Indometacin amphiphilic polymer prodrug as well as preparation method and application thereof

Abstract

The invention discloses an indometacin amphiphilic polymer prodrug as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry, the indometacin amphiphilic polymer prodrug comprises a polyethylene glycol hydrophilic segment and a poly (indometacin) hydrophobic segment, the preparation method comprises the following steps: (1) carrying out coupling reaction on methoxypolyethylene glycol and a small molecular chain transfer agent to prepare a macromolecular chain transfer agent; (2) taking indometacin and 2, 2 '-(propane-2, 2-diylbis (sulfanediyl)) diethanol as raw materials, carrying out single-ended esterification reaction to prepare a first intermediate, and carrying out methylacryloylation treatment on the first intermediate to obtain a second intermediate; and (3) carrying out RAFT free radical polymerization reaction on the macromolecular chain transfer agent and the second intermediate under the action of an initiator to obtain the indometacin amphiphilic polymer prodrug. The indometacin amphiphilic polymer prodrug is long in blood circulation time and good in treatment effect on acute pancreatitis.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to an indomethacin amphiphilic polymer drug precursor and a preparation method and application thereof. Background Art

[0002] Acute pancreatitis (AP) is an acute inflammatory disease of the pancreas. The abnormal activation of pancreatic enzymes leads to autodigestion of the pancreas, which may cause local damage or systemic complications. According to the new Atlanta classification criteria, AP is divided into three categories: mild AP (MAP), moderate AP (MSAP), and severe AP (SAP). MAP patients can recover quickly with symptomatic treatment, but a small number of patients will develop systemic inflammatory response syndrome (SIRS) due to the cascade effect of inflammatory factors, and fluid accumulation will occur in the pancreas or around the pancreas, leading to organ failure, namely MSAP. If the organ failure lasts for more than 48 hours, it will progress to SAP, and the mortality rate will increase significantly. Therefore, inhibiting the production and release of inflammatory factors is crucial for the treatment of acute pancreatitis.

[0003] Indomethacin is a nonsteroidal anti-inflammatory drug that can inhibit prostaglandin synthesis, inhibit serum phospholipase A2 activity, prevent leukocyte aggregation, reduce inflammatory response and damage to important organs. It is mostly used in the prevention of postoperative pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP) and the treatment of MAP. In order to improve the efficacy of indomethacin and its application range, researchers have carried out structural modification on it. For example, Chinese patent document with publication number CN118221668A discloses an indomethacin derivative containing 2-amino-1,3,4-thiadiazole, which is prepared by amide condensation of the free carboxyl group at the terminal of indomethacin with the active small molecules of the thiadiazole series, which has anti-inflammatory activity, enhances the anti-inflammatory activity of indomethacin, and reduces gastrointestinal side effects; Chinese patent document with publication number CN111888344A discloses a method for preparing an inflammation-responsive intelligent controlled-release drug-loaded composite medical membrane, which is composited with a drug precursor containing indomethacin and PCL as raw materials, and the drug precursor containing indomethacin is obtained by the reaction of hydroxyethyl methacrylate and indomethacin, and has inflammation responsiveness.

[0004] The accumulation of drugs in the pancreas is one of the difficulties in treating pancreatitis. In the existing technology, liposomes, polymer nanoparticles, etc. are usually used to encapsulate drugs and passively target the inflammatory site through the enhanced permeability and retention (EPR) effect. However, the relevant nanocarrier technology still needs to optimize the safety of the carrier and the large-scale preparation process during the clinical transformation process. If the blood circulation time of indomethacin can be increased by structural modification, it will be more suitable for industrial production. Summary of the invention

[0005] The invention provides an indomethacin amphiphilic polymer drug precursor, which has a long blood circulation time, can make indomethacin accumulate in the pancreas, enhance the therapeutic effect of acute pancreatitis, and is suitable for industrial production.

[0006] The specific technical solutions adopted are as follows: An indomethacin amphiphilic polymer drug precursor, the structural formula of which is shown in formula (I); (I); In formula (I), m is selected from an integer of 10-500, and n is selected from an integer of 1-100.

[0008] Preferably, in formula (I), m is selected from an integer of 100-500, and n is selected from an integer of 1-10.

[0009] The indomethacin amphiphilic polymer drug precursor includes a polyethylene glycol hydrophilic segment and a poly (indomethacin) hydrophobic segment. By bonding indomethacin to the polymer skeleton, its blood circulation time is increased, so that indomethacin accumulates in the pancreas, thereby enhancing the therapeutic effect of pancreatitis.

[0010] The present invention also provides a method for preparing the indomethacin amphiphilic polymer drug precursor, comprising the following steps: (1) A macromolecular chain transfer agent is prepared by coupling reaction between polyethylene glycol monomethyl ether and a small molecule chain transfer agent; (2) using indomethacin and 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol as raw materials to undergo a single-end esterification reaction to prepare a first intermediate, and subjecting the first intermediate to methacrylation to obtain a second intermediate; (3) The macromolecular chain transfer agent and the second intermediate undergo RAFT free radical polymerization under the action of an initiator. After the polymerization is completed, the indomethacin amphiphilic polymer drug precursor is obtained by dialyzing and freeze-drying.

[0011] Specifically, the coupling reaction in step (1) is carried out under the catalytic action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine and a nitrogen protective atmosphere, and the small molecule chain transfer agent is PETTC (4-cyano-4-(phenylcarbonylthio)pentanoic acid).

[0012] Preferably, the molar ratio of polyethylene glycol monomethyl ether to the small molecule chain transfer agent is 1:1-10, the coupling reaction temperature is 20-30°C, and the coupling reaction time is 18-24 h.

[0013] Specifically, the esterification reaction in step (2) is carried out under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine.

[0014] Preferably, the molar ratio of indomethacin to 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol is 1:1-2, the esterification reaction temperature is 20-30°C, and the esterification reaction time is 6-24 h.

[0015] Specifically, under the action of triethylamine, methyl propyl chloride is reacted with the first intermediate to prepare the second intermediate.

[0016] Specifically, in step (3), the molar ratio of the macromolecular chain transfer agent to the second intermediate is 1:10-20, the RAFT free radical polymerization reaction is carried out under anhydrous and oxygen-free conditions, the free radical polymerization reaction temperature is 75-85° C., and the reaction time is 18-24 h.

[0017] The present invention also provides a nano-micelle, which is formed by self-assembly of the indomethacin amphiphilic polymer drug precursor.

[0018] Preferably, the indomethacin amphiphilic polymer drug precursor is dissolved in an organic solvent, fully mixed, and then deionized water is added dropwise, stirred and dialyzed to obtain the nano-micelles.

[0019] The present invention also provides the use of the indomethacin amphiphilic polymer drug precursor or the nano micelle in the preparation of a drug for treating acute pancreatitis.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) In the indomethacin amphiphilic polymer drug precursor, the chemical bonds binding indomethacin can be efficiently broken in the pancreas, thereby ensuring its anti-inflammatory effect and having low toxicity to normal tissues.

[0021] (2) Indomethacin has poor water solubility. The present invention prepares an indomethacin amphiphilic polymer drug precursor by structural modification. The indomethacin amphiphilic polymer drug precursor can self-assemble to form nanoparticles, which greatly improves the solubility of indomethacin in water, making its in vivo application possible. In addition, the nanomicelles made of the indomethacin amphiphilic polymer drug precursor have a high indomethacin drug loading capacity, which can reduce the amount of drug used. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The synthetic route of the indomethacin amphiphilic polymer drug precursor IndoCP in Example 1.

[0023] Figure 2 This is a tissue section image of the indomethacin amphiphilic polymer drug precursor IndoCP in Example 1 after administration of the nanomicelles.

[0024] Figure 3 This is the tissue score diagram of the nanomicelles made of the amphiphilic polymer drug precursor IndoCP in Example 1 after administration, *** indicates p <0.001, discrete points represent data values ​​from parallel experiments. DETAILED DESCRIPTION

[0025] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description through specific embodiments. In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0026] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The contents not described in detail in this specification belong to the prior art known to professionals in the field.

[0027] Example 1

[0028] In this embodiment, the synthesis route of indomethacin amphiphilic polymer drug precursor IndoCP is as follows: Figure 1 As shown; (1) Synthesis of macromolecular chain transfer agent Take 2.5 g of polyethylene glycol monomethyl ether (0.5 mmol) with a molecular weight of 5000, dissolve it in 50 mL of toluene, reflux and remove water until the distilled toluene becomes clear, pass nitrogen to remove toluene as much as possible, add 50 mL of dichloromethane under nitrogen protection, cool in an ice bath, and add 300 mg of dicyclohexylcarbodiimide (1 mmol) under ice bath conditions, and slowly drop 20 mL of dichloromethane solution containing 4 times the molar amount of small molecule 4-cyano-4-(phenylcarbonylthio)pentanoic acid PETTC (relative to polyethylene glycol monomethyl ether) and 12 mg of 4-dimethylaminopyridine (0.1 mmol). After reacting at 25°C for 18 h, wash it 3 times with 1 N hydrochloric acid aqueous solution, wash it 3 times with saturated brine, dry it over anhydrous sodium sulfate, concentrate it to 3 mL by rotary evaporation, and add it to 100 The precipitate was collected and washed with pre-cooled anhydrous ether for more than three times, and dried in a vacuum oven at room temperature to obtain the product macromolecular chain transfer agent PEG-PETTC (2.1 g, yield 74%).

[0029] The NMR data of the obtained product macromolecular chain transfer agent are: 1 H NMR (500 MHz, DMSO-d6) δ 7.86 (s, 2H), 7.63 (s, 1H), 7.52 – 7.40 (m, 2H), 3.50 (s, 452H), 1.83 (s, 3H). (2) Synthesis of IndoSCSM Weigh 0.9 g (2.51 mmol) of indomethacin, add 0.5 g of 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol (2.55 mmol), mix and dissolve in 20 mL of dichloromethane (DCM), add 93 mg of 4-dimethylaminopyridine (DMAP), and stir in a 100 mL single-necked round-bottom flask. After complete cooling in an ice bath, 5 mL of dichloromethane solution containing 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) was slowly added to the above round-bottom flask through a constant pressure dropping funnel, remove the ice bath, and return to 25°C and stir overnight for 18 h. The solution after the reaction was washed three times with a 1 N dilute hydrochloric acid solution, dried and concentrated by rotary evaporation, and the concentrate was purified by column chromatography on a 200-mesh silica gel column using an ethyl acetate / n-hexane eluent with a volume ratio of 1:4 to obtain 0.34 g (yield 24%) of the intermediate IndoSCS.

[0030] The NMR data of the intermediate IndoSCS are: 1H NMR (500 MHz, Chloroform-d) δ 7.71 –7.64 (m, 2H), 7.50 – 7.44 (m, 2H), 6.97 (d, J = 2.5 Hz, 1H), 6.88 (d, J = 9.0Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 4.28 (t, J = 6.8 Hz, 2H), 3.85 (s,3H), 3.76 (s, 2H), 3.68 (s, 2H), 2.88 (t, J = 6.8 Hz, 2H), 2.80 (t, J = 6.1Hz, 2H), 2.39 (s, 3H), 1.60 (s, 6H). 0.34 g (0.63 mmol) of the intermediate IndoSCS was dissolved in 10 mL of dichloromethane, and 62 μL (0.64 mmol) of methyl propyl chloride and 90 μL of triethylamine (Et3N, 0.65 mmol) were added and stirred together in a 50 mL single-mouth round-bottom flask. After reacting overnight, the solution was concentrated by rotary evaporation, and the concentrate was purified by column chromatography on a 200-mesh silica gel column using an ethyl acetate / n-hexane eluent of 8%:92% by volume to obtain 0.31 g (yield 83%) of the polymerized monomer IndoSCSM.

[0031] The NMR data of polymerized monomer IndoSCSM are: 1 H NMR (500 MHz, Chloroform-d) δ 7.66(d, J = 6.7 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 2.5 Hz, 1H), 6.87(d, J = 9.0 Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.11 (s, 1H), 5.60 –5.54 (m, 1H), 4.28 (q, J = 7.0 Hz, 4H), 3.84 (s, 3H), 3.68 (s, 2H), 2.87 (dt,J = 8.6, 7.0 Hz, 4H), 2.39 (s, 3H), 1.93 (s, 3H), 1.60 (s, 6H). (3) Synthesis of Indomethacin Amphiphilic Polymer Drug Procurator IndoCP The synthesis of indomethacin amphiphilic polymer drug precursor IndoCP was carried out by RAFT free radical polymerization. 213 mg (0.04 mmol) of the macromolecular chain transfer agent PEG-PETTC synthesized in step (1) and 200 mg (0.42 mmol) of the polymer monomer IndoSCSM synthesized in step (2) were added to 20 mL of dry N,N - Dissolve in dimethylformamide (DMF), add azobisisobutyronitrile, and polymerize in an oil bath at 80 °C under argon protection for 18 h. After the polymerization, the solution was placed in a semipermeable membrane dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed with 250 mL of DMSO solution at 40 °C for 3 times, each time for 12 hours, and then dialyzed with 250 mL of DMF solution at 40 °C for 3 times, each time for 12 hours. The DMF was replaced with water and lyophilized to obtain the indomethacin amphiphilic polymer drug precursor IndoCP (280 mg, yield 50%).

[0032] The NMR data of indomethacin amphiphilic polymer drug precursor IndoCP are: 1 H NMR (500 MHz, DMSO-d6) δ 7.64 (s, 9H), 7.03 (s, 3H), 6.92 (s, 2H), 6.72 (s, 3H), 3.76 (s, 11H), 3.51 (s, 452H), 2.22 (s, 7H), 1.53 (s, 34H). The indomethacin amphiphilic polymer drug precursor IndoCP includes a polyethylene glycol hydrophilic segment and a poly (indomethacin) hydrophobic segment. The number of indomethacin residues in the poly (indomethacin) block is 3, and the number of polyethylene glycol repeating units is 114.

[0033] Example 2

[0034] Synthesis of nanoparticles: 5 mg of the indomethacin amphiphilic polymer drug precursor IndoCP powder synthesized in Example 1 was weighed and dissolved in 1 mL of dimethyl sulfoxide DMSO, fully mixed and ultrasonicated for 5 minutes, 5 mL of deionized water was slowly added thereto, and after fully stirring for 15 minutes, it was dialyzed three times in 500 mL of deionized water using a cellulose acetate semipermeable membrane (molecular weight cutoff of 3500 Da), each time for two hours. After the dialysis was completed, centrifuged at 3300 rpm for 5 minutes, and the supernatant was concentrated to a suitable concentration by vacuum distillation at 35°C to obtain a nanomicelle solution.

[0035] Sample analysis Efficacy verification: In the cholecystokinin-induced acute pancreatitis model, mice were intraperitoneally injected with cholecystokinin (50 μg / kg / h), once every hour, for seven consecutive injections. The last injection was co-administered with lipopolysaccharide (LPS; 10 mg / kg; Solebol). The mice were killed 12 hours after the first administration, and blood and tissue samples were collected. The mice were divided into 4 experimental groups (NC group, Cer+LPS group, IND group, and PPIND group). The NC group was the healthy control group, and the Cer+LPS group, IND group, and PPIND group were treated by tail vein injection of PBS, free indomethacin IND, and nanomicelle PPIND prepared in Example 2, respectively. The mouse lung tissue and pancreatic tissue were then isolated for histological evaluation, and the results are as follows. Figure 2 and Figure 3 As shown, it is proved that the nanomicelles made of indomethacin amphiphilic polymer drug precursor IndoCP have anti-pancreatitis effect.

[0036] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An amphiphilic polymer prodrug of indomethacin, characterized in that: The structural formula is shown in formula (I); （I）； In formula (I), m is selected from an integer of 10-500, and n is selected from an integer of 1-100.

2. The indomethacin amphiphilic polymer drug precursor according to claim 1, characterized in that: In formula (I), m is selected from an integer of 100-500, and n is selected from an integer of 1-10.

3. The method for preparing the indomethacin amphiphilic polymer drug precursor according to claim 1 or 2, characterized in that: The following steps are involved: (1) A macromolecular chain transfer agent is prepared by coupling reaction between polyethylene glycol monomethyl ether and a small molecule chain transfer agent; (2) using indomethacin and 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol as raw materials to undergo a single-end esterification reaction to prepare a first intermediate, and subjecting the first intermediate to methacrylation to obtain a second intermediate; (3) The macromolecular chain transfer agent and the second intermediate undergo RAFT free radical polymerization under the action of an initiator. After the polymerization is completed, the indomethacin amphiphilic polymer drug precursor is obtained by dialyzing and freeze-drying.

4. The method for preparing the indomethacin amphiphilic polymer drug precursor according to claim 3, characterized in that: The coupling reaction in step (1) is carried out under the catalytic action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine and a nitrogen protective atmosphere, and the small molecule chain transfer agent is 4-cyano-4-(phenylcarbonylthio)pentanoic acid.

5. The method for preparing the indomethacin amphiphilic polymer drug precursor according to claim 3, characterized in that: In step (1), the molar ratio of polyethylene glycol monomethyl ether to the small molecule chain transfer agent is 1:1-10, the coupling reaction temperature is 20-30°C, and the coupling reaction time is 18-24 h.

6. The method for preparing the indomethacin amphiphilic polymer drug precursor according to claim 3, characterized in that: The esterification reaction in step (2) is carried out under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, the molar ratio of indomethacin to 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol is 1:1-2, the esterification reaction temperature is 20-30°C, and the coupling reaction time is 6-24 h.

7. The method for preparing the indomethacin amphiphilic polymer drug precursor according to claim 3, characterized in that: Under the action of triethylamine, methyl propyl chloride is reacted with the first intermediate to prepare the second intermediate.

8. The method for preparing the indomethacin amphiphilic polymer drug precursor according to claim 3, characterized in that: In step (3), the molar ratio of the macromolecular chain transfer agent to the second intermediate is 1:10-20, the RAFT free radical polymerization reaction is carried out under anhydrous and oxygen-free conditions, the free radical polymerization reaction temperature is 75-85°C, and the reaction time is 18-24 h.

9. A nanomicelle, characterized in that: It is formed by self-assembly of the indomethacin amphiphilic polymer drug precursor described in claim 1 or 2.

10. Use of the indomethacin amphiphilic polymer drug precursor according to claim 1 or 2 or the nanomicelle according to claim 9 in the preparation of a drug for treating acute pancreatitis.

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