According to the H NMR spectrum of compound 8 ( Figure 1 ) It can be seen that the chemical shifts 7.81-7.99 are attributed to the hydrogen on the styridine ring of folic acid, and 6.76-6.66 and 7.30 are attributed to the hydrogen on the benzene ring of folic acid, indicating that folic acid has been successfully connected to the chain of HA. The nuclear magnetic resonance characterization of compound 8 is that the solid compound 8 (10 mg) obtained in Example 1 is dissolved in deuterated water (0.6 mL) for nuclear magnetic hydrogen spectrum characterization.

[0152] According to FA-HA-SS-PPT ( Figure 2 ) and FA-HA-CC-PPT of Example 2 ( Figure 5), the signal peak at the chemical shift of 2.8 belongs to the signal peak of polyethylene glycol (-OCH2-) methylene, indicating that polyethylene glycol is linked to HA. The chemical shift of 5.8-7.25 belongs to the hydrogen signal peak on the PPT benzene ring, proving the presence of PPT in the obtained FA-HA-SS-PPT, while 7.9-8.1 belongs to the hydrogen on the dapoxetine ring of folic acid, and it is concluded that folic acid is successfully introduced into the HA chain. Similarly, for FA-HA-CC-PPT, in addition to the hydrogen signal peak on the PPT benzene ring with chemical shifts of 5.8 to 7.25, the signal peak at 2.8 belongs to the signal peak of polyethylene glycol (-OCH2-) methylene, and there is also a hydrogen on the dapoxetine ring of folic acid at 7.9-8.1. The obtained FA-HA-CC-PPT also successfully introduced PPT and folic acid into the HA chain. The NMR characterization of FA-HA-SS-PPT and FA-HA-CC-PPT was performed by dissolving the FA-HA-SS-PPT prepared in Example 1 or the FA-HA-CC-PPT prodrug compound (10 mg) prepared in Example 2 in deuterated water (0.6 mL) for NMR hydrogen spectrum characterization.

[0153] UV Characterization of FA-HA-SS-PPT and FA-HA-CC-PPT Prodrug Compounds

[0154] The FA-HA-SS-PPT prepared in Example 1 or the FA-HA-CC-PPT prodrug compound prepared in Example 2, hyaluronic acid, and podophyllotoxin were dissolved in ultrapure water, respectively, and then scanned in the range of 200-700 nm and characterized by UV. Fig.12 Both polymer prodrugs have the highest UV absorption at 278-281nm, while PPT has the maximum absorption at 285-290. The two absorptions are similar, with a slight difference, which may be because the PPT connected to HA is modified, indicating that PPT has been successfully connected to the HA chain.

[0155] Infrared Characterization of FA-HA-SS-PPT and FA-HA-CC-PPT Prodrug Compounds

[0156] The FA-HA-SS-PPT prepared in Example 1 or the FA-HA-CC-PPT prodrug compound prepared in Example 2 (1-2 mg) was mixed with potassium bromide solid powder (100-200 mg), ground evenly, and pressed into tablets. -1 Scanning within the range and performing infrared characterization, see Fig.13 The infrared spectrum of FA-HA-SS-PPT is compared with that of HA at 1700cm -1 A new characteristic peak of ester bond appears at 1650cm -1 , 1600cm -1The characteristic peak of amide bond appears at 1260cm -1 , 960cm -1 , 650cm -1 The characteristic peaks of benzene rings of podophyllotoxin and folic acid appear at 2900 cm -1 , 2845cm -1 The peak is the characteristic peak of CH of PEG. Compared with the infrared spectrum of HA, the infrared spectrum of FA-HA-CC-PPT shows a new characteristic peak of ester bond at 1700cm-1 and a new characteristic peak of ester bond at 1650cm-1. -1 ,1420cm -1 The characteristic peak of amide bond appears at 1150cm -1 , 960cm -1 The characteristic peak of benzene ring appears at 2900cm -1 , 2845cm -1 The peak is the characteristic peak of PEG CH, indicating that folic acid and podophyllotoxin have been successfully attached to HA, at 1300-1250cm -1 The characteristic signal peak of ester bond (-CO-O-) appears at 1650cm -1 and 1560cm- 1 The characteristic signal peak of the amide bond (-NH-CO-) appears at 920-720, and the characteristic signal peak of the benzene ring appears at 920-720, indicating that the target product has been synthesized.

[0157] Particle size characterization of FA-HA-SS-PPT prodrug micelles and FA-HA-CC-PPT prodrug micelles

[0158] The FA-HA-SS-PPT prepared in Example 1 or the FA-HA-CC-PPT prepared in Example 2 was prepared into a dilute solution with a concentration of 1 mg / mL using ultrapure water to obtain polymer prodrug micelles. The particle size distribution and potential of the two prodrug micelles were measured by dynamic light scattering method. Fig.14 , among which FA-HA-SS-PPT prodrug micelles, the particle size is about 69.66nm, PDI is 0.252, Zeta potential is -18.7mV, FA-HA-CC-PPT polymer prodrug micelles, the particle size is about 89.18nm, PDI is 0.192, Zeta potential is -15.3mV, this is because there are a large number of carboxyl groups on the hyaluronic acid molecules, these groups are easily dissociated into -COO- groups in neutral solutions, the groups make the complex with a trace negative charge, the nano-microspheres have good stability, and can be degraded for a long time in buffer solutions. This particle size can freely pass through tissue capillaries (the diameter of capillaries is generally 6-8μm). At the same time, by comparing the two polymer prodrug micelles, it can be found that the polymer micelles formed by the polymer prodrug micelles with disulfide bonds are smaller and more evenly distributed.

[0159] Morphological Characterization of HA-NH-CO-PPT and HA-SS-PPT Prodrug Micelle

[0160] The FA-HA-SS-PPT prodrug micelles prepared in Example 1 and the FA-HA-CC-PPT prodrug micelles prepared in Example 2 were characterized by transmission electron microscopy for their morphology and particle size. Fig.15 , FA-HA-SS-PPT and FA-HA-CC-PPT prodrug micelles are basically spherical or ellipsoidal and evenly distributed.

[0161] Synthesis process optimization:

[0162] (1) Screening of conditions for the synthesis of compound 8:

[0163] Scheme 1: Folic acid (FA) (49 mg, 0.112 mmol, 4 eq) and EDCI (21.392 mg, 0.112 mmol, 4 eq) were dissolved in dimethyl sulfoxide (DMSO) in a round-bottom flask and reacted at room temperature for 1 hour. Then, hyaluronic acid (HA) (200 mg, 0.028 mmol, equivalent to 0.486 mmol-COOH group) and DMAP (6.28 mg, 0.056 mmol, 2 eq) were dissolved in formamide and added to the round-bottom flask. The reaction was reacted at room temperature for 12 hours. After the reaction was completed, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 8 as a light yellow solid. The folic acid reaction rate was estimated to be 35% by the nuclear magnetic resonance spectrum, and a large amount of folic acid solid was precipitated during the reaction.

[0164] Scheme 2: Folic acid (FA) (49 mg, 0.112 mmol, 4 eq) and DCC (25 mg, 0.112 mmol, 4 eq) were dissolved in dimethyl sulfoxide (DMSO) in a round-bottom flask and reacted at room temperature for 1 hour. Then, hyaluronic acid (HA) (200 mg, 0.028 mmol, equivalent to 0.486 mmol-COOH group) and triethylamine (7.78 uL, 0.056 mmol, 2 eq) were dissolved in formamide and added to the round-bottom flask. The reaction was reacted at room temperature for 12 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 8, which was a light yellow solid. The folic acid reaction rate was estimated to be 45% by the nuclear magnetic resonance spectrum. Only a small amount of folic acid solid was precipitated during the reaction, but a large amount of white solid was produced, which was a urea byproduct of DCC.

[0165] Scheme 3: Folic acid (FA) (49 mg, 0.112 mmol, 4 eq) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in dimethyl sulfoxide (DMSO) in a round-bottom flask and reacted at room temperature for 1 hour. Then, hyaluronic acid (HA) (200 mg, 0.028 mmol, equivalent to 0.486 mmol-COOH group) and DMAP (6.28 mg, 0.056 mmol, 2 eq) were dissolved in formamide and added to the round-bottom flask. The reaction was reacted at room temperature for 12 hours. After the reaction was completed, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 8 as a light yellow solid. The folic acid reaction rate was estimated to be 50% by the nuclear magnetic resonance spectrum, and no folic acid solid was precipitated.

[0166] Scheme 4: Folic acid (FA) (49 mg, 0.112 mmol, 4 eq) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in dimethyl sulfoxide (DMSO) in a round-bottom flask and reacted at room temperature for 1 hour. Then, hyaluronic acid (HA) (200 mg, 0.028 mmol, equivalent to 0.486 mmol-COOH group) and DMAP (6.28 mg, 0.056 mmol, 2 eq) were dissolved in formamide and added to the round-bottom flask. The reaction was reacted at room temperature for 24 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 8 as a light yellow solid. The folic acid reaction rate was estimated to be 80% by the nuclear magnetic resonance spectrum, and no folic acid solid was precipitated.

[0167] Scheme 5: Folic acid (FA) (49 mg, 0.112 mmol, 4 eq) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in dimethyl sulfoxide (DMSO) in a round-bottom flask and reacted at room temperature for 1 hour. Then, hyaluronic acid (HA) (200 mg, 0.028 mmol, equivalent to 0.486 mmol-COOH group) and DMAP (6.28 mg, 0.056 mmol, 2 eq) were dissolved in formamide and added to the round-bottom flask. The reaction was reacted at room temperature for 36 hours. After the reaction was completed, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 8, which was a light yellow solid. The folic acid reaction rate was estimated to be 85% by nuclear magnetic resonance spectrum, and no folic acid solid was precipitated.

[0168] After comparing the above five schemes, it was found that the commonly used DCC and EDCI ester condensation reagents can only react about 40% of folic acid, and folic acid solids and byproducts will precipitate. The DCC byproducts are not easy to dialyze to remove, while the reaction of folic acid using CDI as a condensation agent can reach about 80%, and there is no folic acid precipitation. In terms of time selection, the amount of folic acid reaction increases with time, but the amount of folic acid reaction at 24h and 36h is basically the same, so the optimal reaction conditions are to use CDI as a condensation agent and react at room temperature for 24h.

[0169] (2) Conditional screening of polymer prodrug compounds:

[0170] Scheme 1: Compound 8 (50 mg, 0.007 mmol, equivalent to 0.126 mmol-COOH) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour to dissolve. Then, compound 7 (128 mg, 0.126 mmol, 1 eq), DMAP (16 mg, 0.126 mmol, 1 eq) and 4-PPy (18 mg, 0.126 mmol, 1 eq) were dissolved in DMSO and added to the reaction bottle. The mixture was reacted at room temperature for 24 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT), which was a light yellow solid with a PTT grafting rate of about 5%.

[0171] Scheme 2: Compound 8 (50 mg, 0.007 mmol, equivalent to 0.126 mmol-COOH) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour to dissolve. Then, compound 7 (128 mg, 0.126 mmol, 1 eq), DMAP (16 mg, 0.126 mmol, 1 eq) and 4-PPy (18 mg, 0.126 mmol, 1 eq) were dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 30°C for 24 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT), which was a light yellow solid with a PTT grafting rate of about 7.5%.

[0172] Scheme 2: Compound 8 (50 mg, 0.007 mmol, equivalent to 0.126 mmol-COOH) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour to dissolve. Then, compound 7 (128 mg, 0.126 mmol, 1 eq) DMAP (16 mg, 0.126 mmol, 1 eq) and 4-PPy (18 mg, 0.126 mmol, 1 eq) were dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 24 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT), which was a light yellow solid with a PTT grafting rate of about 15%.

[0173] Scheme 3: Compound 8 (50 mg, 0.007 mmol, equivalent to 0.126 mmol-COOH) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour to dissolve. Compound 7 (128 mg, 0.126 mmol, 1 eq) DMAP (16 mg, 0.126 mmol, 1 eq) and 4-PPy (18 mg, 0.126 mmol, 1 eq) were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 60°C for 24 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT) as a light yellow solid with a podophyllotoxin grafting rate of 13%.

[0174] Scheme 4: Compound 8 (50 mg, 0.007 mmol, equivalent to 0.126 mmol-COOH) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour to dissolve. Compound 7 (128 mg, 0.126 mmol, 1 eq) DMAP (16 mg, 0.126 mmol, 1 eq) and 4-PPy (18 mg, 0.126 mmol, 1 eq) were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT) as a light yellow solid with a podophyllotoxin grafting rate of 24%.

[0175] Scheme 5: Compound 8 (50 mg, 0.007 mmol, equivalent to 0.126 mmol-COOH) and CDI (18 mg, 0.112 mmol, 4 eq) were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour to dissolve. Compound 7 (128 mg, 0.126 mmol, 1 eq) DMAP (16 mg, 0.126 mmol, 1 eq) and 4-PPy (18 mg, 0.126 mmol, 1 eq) were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 60 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 9 (FA-HA-SS-PPT) as a light yellow solid with a grafting rate of 25%.

[0176] Comparison of the above five schemes revealed that: H NMR characterization revealed that with the increase of reaction time and temperature, the grafting rate of podophyllotoxin would increase, but when heated at 60°C, folic acid and podophyllotoxin prodrug might break, resulting in a decrease in the grafting rate of podophyllotoxin. At the same time, after 48 hours, the grafting rate of podophyllotoxin did not increase significantly, so the optimal conditions were selected, that is, after reacting at 40°C for 48 hours, the polymer prodrug was obtained after dialysis and freeze-drying.

[0177] Screening of preparation conditions for polymer prodrug micelles:

[0178] Scheme 1: Compound 9 (1 mg) was dissolved in ultrapure water (2 mL) to make it uniformly dispersed and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles. Figure 23-1 .

[0179] Solution 2: Compound 9 (1 mg) was dissolved in ultrapure water (2 mL), and the mixture was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 2 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles. The particle size diagram is shown in Figure 23-2 .

[0180] Scheme 3: Compound 9 (2.5 mg) was dissolved in ultrapure water (2 mL), and the mixture was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 3 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles. The particle size diagram is shown in Figure 23-3 .

[0181] Scheme 4: Compound 9 (2.5 mg) was dissolved in ultrapure water (3 mL), and the mixture was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 3 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles. The particle size diagram is shown in Figure 23-4 .

[0182] Scheme 5: Compound 9 (2.5 mg) was dissolved in ultrapure water (2 mL), and the mixture was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 5 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles. The particle size diagram is shown in Figure 23-5 .

[0183] Scheme 6: Compound 9 (2.5 mg) was dissolved in ultrapure water (2 mL), and the mixture was evenly dispersed using an ultrasonic cell disruptor (power 20 W, treatment 6 min, once every five seconds), and allowed to stand for half an hour to obtain FA-HA-SS-PPT prodrug micelles. The particle size diagram is shown in Figure 23-6 .

[0184] By comparing the polymer micelle preparation methods made by the above five schemes, it was found that the polymer micelles obtained without ultrasonic crushing generally had a larger particle size. After ultrasonic crushing, the particle size became smaller, but the particle size would become larger again with the increase of power and time. This may be due to the aggregation of the micelles and the breakup of the micelles. Therefore, the best condition was to dissolve 2.5 mg of polymer prodrug in ultrapure water (2 mL) and use an ultrasonic cell disruptor (power of 20 w, treatment for 5 min, once every five seconds).

[0185] Comparative Example 1:

[0186]

[0187] The specific method is as follows:

[0188] Synthesis of compound 30: FMOC-valine (1 g, 2.9 mmol, 1 eq) and N-hydroxysuccinimide (NHS) (980 mg, 2.9 mmol, 1 eq) were dissolved in a round-bottom flask with dichloromethane, and EDCI (2215 mg, 8.7 mmol, 4 eq) was added after stirring for three minutes. Stir at room temperature for 8 hours. The reaction was completed by spot plate and LC-MS detection, and the target compound 30 was obtained after slurrying with methyl tert-butyl ether. HRMS (ESI) m / z caled for C 24 H 24 N2O6H[M+H] + :437.1714, found 437.1716.

[0189] Synthesis of compound 31: Compound 30 (2.0 g) was dissolved in 10 mL THF and added to 15 mL aqueous solution of alanine (0.43 g, 4.81 mmol, 1.05 eq) and sodium bicarbonate (0.40 g, 4.81 mmol, 1.05 eq). The colorless solution immediately became turbid and refluxed at 40 ° C for 24 h. The reaction was completed by spot plate and LC-MS detection. It was extracted with dichloromethane and brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and concentrated to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 30 as a white solid. HRMS (ESI) m / z caled for C 23 H 25 N2O5H[M+H] + :411.1922,found 411.1926

[0190] Synthesis of compound 32: Under nitrogen, compound 31 (0.36 g, 0.88 mmol, 1 eq), p-aminobenzyl alcohol (0.13 g, 1.06 mmol, 1.2 eq) and EEDQ (0.33 g, 1.33 mmol, 1.5 eq) in dichloromethane were treated with methanol until a transparent solution was obtained. Over the next two days, a precipitate was precipitated from the reaction. After filtration, the precipitate was washed with dichloromethane and methyl tert-butyl ether and sonicated at room temperature for 15 min. This process was repeated twice to obtain compound 32 as a white solid. HRMS (ESI) m / z caled for C 30 H 33 N3O5Na[M+Na] + :538.2317,found 538.2320

[0191] Synthesis of compound 33: Under nitrogen conditions, N,N-diisopropylethylamine (DIEA) (4.2mL, 4.55mmol, 3eq) was slowly added dropwise to a 100mL dichloromethane suspension of compound 32 (4.22g, 8.18mmol, 1eq) and bis(p-nitrobenzene) carbonate (3.73g, 18.2mmol, 12.28eq,). The resulting yellow mixture was treated with dry N,N-dimethylformamide until a transparent solution was obtained and stirred at room temperature overnight. The solution was then washed with water and the organic phase was separated. The aqueous layer was extracted 4 times with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by silica gel column to obtain the target compound 33 as a white solid. HRMS (ESI) m / z caled for C 37 H 36 N4O9Na[M+Na] +:703.2379,found 703.2381

[0192] Synthesis of compound 34: Under nitrogen conditions, 4-dimethylaminopyridine (448 mg, 3.66 mmol, 2 eq) was dissolved in dichloromethane in a round-bottom flask, and then compound 33 (800 mg, 2.01 mmol, 1.1 eq eq) and podophyllotoxin (760 mg, 1.83 mmol, 1 eq) were slowly added dropwise into the round-bottom flask using a syringe. The reaction was stirred at room temperature. No desired product was detected by LC-MS, and the reaction was unsuccessful.

[0193] It can be seen from this comparative example that the desired polymer prodrug compound cannot be synthesized using a dipeptide bond.

[0194] Comparative Example 2:

[0195]

[0196] Synthesis of compound 35: Compound 8 (50 mg, 0.046 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Then, dihydroxy disulfide, DMAP was dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the resulting mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times, and then filtered and freeze-dried to obtain compound 36 as a light yellow solid.

[0197] Synthesis of compound 36: Compound 35 (50 mg, 0.046 mmol) and CDI were dissolved in formamide in a round-bottom flask and stirred at room temperature for one hour. Compound 22 and DMAP were then dissolved in DMSO and added to the reaction bottle. The mixture was reacted at 40°C for 48 hours. After the reaction, the mixture was placed in a dialysis bag (3000 Da) and dialyzed with deionized water. The water was changed every six hours for three times. The solid was then filtered and freeze-dried to obtain a light yellow solid. Deuterated water was used for NMR identification, but no peak of podophyllotoxin was seen in the NMR spectrum, indicating that the reaction was unsuccessful.

[0198] It can be seen from this comparative example that the desired polymer prodrug compound cannot be obtained by changing the preparation steps provided in the examples of the present invention.

[0199] Experimental Example 1: Drug release behavior of FA-HA-SS-PPT and FA-HA-CC-PPT prodrug micelles

[0200] The release behavior of the FA-HA-SS-PPT prodrug micelles prepared in Example 1 and the FA-HA-CC-PPT prodrug micelles prepared in Example 2 was studied by dialysis method. 2mL of prodrug micelles were added to a dialysis bag (molecular weight cutoff of 3000Da), and then the dialysis bag was immersed in a 50mL PBS (pH5.0, pH 5.0+20mM GSH, pH 7.4 and pH 7.4+20mM GSH) centrifuge tube of pH 5.0 and pH 7.4. Then it was placed in a constant temperature shaker with an oscillation speed of 200r / min and a temperature control of 37°C. At 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, 2.0mL of liquid was taken out from the release medium and replaced with 2.0mL of PBS of the corresponding pH to maintain the volume unchanged. The concentration of podophyllotoxin released into the PBS solution was measured by HPLC.

[0201] Results Fig.16 For FA-HA-CC-PPT micelles, less than 24.3% of PPT was slowly released after incubation at pH 7.4 for 24 h, while the cumulative release increased to 65.4% after incubation at pH 5.0 for 24 h. For FA-HA-SS-PPT micelles, only 25.3% of PPT was released when incubated at pH 7.4, and the cumulative release of PPT increased from 66.5% to 66.5% when the pH value was 5.0. In particular, when 20 mM GSH was added to PBS at pH 5.0, the cumulative release of PPT increased significantly to 88.3%.

[0202] Experimental Example 2: Hemolytic evaluation of FA-HA-SS-PPT and FA-HA-CC-PPT prodrug micelles

[0203] First, fresh mouse plasma was defibrinated. 2 mL of mouse blood was taken and gently stirred in a clockwise direction with a glass rod for 15 minutes to remove the fibrin in the blood. Then 8 mL of PBS solution was added, placed in a centrifuge, centrifuged at 2500 r / min for 10 minutes, the supernatant was removed, and the centrifugation was repeated three times until the supernatant was colorless to obtain a red blood cell suspension. The cell suspension was diluted with PBS, vortexed for 5 minutes to mix the suspension evenly, and a red blood cell suspension with a concentration of about 5% (v / v) was obtained. The red blood cell suspension was mixed with 5 mg / mL of FA-HA-SS-PPT, FA-HA-CC-PPT prodrug micelles, and Tween 80 solution, respectively, so that the final concentration range of the two types of prodrug micelles or Tween 80 in the mixture was 0.1 to 2 mg / mL (0.1, 0.25, 0.5, 1, 2 mg / mL, respectively). At the same time, 1 mL of red blood cell suspension was mixed with equal amounts of ultrapure water and PBS as positive and negative controls. After incubation on a 37°C constant temperature shaker for 4 h and 12 h, centrifuge at 2500 r / min for 10 min. Detect the absorbance of the supernatant at 541 nm using an ELISA reader, and calculate the degree of hemolysis using the following equation:

[0204] Hemolysis rate (%) = (Atest-Aneg) / (Apos-Aneg) × 100

[0205] Where Atest, Aneg, and Apos are the absorbance values ​​of the sample, negative control (PBS), and positive control (water), respectively. Fig.17 ,according to Fig.17 It can be seen that when the concentration increased from 0.1 to 2.0 mg / mL, the hemolysis rate induced by Tween 80 increased significantly from 11.5% to 75.5%. However, FA-HA-SS-PPT and FA-HA-CC-PPT prodrug micelles showed a hemolysis rate of no more than 7% at the same concentration, which was significantly lower than that of Tween 80, indicating that the prepared prodrug micelles had good blood

[0206] Experimental Example 3: Study on drug targeting of FA-HA-SS-PPT polymer prodrug

[0207] HeLa and 4T1 cells were cultured at 6×10 4The cell density was inoculated on a 12-well plate. After incubation for 24 hours, it was observed that the cells occupied about 80% of the flask wall. 10 mg / mL hyaluronic acid (molecular weight 7000Da) and 10 mg / mL folic acid were added respectively, and cultured for 2 hours. The control group was treated with no hyaluronic acid or folic acid. After that, the culture medium (DMEM) was removed, and fresh DMEM containing 5 μg / mL of FA-HA-SS-PPT prodrug micelles prepared in Example 1 was added respectively, and culture was continued for 4 hours. The cells were washed three times with 1×PBS (pH 7.4), digested with trypsin, blown and collected, and sieved. Flow cytometry was selected for detection.

[0208] Results Fig.18 ,according to Fig.18 It can be seen that when FA or HA was co-incubated with 4T1 and HeLa cells with high expression of CD44 and FR dual receptors, the cell uptake of rhodamine-labeled FA-HA-SS-PPT was reduced, which proved that FA-HA-SS-PPT entered the tumor cells through CD44 and FR-mediated endocytosis, which confirmed that FA-HA-SS-PPT interacted with CD44 and FR, and CD44 targeted hyaluronic acid, and FA targeted folate receptor FR. Therefore, FA-HA-SS-PPT had a targeting effect with CD44 and FR.

[0209] Experimental Example 4: Study on the antitumor activity of FA-HA-SS-PPT polymer prodrug

[0210] The toxicity of FA-HA-SS-PPT polymer prodrug, FA-HA-CC-PPT polymer prodrug, HA-PPT polymer prodrug, and free PPT to cervical cancer cells (HeLa) and breast cancer cells (4T1) was determined by MTT method. HeLa and 4T1 cells with good growth status were taken, and the cells were subjected to the same cell passage experiment. After the cells were resuspended, the cells were counted with a blood cell counting plate, and 2500 cells / well were planted in a 96-well plate. After the cells were cultured for 24 hours, the original culture medium was removed, and culture medium containing different concentrations of drugs was added to continue culturing for 48 hours. After the drug effect was over, the original culture medium was removed, 100 μL of fresh culture medium was added, 10 μL of MTT solution was added, and after incubation in an incubator for 2 hours, 150 mL of DMSO was added, and the shaking table was incubated for 15 minutes. At 490 nm, the OD value was measured with an enzyme marker, and the cell proliferation inhibition rate was calculated. Repeat the above steps three times.

[0211] Results Fig.19 ,Depend on Fig.19It can be seen that the antitumor activity of FA-HA-SS-PPT polymer prodrug and HA-SS-PPT polymer prodrug on HeLa and 4T1 expressing both CD44 and FR is higher than that of PPT. The antitumor activity of FA-HA-CC-PPT polymer prodrug connected by carbonate bond is lower than that of free PPT, which proves that drugs connected by disulfide bond can release more PPT in the tumor microenvironment, and the tumor microenvironment responsiveness of disulfide bond is higher than that of carbonate bond. At the same time, the antitumor activity of FA-HA-SS-PPT polymer prodrug is higher than that of HA-SS-PPT polymer prodrug, which proves that compared with single targeting, dual-targeted PPT nanomicelles have better antitumor effect and higher uptake of tumor cells.

[0212] Experimental Example 5: EdU method to determine the cell proliferation inhibitory effect of FA-HA-SS-PPT polymer prodrug on HeLa and 4T1 cells

[0213] HeLa and 4T1 cells were cultured at 6×10 4 The cell density was inoculated on a 12-well plate with a cell slide placed in advance, and incubated for 24 hours. Drugs containing the same concentration of PPT were added and cultured for 24 hours. Prepare 2X EdU working solution, add equal volumes of 2X EdU working solution (20μM) preheated at 37°C to the 12-well plate, so that the final concentration of EdU in the 12-well plate becomes 1X. For example, the final concentration is designed to be 10μM. Continue to incubate the cells for 2 hours. After EdU labeling of cells is completed, remove the culture medium and add 1mL of fixative. Remove the fixative and wash the cells 3 times with 1ml of washing solution per well, each for 3-5 minutes. Remove the washing solution, use 1mL of permeabilization solution per well, and incubate at room temperature for 10-15min. Remove the permeabilization solution, wash the cells 1-2 times with 1mL of washing solution per well, each for 3-5min. After the end, seal the slides and store them, and observe them under a laser confocal microscope.

[0214] The results are as follows Fig. 20As shown in the figure, FA-HA-SS-PPT has a good cell proliferation inhibitory effect on 4T1 and HeLa cells with high expression of CD44 and FR receptors. Under normal conditions, the proportion of 4T1 cells in the cell proliferation state is about 96%, and FA-HA has almost no obvious effect on cell proliferation, and the proportion of 4T1 cells in the proliferating state is about 95%. When the cells were treated with free PPT, FA-HA-SS-PPT, FA-HA-CC-PPT and HA-SS-PPT, the number of proliferating cells decreased. Among them, FA-HA-SS-PPT had the strongest cell proliferation inhibitory effect, and the number of cells in the proliferating state was as low as 9%, followed by single-targeted HA-PPT and free PPT, and the FA-HA-CC-PPT drug with non-disulfide bond linkage had the worst effect. The same phenomenon was observed in the EdU cell proliferation experiment of human cervical cancer cells. This confirms that the dual-targeted and disulfide-linked FA-HA-SS-PPT has a better effect. The targeting effect of FA and HA enhances the inhibitory effect of free PPT on tumor cell proliferation. The dual-targeted anti-tumor effect is better than that of single-targeted drugs. At the same time, the disulfide bond GSH-responsive disulfide bond gives the drug stronger tumor microenvironment responsiveness, which can significantly enhance the drug's anti-tumor effect.

[0215] Experimental Example 6: Annexin V-FITC / PI double staining method to determine the effect of FA-HA-SS-PPT polymer prodrug on apoptosis of HeLa and 4T1 cells

[0216] HeLa and 4T1 cells in the logarithmic growth phase were taken, centrifuged and inoculated in 6-well plates at 1x105 / well, and the cells were cultured for 24 hours. FA-HA, free PPT, FA-HA-SS-PPT, HA-SS-PPT, and FA-HA-CC-PPT with an effective concentration of podophyllotoxin equal to that of PPT were applied to HeLa and 4T1 cells respectively, and culture was continued for 48 hours. The cells were washed with PBS buffer, then digested with trypsin, and the digested cells were centrifuged at 1000r for 5 minutes, the supernatant was discarded, and the cells were collected. The supernatant was removed, and 195μL Annexin V-FITC binding solution was added to gently resuspend the cells. 5μL Annexin V-FITC was added and gently mixed. 10μL propidium iodide staining solution was added and gently mixed. Incubate at room temperature in the dark for 15min, and then place in an ice bath. Flow cytometry was used to detect cell apoptosis after the drug action ended.

[0217] The results are as follows Fig.21As shown in the figure, in order to further clarify the mechanism of action of FA-HA-SS-PPT on FR and CD44 overexpressing tumor cells, we used the Annexin V-FITC / PI double staining method to detect the cell apoptosis rate after the drug acted on HeLa and 4T1 cells. HA-FA had no significant effect on the apoptosis of HeLa and 4T1 cells, and their apoptosis rates were all lower than 5%, compared with the normal group, P<0.05. When FA-HA-CC-PPT acted on the cells, it induced apoptosis of cells with high expression of FR and CD44, causing 32% of HeLa cells and 23% of 4T1 cells to apoptosis. At the same time, when the same concentration of free PPT was applied to the cells, the apoptosis rates of HeLa cells and 4T1 cells were 43% and 34%, respectively. At the same concentration, HA-SS-PPT and FA-HA-SS-PPT had the strongest ability to induce tumor cell apoptosis. HA-SS-PPT induced apoptosis in 62% of HeLa cells and 61% of 4T1 cells, but its anti-tumor ability was still lower than that of FA-HA-SS-PPT (P<0.05), which induced apoptosis in 3 / 4 of HeLa cells and 4T1 cells. This proves that FA-HA-SS-PPT has a stronger ability to promote apoptosis in tumor cell lines with high expression of CD44 and FR dual receptors than single-targeted HA-SS-PPT and free PPT, and the tumor cell killing ability of GSH-responsive disulfide-bonded nanoprodrugs is stronger than that of non-disulfide-bonded drugs.

[0218] Experimental Example 7: Annexin V-FITC / PI double staining method to determine the effect of FA-HA-SS-PPT polymer prodrug on apoptosis of HeLa and 4T1 cells

[0219] In order to evaluate the effect of drugs on solid tumor tissues in vivo, FA-HA, free PPT, FA-HA-SS-PPT, FA-HA-CC-PPT and etoposide with an effective concentration of equal amount of PPT were respectively applied to the tail vein for administration, once every 2 days, for a total of 8 times, and the control group was injected with the same dose of saline solution. After each administration, the weight of the mice was weighed by an analytical balance, and the long diameter (L) and short diameter (W) of the tumor were measured by a vernier caliper, and the tumor volume was calculated according to V = (L × W^2) / 2. After the administration, the blood of the mice was obtained by eyeball blood sampling and placed in an anticoagulant tube, which was stored in a -80℃ refrigerator for standby use. The mice were then dissected and their heart, liver, spleen, lung, kidney and tumor tissues were taken. The weight of the tumors and organs in each administration group was accurately weighed by an analytical balance and immersed in a 4% paraformaldehyde solution for standby use. Subsequently, based on the data recorded during the administration process, the tumor growth curve and the weight change curve of each administration group were drawn, and the tumor growth inhibition rate was calculated according to [(average tumor weight of the control group - average tumor weight of the treatment group) / average tumor weight of the control group] × 100%.

[0220] like Fig. 22 As shown, in order to evaluate the in vivo anti-tumor effect of FA-HA-SS-PPT on tumor tissues with high expression of CD44 and FR, we used 4T1 cells to establish an in vivo tumor model, and determined the in vivo anti-tumor effect of different drugs by tail vein injection. As can be seen from the figure, after administration of FA-HA-SS-PPT, the mouse tumor volume growth volume was the smallest and the tumor tissue weight was the lightest, followed by etoposide and free PPT. The mouse tumor volume of FA-HA-CC-PPT was the largest and the tumor was heavier, which proved that the in vivo anti-tumor effect of FA-HA-CC-PPT was poor. This proves that the in vivo anti-tumor effect of FA-HA-SS-PPT is much better than that of FA-HA-CC-PPT, which is due to its disulfide bond connection. At the same time, through the targeting effect of FA and HA, the anti-tumor effect of FA-HA-SS-PPT is better than that of free PPT and PP derivative etoposide.

[0221] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polymer prodrug compound, characterized in that: It is selected from any one of the compounds represented by the following structural formulas: Among them, n is 1-14 and m is 8-17.

2. The polymer prodrug compound according to claim 1, characterized in that n is 1-7.

3. A method for preparing the polymer prodrug compound according to claim 1, characterized in that: include: The synthesis was carried out according to the following synthesis route: wherein Y is selected from N, O or C; A is selected from -CH2-(OCH2)n-CH2-, -CO-(CH2)n-CO-, -CH=CH-CO-, -CH=CH-(CH2)n-CO-, -CH(OH)-Ph-CO-, -CH(O H)-Ph-(CH2)n-CO-, -CH2-Ph-(CH2)n-CO-, -CH(OCH3)-Ph-CO-, -CH(OCH3)-Ph-(CH2)n-CO-, -CO-NH-C O-, -CO-NH-(CH2)n-CO-, -CH2-CH=CH-CO-, -CH2-CH=CH-(CH2)n-CO-, -CO-(CH2)n-, -(CH2)n-CO-, -CO -O-(CH2)n-CO-, -SO2-Ph-CO-, -SO2-Ph-(CH2)n-CO-, -CO-O-PAB-Cit-Val-CO-(CH2)n-CO-, -CO-O-PA B-Lys-Phe-CO-(CH2)n-CO-, -CO-(CH2)n-Su-, -CH=CH-Su-, -CH=CH-(CH2)n-Su-, -CH(OH)-Ph-Su-, -C H(OH)-Ph-(CH2)n-Su-, -CH2-Ph-(CH2)n-Su-, -CO-NH-Su-, -CO-NH-(CH2)n-Su-, -CH2-CH=CH-Su-, -C H2-CH=CH-(CH2)n-Su-, -CO-(CH2)n-, -(CH2)n-Su-, -CO-O-(CH2)n-Su-, -SO2-Ph-Su-, -SO2-Ph-(CH2)n-Su-, -CO-O-PAB-Cit-Val-CO-(CH2)n-Su-, -CO-O-PAB-Lys-Phe-CO-(CH2)n-Su-, any one of which 1≤n≤14; B is selected from any one of -SSS-, -SS-, -S-, -Se-Se-, -Se-, -C=N-NH- and -CO-O-PAB-Cit-Val-CO-(CH2)n-CO-; U is selected from existing fat-soluble anti-tumor compounds; A, B, U and Y correspond to the corresponding groups in the polymer prodrug compound of claim 1.

4. The preparation method according to claim 3, characterized in that: include: Reacting folic acid with hyaluronic acid to form intermediate 1; The antitumor compound is reacted with a substance containing a B group, and then reacted with a compound containing an A group to form an intermediate 2; The intermediate 1 and the intermediate 2 react to form the polymer prodrug compound.

5. The preparation method according to claim 4, characterized in that: The step of forming the intermediate 1 comprises: mixing folic acid, CDI and DMSO for reaction, and then mixing with hyaluronic acid, DMAP and amide solution for reaction for 24-26 hours.

6. The preparation method according to claim 5, characterized in that: After the reaction, dialysis was performed.

7. The preparation method according to claim 4, characterized in that: The step of forming the polymer prodrug compound comprises: mixing the intermediate 1 and an amide solution, and then reacting with the intermediate 2, DMSO, 4-PPy, CDI and DMAP at 40-45° C. for 48-52 hours.

8. A polymer prodrug micelle, characterized in that: It is prepared by the polymer prodrug compound described in claim 1.

9. A method for preparing the polymer prodrug micelle according to claim 8, characterized in that: include: The polymer prodrug compound according to claim 1 is used to form the polymer prodrug micelle.

10. The preparation method according to claim 9, characterized in that: include: The polymer prodrug compound and water are mixed, then sonicated, and then allowed to stand.

11. The preparation method according to claim 10, characterized in that: 2.5 mg of polymer prodrug compound corresponds to 2.2-2.5 ml of water, the ultrasonic time is 5-6 minutes, and the ultrasonic power is 18-22W.

12. An anti-tumor agent, characterized in that: It comprises the polymer prodrug compound according to claim 1 or the polymer prodrug micelle according to claim 8.

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