A tetravalent platinum ester alkyne complex, degradable high polymer, nanomicelle and preparation method and application thereof

Abstract

The application discloses a tetravalent platinum ester alkyne complex, a degradable high polymer, a nanomicelle and a preparation method and application thereof; the structural formula of the tetravalent platinum ester alkyne complex is shown as formula (I); the preparation method of the degradable high polymer is as follows: the tetravalent platinum ester alkyne complex is prepared first; then the obtained tetravalent platinum ester alkyne complex and diamine and monomethyl ether polyethylene glycol mPEG-NH2 are clicked to obtain a triblock high polymer. The nanomicelle of the degradable high polymer is also disclosed. The tetravalent platinum ester alkyne complex disclosed by the application can specifically respond to reducing substances in a tumor microenvironment, and the enamine ester structure generated by the reaction of the tetravalent platinum ester alkyne complex with diamine also has the property of responding to an acidic environment; the high polymer has mild and simple preparation conditions, the molecular weight can reach 35200, and the platinum drug loading capacity is as high as 33.91%; the nanomicelle can efficiently degrade and release anticancer active drugs in the tumor microenvironment, and has high antitumor activity and low cytotoxicity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical synthetic drugs, and particularly relates to a tetravalent platinum complex, a degradable high polymer, a nanomicelle, and a preparation method and application thereof. BACKGROUND

[0002] Platinum drugs are indispensable first-line drugs in cancer chemotherapy. Traditional platinum chemotherapy drugs such as cisplatin, carboplatin and oxaliplatin account for half of the currently used cancer chemotherapy drugs, but their systemic toxicity and tumor cell drug resistance limit their therapeutic effect and clinical application. Therefore, it is necessary to explore alternative strategies to reduce side effects and improve the pharmacokinetic characteristics of platinum drugs.

[0003] Tetravalent platinum prodrugs have attracted widespread attention from researchers due to their high degree of inertness, easy modification of axial ligands, ability to release highly active divalent platinum inside cancer cells to hinder cell division, longer body circulation time and lower toxicity. Subsequently, thousands of new platinum drugs have been developed. However, due to the limitations of small molecule single treatment mode, insufficient stability and body circulation time, and the need to improve controllability and selectivity, the clinical application of small molecules is greatly limited.

[0004] Nanodelivery carriers not only improve the solubility of anticancer drugs, prolong the body circulation time, controllably release and degrade, flexibly embed targeting groups and different drugs to realize multifunctional diagnosis and treatment, and realize the maximum accumulation of drugs in tumor sites through the difference between normal tissues and tumors (EPR effect), thereby greatly improving the off-target toxicity and tumor cell drug resistance of traditional platinum drugs. Therefore, the development of new nanocarrier systems, especially the design of tetravalent platinum prodrug polymer drug delivery systems, is of great significance to the development and clinical transformation of platinum drugs, and has become a hot research topic for scientists.

[0005] Anticancer drugs are typically embedded into drug-loaded systems through physical encapsulation, polymer grafting, or electrostatic complexation. These methods often suffer from drawbacks such as premature release, low drug loading, and uncontrolled release. However, designing tetravalent platinum prodrugs as monomers to participate in polymer synthesis can effectively overcome these shortcomings. For example, Chinese patent document CN105254867A discloses a copolymer of a tetravalent platinum prodrug, where the prodrug is designed as a dicarboxylic acid derivative, further coupled with diamine and polyethylene glycol to generate an amphiphilic triblock coordinated linear polymer. Its nanoparticles exhibit extremely high drug loading and precise chain segment distribution. However, its synthesis requires a long reaction time, multiple catalysts, low monomer conversion, and difficulty in preparing high molecular weight polypeptides. Chinese patent document CN105254836A... Patent literature discloses a copolymer of photosensitive tetravalent platinum prodrugs. The preparation method involves oxidizing with hydrogen peroxide to obtain a photosensitive tetravalent platinum prodrug with dihydroxyl coordination, which is then copolymerized with isonitriles and polyethylene glycol to obtain an amphiphilic triblock polymer. However, the monomer functional groups have insufficient tolerance, the isonitriles have a strong pungent odor, and although the monomer conversion rate has been improved, the polymer's controllability is weak, and the molecular weight is not optimistic. Huang Yubin's research group reported a tetravalent platinum prodrug monomer of diazid, which reacts with ester alkyne at 60°C for 48 h to obtain an amphiphilic block copolymer (J. Mater. Chem. B, 2017, 5, 2115-2125). However, the preparation, storage, and use of the diazid monomer pose a potential explosion risk, and the reaction requires high temperature and long reaction time, further limiting its application range.

[0006] In 2017, Tang Benzhong's research group reported a catalytic-free spontaneous click polymerization reaction of ester-activated diacetyl monomers with diamine monomers. This reaction is characterized by mild conditions, readily available monomers, high polymer yield, high molecular weight, high atom economy, and excellent regioselectivity and stereoselectivity. High-molecular-weight (weight-average molecular weight up to 64,400) anti-Markovnikov E-addition polymers can be obtained in high yields (up to 99%) within 3 hours at room temperature without a catalyst (J. Am. Chem. Soc. 2017, 139, 5437-5443). Inspired by this, we designed and synthesized an ester-acetylene monomer for a tetravalent platinum prodrug, which was copolymerized with a diamine monomer and monomethyl ether polyethylene glycol mPEG-NH2 to obtain a triblock copolymer with a molecular weight as high as 40,000. Furthermore, this polyβ-enamine ester structure exhibits acidic and reactive oxygen species responsive properties, providing a new reference and approach for the design and synthesis of multifunctional tetravalent platinum prodrug polymer supports. Summary of the Invention

[0007] The present invention aims to provide a tetravalent platinum ester acetylene complex, a biodegradable polymer, nanomicelles, and their preparation methods and applications. The tetravalent platinum ester acetylene complex can specifically respond to reducing substances in the tumor microenvironment, and the enamine ester structure formed by the reaction with diamine also has the property of responding to an acidic environment. The polymer is prepared under mild and simple conditions, with a molecular weight of up to 35,200 and a platinum drug loading of up to 33.91%. The nanomicelles can efficiently degrade and release anticancer active drugs in the tumor microenvironment, exhibiting high antitumor activity and low cytotoxicity.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention first provides a tetravalent platinum ester yne complex having the structure shown in formula (Ⅰ):

[0010]

[0011] In formula (Ⅰ), the Selected from:

[0012] This invention also provides a method for preparing a tetravalent platinum ester yne complex, comprising the following:

[0013] Step 1: React the divalent platinum complex with hydrogen peroxide to obtain the tetravalent platinum complex;

[0014] Step 2: React the tetravalent platinum complex obtained in Step 1 with triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride to obtain a tetravalent platinum ester acetylene complex intermediate.

[0015] Step 3: Add the tetravalent platinum ester yne complex intermediate obtained in Step 2 to TBAF and AcOH to react and obtain the tetravalent platinum ester yne complex;

[0016] Preferably, the divalent platinum complex is at least one of cisplatin, carboplatin, and oxaliplatin.

[0017] Preferably, the reaction temperature in step two is 40-70℃, and the reaction time is 12-24h.

[0018] Preferably, the molar ratio of the tetravalent platinum complex to triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride is 1:(2-6).

[0019] Preferably, the reaction temperature in step three is room temperature, and the reaction time is 12-24 hours.

[0020] Preferably, the molar ratio of the tetravalent platinum ester yne complex intermediate, TBAF and AcOH is 1:(2-4):(4-8).

[0021] The present invention also provides a biodegradable polymer having the structure shown in formula (II):

[0022]

[0023] in, This indicates monomethyl ether polyethylene glycol mPEG-NH2;

[0024] It represents a diamine;

[0025] The above refers to the tetravalent platinum ester acetylide complex, wherein the number of the tetravalent platinum ester acetylide complex is 10-40;

[0026] The They are connected by enamine bonds.

[0027] Preferably, the number average molecular weight of the monomethyl ether polyethylene glycol mPEG-NH2 is 1000-10000.

[0028] Preferably, the diamine is at least one of N,N'-dimethyl-1,6-hexanediamine, 1,3-di(4-piperidinyl)propane, or 4,4'-((1,2-diphenyl ether-1,2-diyl)bis(4,1-phenyl ether))bis(oxy))bis(N-n-propylbutane-1-amine).

[0029] Preferably, the tetravalent platinum ester ytylene complex has the following structure:

[0030]

[0031] The present invention also provides a method for preparing a biodegradable polymer, comprising the following:

[0032] The above tetravalent platinum ester acetylene complex was reacted with diamine, and then reacted with monomethyl ether polyethylene glycol mPEG-NH2 to obtain a biodegradable polymer.

[0033] Preferably, the reaction of the tetravalent platinum ester acetylene complex with diamine and monomethyl ether polyethylene glycol mPEG-NH2 involves the following steps:

[0034] ① The diamine is added to the tetravalent platinum ester ytylene complex to react and a reaction solution is obtained;

[0035] ② Add monomethyl ether polyethylene glycol mPEG-NH2 to the above solution and react to obtain a biodegradable polymer.

[0036] Preferably, the molar ratio of the tetravalent platinum ester acetylene complex, the diamine, and the monomethyl ether polyethylene glycol mPEG-NH2 is 1:(0.8-0.9):(0.2-0.3).

[0037] Preferably, the reaction temperature in ① is room temperature, and the reaction time is 1-3 hours.

[0038] Preferably, the reaction temperature in step ② is room temperature, and the reaction time is 6-12 hours.

[0039] The present invention also provides a nanomicelle prepared from the above-mentioned biodegradable polymer.

[0040] This invention also provides a method for preparing nanomicelles, comprising the following:

[0041] The biodegradable polymer was dissolved in an organic solvent, and distilled water was added dropwise under ultrasonication. The mixture was stirred and allowed to evaporate naturally to remove the organic solvent, thus obtaining nanomicelles.

[0042] The present invention also provides the application of the above-mentioned tetravalent platinum ester acetylene complex, the above-mentioned biodegradable polymer and the above-mentioned nanomicelles in the preparation of anticancer drugs.

[0043] The beneficial effects of this invention are:

[0044] The present invention first provides a tetravalent platinum ester acetylene complex with the structural formula shown in formula (I), which can specifically respond to reducing substances in the tumor microenvironment, and the enamine ester structure generated by the reaction with diamine also has the property of responding to acidic environment.

[0045] The present invention also provides a biodegradable polymer with the structural formula shown in formula (II). This biodegradable polymer introduces a reduction-responsive tetravalent platinum ester acetylene complex into the polymer backbone, with long alkyl chains or aromatic rings as hydrophobic segments and monomethyl ether polyethylene glycol mPEG-NH2 end caps as hydrophilic segments, and is connected by an acid-responsive enamine ester structure. The polymer molecular weight can reach 35,200, and the platinum drug loading is as high as 33.91%.

[0046] This invention also provides a method for preparing a biodegradable polymer. This method uses alkyne-amine click polymerization to simply stir tetravalent platinum ester alkyne complex, diamine, and monomethyl ether polyethylene glycol mPEG-NH2 in a one-pot process to obtain a polymer with a molecular weight as high as 35,200. The method is mild and can be carried out spontaneously at room temperature without a catalyst. The monomers are simple and readily available, and the polymer yield, molecular weight, and atom economy are high.

[0047] The present invention also provides nanomicelles prepared from the above-mentioned biodegradable polymers. These nanomicelles can efficiently degrade and release anticancer active drugs in the tumor microenvironment, exhibiting high antitumor activity and low cytotoxicity. Attached Figure Description

[0048] Figure 1 The NMR spectrum of the tetravalent platinum ester yne complex obtained in Example 3 of this invention;

[0049] Figure 2 This is the mass spectrum of the tetravalent platinum ester yne complex obtained in Example 3 of the present invention;

[0050] Figure 3 The elemental analysis diagram of the tetravalent platinum ester yne complex prepared in Example 3 of this invention is shown below.

[0051] Figure 4 The biodegradable polymer mPEG prepared in Example 6 of this invention 5k -Pt-mPEG 5k ICP-OES diagram;

[0052] Figure 5 The tetravalent platinum ester yne complex prepared in Example 3 and the biodegradable polymer mPEG prepared in Example 6 are examples of the present invention. 5k -Pt-mPEG 5k Infrared spectrum;

[0053] Figure 6 The biodegradable polymer mPEG prepared in Example 6 of this invention 5k -Pt-mPEG 5k GPC spectrum;

[0054] Figure 7 mPEG, a biodegradable polymer nanomicelle prepared in Example 9 of this invention. 5k -Pt-mPEG 5k DLS diagram;

[0055] Figure 8 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Transmission electron microscopy image of nanomicelles;

[0056] Figure 9 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Absorption spectrum of nanomicelles;

[0057] Figure 10 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Emission spectrum of nanomicelles;

[0058] Figure 11 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k DLS images of nanomicelles incubated for 9 hours under different conditions;

[0059] Figure 12 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k GPC spectra of nanomicelles incubated for 9 hours under different conditions;

[0060] Figure 13 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Transmission electron microscopy images of nanomicelles incubated for 9 hours under different environments;

[0061] Figure 14 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Cumulative release of platinum content from nanomicelles after 24 hours of incubation under different environments;

[0062] Figure 15 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Cytotoxicity curves of 4T1 cells treated with nanomicelles, the tetravalent platinum ester acetylene complex prepared in Example 3, and oxaliplatin for 48 h. Detailed Implementation

[0063] This invention first provides a tetravalent platinum ester yne complex having the structure shown in formula (Ⅰ):

[0064]

[0065] In formula (Ⅰ), the Selected from:

[0066] This invention also provides a method for preparing a tetravalent platinum ester yne complex, comprising the following:

[0067] Step 1: React the divalent platinum complex with hydrogen peroxide to obtain the tetravalent platinum complex;

[0068] Step 2: React the tetravalent platinum complex obtained in Step 1 with triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride to obtain a tetravalent platinum ester acetylene complex intermediate.

[0069] Step 3: Add the tetravalent platinum ester yne complex intermediate obtained in Step 2 to TBAF and AcOH to react and obtain the tetravalent platinum ester yne complex;

[0070] According to the present invention, a divalent platinum complex is first reacted with hydrogen peroxide to obtain a tetravalent platinum complex. The reaction temperature is preferably room temperature, the reaction time is preferably 12 hours, and the ratio of the divalent platinum complex to 30% hydrogen peroxide is preferably 1 g of divalent platinum complex / 10 ml of hydrogen peroxide (mass / volume). The divalent platinum complex is preferably cisplatin, carboplatin, or oxaliplatin.

[0071] According to the present invention, triisopropylsilylacetylene or tert-butyldimethylsilylacetylene is first reacted with n-butyllithium, preferably at -78°C under nitrogen conditions in a tetrahydrofuran solvent for 1 hour. Then, carbon dioxide is introduced and the reaction is carried out for 2 hours. The reaction is then quenched by adding 1M hydrochloric acid aqueous solution. After extraction and rotary evaporation, triisopropylsilylpropyonic acid or tert-butyldimethylsilylpropyonic acid is obtained. The ratio of triisopropylsilylacetylene or tert-butyldimethylsilylacetylene to n-butyllithium is preferably 1 / 1.2 (mol / mol), and the order of addition is triisopropylsilylacetylene or tert-butyldimethylsilylacetylene, n-butyllithium, and carbon dioxide.

[0072] According to the present invention, the above-obtained triisopropylsilylpropiodic acid or tert-butyldimethylsilylpropiodic acid is reacted with N,N'-dicyclohexylcarbodiimide (DCC) to obtain triisopropylsilylpropiodic anhydride or tert-butyldimethylsilylpropiodic anhydride. The preferred specific steps are: N,N'-dicyclohexylcarbodiimide (DCC) is added dropwise to the above-obtained triisopropylsilylpropiodic acid or tert-butyldimethylsilylpropiodic acid DCM solution, preferably at 0°C for 30-120 min, and then preferably reacted at room temperature for 12 h. The molar ratio of the triisopropylsilylpropiodic acid or tert-butyldimethylsilylpropiodic acid to N,N'-dicyclohexylcarbodiimide (DCC) is preferably 1 / (0.5-0.75).

[0073] According to the present invention, the triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride obtained above is dissolved in a solvent, preferably N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), preferably at 40-70°C for 12-24 hours. The solvent is then removed by vacuum drying, the remaining substance is dissolved in methanol, and insoluble matter is filtered off. The filtrate is concentrated and precipitated in diethyl ether and n-hexane, preferably placed in a refrigerator for 4-6 hours. After centrifugation, filtration and drying are performed to obtain the tetravalent platinum ester acetylene complex intermediate. The molar ratio of the triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride to the tetravalent platinum complex is preferably 1:(2-6); the volume ratio of diethyl ether to n-hexane is 1:2; the structural formula of the tetravalent platinum ester acetylene complex intermediate is as follows:

[0074]

[0075] According to the present invention, the above-mentioned tetravalent platinum ester yne complex intermediate is dissolved in methanol, preferably by adding a mixed solution of TBAF and AcOH at 0°C, and then reacting at room temperature for 12 h. The mixture is filtered to obtain a white solid, which is washed with a 1% AcOH aqueous solution and dried under vacuum to obtain the tetravalent platinum ester yne complex. The molar ratio of the tetravalent platinum ester yne complex intermediate, TBAF and AcOH is 1:(2-4):(4-8).

[0076] The present invention also provides a biodegradable polymer having the structure shown in formula (II):

[0077]

[0078]

[0079] in, This refers to monomethyl ether polyethylene glycol mPEG-NH2, wherein the number average molecular weight of the monomethyl ether polyethylene glycol mPEG-NH2 is preferably 1000-10000;

[0080] The diamine is indicated, preferably N,N'-dimethyl-1,6-hexanediamine, 1,3-di(4-piperidinyl)propane or 4,4'-((1,2-diphenyl ether-1,2-diyl)bis(4,1-phenyl ether))bis(oxy))bis(N-n-propylbutane-1-amine);

[0081] This refers to a tetravalent platinum ester yne complex, wherein the number of tetravalent platinum ester yne complexes is 10-40; preferably, the tetravalent platinum ester yne complex has the following structure:

[0082]

[0083] The present invention also provides a method for preparing a biodegradable polymer, comprising the following:

[0084] A biodegradable polymer was obtained by reacting a tetravalent platinum ester acetylene complex with diamine and monomethyl ether polyethylene glycol mPEG-NH2.

[0085] According to the present invention, the above-mentioned tetravalent platinum ester acetylene complex is reacted with diamine and monomethyl ether polyethylene glycol mPEG-NH2. The preferred steps are as follows: the diamine is added to the DMF solution of the above-mentioned tetravalent platinum ester acetylene complex, preferably stirred at room temperature for 1-3 h, end-capped with monomethyl ether polyethylene glycol mPEG-NH2, preferably stirred at room temperature for 6-12 h, then dialyzed with distilled water, and finally freeze-dried to obtain a biodegradable polymer. The diamine is preferably N,N'-dimethyl-1,6-hexanediamine, 1,3-di(4-piperidinyl)propane or 4,4'-((1,2-diphenyl ether-1,2-diyl)bis(4,1-phenyl ether))bis(oxy))bis(N-n-propylbutane-1-amine); the molar ratio of the tetravalent platinum ester acetylene complex, the diamine and the monomethyl ether polyethylene glycol mPEG-NH2 is preferably 1:(0.8-0.9):(0.2-0.3).

[0086] This invention also provides nanomicelles prepared from the above-mentioned biodegradable polymer. The method for preparing the nanomicelles preferably includes the following steps:

[0087] (1) Dissolve the biodegradable polymer in THF, wherein the mass / volume concentration of the biodegradable polymer is 1-10 mg / ml;

[0088] (2) Under ultrasound, add double-distilled water dropwise to the biodegradable polymer solution to form a micelle solution, with the water volume being 5-10 times the volume of THF.

[0089] (3) Stir the formed micelle solution overnight to allow the residual solvent THF to evaporate naturally;

[0090] (4) Concentrate the micelle solution after removing the solvent to a concentration of 1-5 mg / mL.

[0091] (5) Freeze-dry to obtain flocculent nanomicelles.

[0092] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the compounds with anticancer activity provided by the present invention and their preparation methods.

[0093] Example 1

[0094] In this embodiment, the structural formula of the obtained tetravalent platinum ester acetylene complex is shown below:

[0095]

[0096] The synthesis route is as follows:

[0097]

[0098]

[0099] (1) Dissolve triisopropylsilylacetylene (1 ml, 4.46 mmol) in 40 ml THF, place in a 250 ml flask and cool to -78 °C. Add n-butyllithium (2.5 M n-hexane solution, 4.8 ml, 10 mmol) under nitrogen protection, stir for 1 h, bubble carbon dioxide gas into the reaction solution, quench the reaction with 30 ml 1 M hydrochloric acid aqueous solution after 2 h, restore the solution to room temperature, extract with ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, and remove the solvent under reduced pressure to obtain a yellow sugar-oil-like liquid with a pungent odor. After cooling, a white layered solid intermediate 1 is formed with a yield of 100%.

[0100] (2) The intermediate 1 (565 mg, 2.5 mmol) obtained in step (1) was dissolved in 10 ml of ultra-dry DCM and placed in a 100 ml flask and cooled to 0 °C. N,N'-dicyclohexylcarbodiimide (DCC) (258 mg, 1.25 mmol) was dissolved in 10 ml of ultra-dry DCM and added dropwise to the reaction solution over a period of 30 min. The reaction solution was then brought back to room temperature and stirred for 12 h. The white insoluble solid was filtered off and the solution was evaporated to dryness to obtain intermediate 2, which was a yellow syrupy liquid with a yield of 100%.

[0101] (3) Cisplatin (1g, 3.3mmol) was placed in a flask, 10ml of 30% hydrogen peroxide was added, and the mixture was stirred at 40℃ in the dark for 12h. The hydrogen peroxide was removed by filtration to obtain intermediate 3 as a yellow solid powder with a yield of 88%.

[0102] (4) The obtained intermediate 2 (1.25 mmol) and intermediate 3 (102.6 mg, 0.31 mmol) were dissolved in 5 ml of ultra-dry DMF, placed in a 100 ml flask, and stirred thoroughly at 50 °C for 24 h. Then the solvent of the system was dried, the remaining substances were dissolved in methanol, and the insoluble matter was filtered off. The filtrate was concentrated and precipitated in diethyl ether and n-hexane. It was placed in a refrigerator for 4-6 h, centrifuged, filtered, and dried to obtain intermediate 4 with a yield of 51%.

[0103] (5) The intermediate 4 (26.2 mg, 0.035 mmol) obtained above was dissolved in methanol (2 ml) to form a saturated solution, placed in a 25 ml flask, and a mixed solution of TBAF (1.0 M THF solution, 105 μL, 0.105 mmol) and AcOH (12.6 mg, 0.21 mmol) was added at 0 °C. The mixture was then reacted at room temperature for 12 h. The mixture was filtered to obtain a white solid, which was washed with 1% AcOH aqueous solution and dried under vacuum to obtain a tetravalent platinum ester yne complex with a yield of 80%.

[0104] Example 2

[0105] In this embodiment, the structural formula of the obtained tetravalent platinum ester acetylene complex is shown below:

[0106]

[0107] The synthesis route is as follows:

[0108]

[0109] (1) Dissolve triisopropylsilylacetylene (1 ml, 4.46 mmol) in 40 ml THF, place in a 250 ml flask and cool to -78 °C. Add n-butyllithium (2.5 M n-hexane solution, 4.8 ml, 10 mmol) under nitrogen protection, stir for 1 h, bubble carbon dioxide gas into the reaction solution, quench the reaction with 30 ml 1 M hydrochloric acid aqueous solution after 2 h, restore the solution to room temperature, extract with ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, and remove the solvent under reduced pressure to obtain a yellow sugar-oil-like liquid with a pungent odor. After cooling, a white layered solid intermediate 1 is formed with a yield of 100%.

[0110] (2) The intermediate 1 (565 mg, 2.5 mmol) obtained in step (1) was dissolved in 10 ml of ultra-dry DCM and placed in a 100 ml flask and cooled to 0 °C. N,N'-dicyclohexylcarbodiimide (DCC) (258 mg, 1.25 mmol) was dissolved in 10 ml of ultra-dry DCM and added dropwise to the reaction solution over a period of 30 min. The reaction solution was then brought back to room temperature and stirred for 12 h. The white insoluble solid was filtered off and the solution was evaporated to dryness to obtain intermediate 2, which was a yellow syrupy liquid with a yield of 100%.

[0111] (3) Place cisplatin (1g, 2.69mmol) in a flask, add 10ml of 30% hydrogen peroxide, stir at 40℃ in the dark for 12h, filter to remove hydrogen peroxide to obtain intermediate 6 as a yellow solid powder with a yield of 86%.

[0112] (4) The obtained intermediate 2 (1.25 mmol) and intermediate 6 (125 mg, 0.31 mmol) were dissolved in 5 ml of ultra-dry DMF, placed in a 100 ml flask, and stirred thoroughly at 50 °C for 24 h. Then the solvent of the system was dried, the remaining substances were dissolved in methanol, and the insoluble matter was filtered off. The filtrate was concentrated and precipitated in diethyl ether and n-hexane. It was placed in a refrigerator for 4-6 h, centrifuged, filtered, and dried to obtain intermediate 7 with a yield of 47%.

[0113] (5) The intermediate 7 (28.8 mg, 0.035 mmol) obtained above was dissolved in methanol (2 ml) to form a saturated solution, placed in a 25 ml flask, and a mixed solution of TBAF (1.0 M THF solution, 105 μL, 0.105 mmol) and AcOH (12.6 mg, 0.21 mmol) was added at 0 °C. The mixture was then reacted at room temperature for 12 h. The mixture was filtered to obtain a white solid, which was washed with 1% AcOH aqueous solution and dried under vacuum to obtain a tetravalent platinum ester yne complex with a yield of 85%.

[0114] Example 3

[0115] In this embodiment, the structural formula of the obtained tetravalent platinum ester acetylene complex is shown below:

[0116]

[0117] The synthesis route is as follows:

[0118]

[0119] (1) Dissolve triisopropylsilylacetylene (1 ml, 4.46 mmol) in 40 ml THF, place in a 250 ml flask and cool to -78 °C. Add n-butyllithium (2.5 M n-hexane solution, 4.8 ml, 10 mmol) under nitrogen protection, stir for 1 h, bubble carbon dioxide gas into the reaction solution, quench the reaction with 30 ml 1 M hydrochloric acid aqueous solution after 2 h, restore the solution to room temperature, extract with ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, and remove the solvent under reduced pressure to obtain a yellow sugar-oil-like liquid with a pungent odor. After cooling, a white layered solid intermediate 1 is formed with a yield of 100%.

[0120] (2) The intermediate 1 (565 mg, 2.5 mmol) obtained in step (1) was dissolved in 10 ml of ultra-dry DCM and placed in a 100 ml flask and cooled to 0 °C. N,N'-dicyclohexylcarbodiimide (DCC) (258 mg, 1.25 mmol) was dissolved in 10 ml of ultra-dry DCM and added dropwise to the reaction solution over a period of 30 min. The reaction solution was then brought back to room temperature and stirred for 12 h. The white insoluble solid was filtered off and the solution was evaporated to dryness to obtain intermediate 2, which was a yellow syrupy liquid with a yield of 100%.

[0121] (3) Place Oxaliplatin (1g, 2.52mmol) in a flask, add 10ml of 30% hydrogen peroxide, stir at 40℃ in the dark for 12h, filter to remove hydrogen peroxide to obtain intermediate 9 as a yellow solid powder, with a yield of 86%.

[0122] (4) The obtained intermediate 2 (1.25 mmol) and intermediate 9 (133.6 mg, 0.31 mmol) were dissolved in 5 ml of ultra-dry DMF, placed in a 100 ml flask, and stirred thoroughly at 50 °C for 24 h. Then the solvent of the system was dried, the remaining substances were dissolved in methanol, and the insoluble matter was filtered off. The filtrate was concentrated and precipitated in diethyl ether and n-hexane. It was placed in a refrigerator for 4-6 h, centrifuged, filtered, and dried to obtain intermediate 10 with a yield of 50%.

[0123] (5) The intermediate 10 (29.7 mg, 0.035 mmol) obtained above was dissolved in methanol (2 ml) to form a saturated solution, placed in a 25 ml flask, and a mixed solution of TBAF (1.0 M THF solution, 105 μL, 0.105 mmol) and AcOH (12.6 mg, 0.21 mmol) was added at 0 °C. The mixture was then reacted at room temperature for 12 h. The mixture was filtered to obtain a white solid, which was washed with 1% AcOH aqueous solution and dried under vacuum to obtain a tetravalent platinum ester yne complex with a yield of 90%.

[0124] Figure 1 This is the NMR spectrum of the tetravalent platinum ester yne complex obtained in Example 3 of the present invention. Figure 2 This is the mass spectrum of the tetravalent platinum ester yne complex obtained in Example 3 of the present invention. Figure 1 and Figure 2 This indicates that each peak has a clear affiliation, and the integral area ratio also corresponds to the proportion of H in the structure, demonstrating that the present invention has successfully synthesized a tetravalent platinum ester acetylene complex.

[0125] Figure 3 The image shows the elemental analysis of the tetravalent platinum ester acetylene complex obtained in Example 3 of this invention. The actual measured values ​​of each element are consistent with the theoretical values.

[0126] Example 4

[0127] The tetravalent platinum ester acetylene complex (24.5 mg, 0.056 mmol) from Example 1 and N,N'-dimethyl-1,6-hexanediamine (6.5 mg, 0.045 mmol) were dissolved in 225 μL of DMF solution and stirred at room temperature for 3 h. Then, 60 mg of monomethyl ether polyethylene glycol mPEG was added. 5k -NH2 was dissolved in 1 ml of DMF and added to the reaction mixture. The mixture was stirred at room temperature for 12 h, then dialyzed with distilled water, and finally freeze-dried to obtain the biodegradable polymer.

[0128] Example 5

[0129] The tetravalent platinum ester acetylene complex (28.5 mg, 0.056 mmol) from Example 2 and 1,3-bis(4-piperidinyl)propane (9.5 mg, 0.045 mmol) were dissolved in 225 μL of DMF solution and stirred at room temperature for 3 h. Then, 60 mg of monomethyl ether polyethylene glycol mPEG was added. 5k -NH2 was dissolved in 1 ml of DMF and added to the reaction mixture. The mixture was stirred at room temperature for 12 h, then dialyzed with distilled water, and finally freeze-dried to obtain the biodegradable polymer.

[0130] Example 6

[0131] The tetravalent platinum ester acetylene complex (30 mg, 0.056 mmol) from Example 3 and 4,4'-((1,2-diphenyl ether-1,2-diyl)bis(4,1-phenyl ether))bis(oxy))bis(N-n-propylbutane-1-amine) (26.5 mg, 0.045 mmol) were dissolved in 225 μL of DMF solution and stirred at room temperature for 3 h. Then, 60 mg of monomethyl ether polyethylene glycol mPEG was added. 5k -NH2 was dissolved in 1 ml of DMF and added to the reaction mixture. The mixture was stirred at room temperature for 12 h, then dialyzed against distilled water, and finally freeze-dried to obtain the biodegradable polymer mPEG synthesized from the tetravalent platinum ester ytylene complex. 5k -Pt-mPEG 5k .

[0132] Figure 4 The biodegradable polymer mPEG containing a tetravalent platinum ester yne complex was prepared in Example 6 of this invention. 5k -Pt-mPEG 5k The ICP-OES plot showed that the platinum content in the polymer was as high as 33.91%.

[0133] Figure 5 The tetravalent platinum ester yne complex prepared in Example 3 of this invention and the biodegradable polymer mPEG containing the tetravalent platinum ester yne complex prepared in Example 6 are examples of the present invention. 5k -Pt-mPEG 5k The infrared spectrum shows that, compared with small molecule monomers, the disappearance of the triple bond peak and the formation of the double bond peak in the polymer indicate that the present invention has successfully synthesized a biodegradable polymer.

[0134] Figure 6 The biodegradable polymer mPEG prepared in Example 6 of this invention 5k -Pt-mPEG 5k The GPC spectrum, from Figure 6 It can be seen that the molecular weight of the polymer is 35200.

[0135] Example 7

[0136] Dissolve 7.5 mg of the biodegradable polymer obtained in Example 4 in 3 ml of THF, place it in a 50 ml centrifuge tube and sonicate it. Slowly add 15 ml of double-distilled water to form a micelle solution. Stir the formed micelle solution overnight and allow it to evaporate naturally to remove the residual solvent THF. Concentrate the solvent-free micelle solution to 2 mg / ml and freeze-dry it to obtain biodegradable polymer nanomicelle flocs.

[0137] Example 8

[0138] Dissolve 7.5 mg of the biodegradable polymer obtained in Example 5 in 3 ml of THF, place it in a 50 ml centrifuge tube and sonicate it. Slowly add 15 ml of double-distilled water to form a micelle solution. Stir the formed micelle solution overnight and allow it to evaporate naturally to remove the residual solvent THF. Concentrate the solvent-free micelle solution to 2 mg / ml and freeze-dry it to obtain biodegradable polymer nanomicelle flocs.

[0139] Example 9

[0140] 7.5 mg of the biodegradable polymer obtained in Example 6 was dissolved in 3 ml of THF, placed in a 50 ml centrifuge tube, and sonicated. 15 ml of double-distilled water was slowly added dropwise to form a micelle solution. The micelle solution was stirred overnight and allowed to evaporate naturally to remove the residual solvent THF. The solvent-free micelle solution was concentrated to 2 mg / ml and freeze-dried to obtain biodegradable polymer nanomicelle flocs, referred to as mPEG. 5k -Pt-mPEG 5k Nano micelles.

[0141] Figure 7 This is a DLS image of the biodegradable polymer nanomicelles prepared in Example 9 of this invention. Figure 7 It can be seen that the average particle size of the micelles is 44 nm.

[0142] Figure 8 This is a transmission electron microscope (TEM) image of the biodegradable polymer nanomicelles prepared in Example 9 of this invention. Figure 8 It can be seen that the nanomicelles are spherical and do not aggregate, with an average particle size of 38 nm.

[0143] Figure 9 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k The absorption spectrum of the nanomicelles shows that the absorption of the TPE unit in the polymer is around 330 nm.

[0144] Figure 10 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG5k The emission spectrum of the nanomicelles shows that the TPE units in the polymer emit near 460 nm.

[0145] Figure 11 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k DLS images of nanomicelles after 9 hours of incubation under different conditions.

[0146] Figure 12 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k GPC spectra of nanomicelles after incubation for 9 hours under different conditions.

[0147] Figure 13 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Transmission electron microscopy images of nanomicelles incubated for 9 hours under different conditions.

[0148] Figure 14 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Cumulative release of platinum content from nanomicelles after 24 hours of incubation under different environments.

[0149] from Figures 11-14 It can be seen that nanomicelles have the ability to respond quickly, degrade efficiently, and release under acidic and reducing environments.

[0150] Example 10

[0151] The mPEG obtained in Example 9 of this invention 5k -Pt-mPEG 5k Cytotoxicity testing of nanomicelles was performed using 4T1 cells as a model and the degradable polymer nanomicelles obtained in Example 9 as the test substance. After the test substance was applied to the cells, the cell viability was observed. The cytotoxicity of the degradable polymer nanomicelles of the present invention was examined using the MTT assay. The specific operation steps are as follows:

[0152] 1) Collect 4T1 cells, adjust the concentration of the cell suspension, and add 100 μL to each well of a 96-well plate, with approximately 5000 cells per well;

[0153] 2) Place the above 96-well plate in a cell culture incubator with a CO2 concentration of 5% and culture it at 37°C and saturated humidity for 24 hours to allow the cells to fully adhere to the plate.

[0154] 3) The biodegradable polymer nanomicelles were diluted in a certain gradient to a concentration of 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM and 0.6125 μM, and then added to a 96-well plate containing cells. Each concentration was used to make 6 replicates, and the incubation time was set to 48 h.

[0155] 4) Add 100 μL of MTT solution (0.5 mg / mL) to each well, continue culturing for 4 h, remove the culture medium, and add 100 μL of DMSO to each well;

[0156] 5) Place the above-mentioned plate into the microplate reader and shake it thoroughly to dissolve the crystals completely. Measure the absorbance of each well at 490 nm using the microplate reader.

[0157] 6) Simultaneously set up a control group (culture medium, cells, MTT, DMSO) and apoptosis wells (culture medium, DMSO, MTT). Calculate cell viability using the following formula:

[0158]

[0159] Where Abs(sample) is the absorbance value of cells in the sample group; Abs(blank) is the absorbance value of the liquid in the culture well of the blank control group; and Abs(control) is the absorbance value of cells in the untreated experimental group.

[0160] The biodegradable polymeric nanomicelles were obtained by replacing oxaliplatin and the tetravalent platinum ester acetylene complex of Example 3 with those of Example 9, and were subjected to the same cytotoxicity test.

[0161] Figure 15 The biodegradable polymer mPEG prepared in Example 9 of this invention 5k -Pt-mPEG 5k Cytotoxicity curves of 4T1 cells treated with nanomicelles, the tetravalent platinum ester acetylene complex prepared in Example 3, and oxaliplatin for 48 h.

[0162] from Figure 15 The curves in the figure can be used to determine oxaliplatin, the tetravalent platinum ester acetylene complex in Example 3, and the biodegradable polymer mPEG in Example 9. 5k -Pt-mPEG 5k IC50 of 4T1 cells treated with nanomicelles for 48 h 50 Value data (see Table 1).

[0163] Table 1

[0164] Sample IC 50 values (μM) Oxaliplatin 4.74 Tetravalent platinum alkynyl complex 8.50 Nanomicelle 23.66

[0165] Oxaliplatin has the smallest IC 50The value was highest for tetravalent platinum ester acetylene complexes and mPEG. 5k -Pt-mPEG 5k Nanomicelles demonstrate the safety of the nanomicelles provided by this invention. This is likely due to the tetravalent platinum ester acetylene and mPEG. 5k -Pt-mPEG 5k All nanomicelles are tetravalent and require a reduction process after entering cells. However, as the concentration continues to increase, mPEG... 5k -Pt-mPEG 5k The cytotoxicity curve of the nanomicelles showed a more pronounced downward trend, exhibiting stronger cytotoxicity than oxaliplatin at higher drug concentrations. This may be due to the higher uptake of the nanomicelles, demonstrating the good biocompatibility of the nanomicelles provided by this invention.

[0166] The method for preparing biodegradable polymers provided by this invention has mild conditions, simple and readily available monomers, high polymer yield, high molecular weight, and high atom economy. Experimental results show that the polymers provided by this invention have significant acid and reduction condition responses, can efficiently release anticancer active drugs, and the nanomicelles have good anticancer activity and low cytotoxicity.

[0167] The above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A tetravalent platinum ester yne complex, characterized in that, The structural formula (Ⅰ) is as follows: （Ⅰ） In formula (Ⅰ), the Selected from: , or .

2. The method for preparing the tetravalent platinum ester yne complex according to claim 1, characterized in that, Includes the following steps: (1) The divalent platinum complex was reacted with hydrogen peroxide to obtain the tetravalent platinum complex; (2) The tetravalent platinum complex obtained in step (1) is reacted with triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride to obtain a tetravalent platinum ester acetylene complex intermediate. (3) Add the tetravalent platinum ester yne complex intermediate obtained in step (2) to TBAF and AcOH to react and obtain the tetravalent platinum ester yne complex.

3. The preparation method according to claim 2, characterized in that, The divalent platinum complex mentioned in step (1) is at least one of cisplatin, carboplatin, and oxaliplatin; The reaction temperature in step (2) is 40-70°C. o C, the reaction time is 12-24 h; The molar ratio of the tetravalent platinum complex to triisopropylsilylpropynic anhydride or tert-butyldimethylsilylpropynic anhydride in step (2) is 1:(2-6). The reaction in step (3) is carried out at room temperature for 12-24 hours. The molar ratio of the tetravalent platinum ester acetylene complex intermediate, TBAF and AcOH in step (3) is 1:(2-4):(4-8).

4. A method for preparing a biodegradable polymer, characterized in that, The tetravalent platinum ester acetylene complex of claim 1 is reacted with a diamine, and then reacted with mPEG-NH2 to obtain a biodegradable polymer; wherein the diamine is at least one of N,N'-dimethyl-1,6-hexanediamine and 1,3-di(4-piperidinyl)propane.

5. The preparation method according to claim 4, characterized in that, The number-average molecular weight of the mPEG-NH2 is 1000-10000.

6. The preparation method according to claim 4, characterized in that, The molar ratio of the tetravalent platinum ester acetylene complex, diamine, and mPEG-NH2 is 1:(0.8-0.9):(0.2-0.3). The reaction of the tetravalent platinum ester acetylene complex with the diamine was carried out at room temperature for 1-3 hours. The reaction with mPEG-NH2 was carried out at room temperature for 6-12 hours.

7. A biodegradable polymer prepared by the method described in claim 4.

8. A type of nanomicelle, characterized in that, It is prepared from the biodegradable polymer described in claim 7.

9. The method for preparing nanomicelles according to claim 8, characterized in that, The biodegradable polymer was dissolved in an organic solvent, and distilled water was added dropwise under ultrasonication. The mixture was stirred and allowed to evaporate naturally to remove the organic solvent, thus obtaining nanomicelles.

10. The use of the tetravalent platinum ester acetylene complex of claim 1, the biodegradable polymer of claim 7, and the nanomicelles of claim 8 in the preparation of anticancer drugs.

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