A polyethylene glycol-polyunsaturated fatty acid derivative, its preparation method and application

Abstract

This invention relates to a polyethylene glycol-polyunsaturated fatty acid derivative and its preparation method. The method involves dissolving methoxy polyethylene glycol amino 2000 and 3,5-dihydroxybenzoic acid in anhydrous N,N-dimethylformamide to obtain intermediate 1. Intermediate 1 is then dissolved in anhydrous N,N-dimethylformamide with 3-(2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid to obtain intermediate 2. Intermediate 2 is then dissolved in dichloromethane, and trifluoroacetic acid is added to obtain intermediate 3. Finally, intermediate 3 is dissolved in N,N-dimethylformamide with polyunsaturated fatty acids to obtain the final product. The polyethylene glycol-polyunsaturated fatty acid derivative prepared by this invention exhibits good resistance to multidrug resistance and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a polyethylene glycol-polyunsaturated fatty acid derivative, its preparation method, and its application. Background Technology

[0002] The development and progression of tumors are closely related to lipid metabolism disorders in the body, with lipids playing a key regulatory role in tumor cell proliferation, apoptosis, invasion, and metastasis. Phospholipids, as core structural components of biological membranes, are currently widely used as lipid drug carriers due to their good biocompatibility and amphiphilicity to enhance the solubility and targeted accumulation of anti-tumor drugs. However, phospholipids themselves have almost no direct inhibitory activity against tumor cells; their role is mainly limited to excipients or membrane structural support.

[0003] In contrast, polyunsaturated fatty acids (PUFAs) are a class of lipid molecules with well-defined biological activities. They mainly include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), alpha-linolenic acid, linoleic acid, arachidonic acid, and adrenaline, which can directly exert anti-tumor effects through multiple pathways, such as regulating tumor lipid metabolism, inducing ferroptosis, and inhibiting inflammatory signaling. Studies have shown that PUFAs can intervene in disease progression by regulating lipid composition and peroxidation levels: in the acidic tumor microenvironment, their induced lipid peroxidation can effectively trigger ferroptosis, achieving selective killing of tumor cells. Mechanistically, PUFAs are regulated by fatty acid acyltransferases, affecting fatty acid storage and utilization, thereby altering membrane lipid composition, redox balance, and cell survival signals. For example, DHA has been shown to induce cancer cell death by consuming glutathione to inactivate the lipid peroxidation inhibitor GPX4. Furthermore, PUFAs can also replenish the cell membrane's easily peroxidized lipid pool, promote ferroptosis, release damage-related molecular patterns, and initiate anti-tumor immune responses.

[0004] Based on the above mechanisms, the application prospects of PUFAs continue to expand: combined use with lipid metabolism regulating drugs is expected to synergistically enhance anti-inflammatory and anti-tumor effects; by regulating immune cell function, they can provide new strategies for tumor immunotherapy; and targeted drug delivery combined with nanodelivery systems has also become an important direction for precision medicine. However, natural PUFA molecules contain multiple unsaturated double bonds, making them extremely sensitive to light, heat, and oxygen, prone to auto-oxidation, and exhibiting poor stability, which severely restricts the full realization of their pharmacological effects.

[0005] To address this bottleneck, polyethylene glycol (PEG), a pharmaceutical polymer with good water solubility and high biocompatibility, has been used for the structural modification of polyunsaturated fatty acids (PUFAs). By linking PEG to PUFAs through tumor microenvironment-sensitive bonds, PEG-polyunsaturated fatty acid derivatives were constructed, exhibiting dual functions: on the one hand, PEG significantly improves the chemical stability of PUFAs; on the other hand, the sensitive bonds respond to the tumor microenvironment and release PUFAs at specific sites, thereby inducing lipid peroxidation, inactivating GPX4, and synergistically promoting ferroptosis, achieving targeted anti-tumor effects.

[0006] However, systematic research on the antitumor activity and mechanism of action of such PEGylated tumor microenvironment-sensitive linked polyunsaturated fatty acid derivatives is still lacking. Therefore, developing a highly stable PUFA derivative with tumor microenvironment-responsive release properties and direct antitumor activity, and constructing an intelligent responsive lipid drug delivery system based on it, is of great research significance and application value for promoting lipid metabolism-based tumor treatment strategies. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing a polyethylene glycol-polyunsaturated fatty acid derivative. This polyethylene glycol-polyunsaturated fatty acid derivative exhibits good resistance to multidrug resistance, and its anti-multidrug resistance effect has been verified through in vitro tumor cell experiments, indicating its promising application prospects in multidrug resistance research.

[0008] The present invention also provides a method for preparing the above-mentioned polyethylene glycol-polyunsaturated fatty acid derivative and its application.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A polyethylene glycol-polyunsaturated fatty acid derivative (PEG-PUFA, a polyethylene glycol-PUFA small molecule prodrug) has any of the following structural formulas: .

[0010] The aforementioned polyethylene glycol-polyunsaturated fatty acid derivative has a polyethylene glycol-polyunsaturated fatty acid core and connects bonds that are redox-sensitive or easily hydrolyzed by enzymes. Preferably, the redox-sensitive bond can be a disulfide bond, etc.; the easily hydrolyzed bond can be an ester bond, amide bond, etc.

[0011] This invention provides a method for preparing the above-mentioned polyethylene glycol-polyunsaturated fatty acid derivative, wherein when the derivative has the structural formula b, the method includes the following steps: Methoxy polyethylene glycol amino 2000 and 3,5-dihydroxybenzoic acid were dissolved in anhydrous N,N-dimethylformamide and reacted to obtain intermediate product 1. Intermediate product 1 and 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid were then dissolved in anhydrous N,N-dimethylformamide and reacted to obtain intermediate product 2. Intermediate product 2 was then dissolved in dichloromethane and trifluoroacetic acid was added dropwise to react and obtain intermediate product 3. Intermediate product 3 and polyunsaturated fatty acids were dissolved in anhydrous N,N-dimethylformamide and reacted to obtain polyethylene glycol-polyunsaturated fatty acid derivatives.

[0012] As a preferred technical solution, the preparation method of the above-mentioned polyethylene glycol-polyunsaturated fatty acid derivative, when the derivative has the structural formula b, includes the following steps: (1) Dissolve 3,5-dihydroxybenzoic acid, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in anhydrous N,N-dimethylformamide to obtain solution A; dissolve methoxy polyethylene glycol amino 2000 (mPEG-NH2) in anhydrous N,N-dimethylformamide, and then add it to solution A. Stir and react at 25~30℃ for 48~72 h under an inert gas atmosphere to obtain intermediate product 1; (2) Dissolve 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid and N,N,N',N-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (TSTU) in anhydrous N,N-dimethylformamide, then add N,N'-diisopropylethylamine (DIEA), and stir the reaction under an inert gas atmosphere at 0±5℃ for 2~4 h. Then add intermediate product 1 and continue stirring the reaction under an inert gas atmosphere at 0±5℃ for 72~96 h to obtain intermediate product 2; (3) Dissolve intermediate product 2 in dichloromethane, then add trifluoroacetic acid, stir at 25~30℃ for 4~6 h to obtain intermediate product 3; (4) Dissolve intermediate product 3, polyunsaturated fatty acid, NHS and EDCI in anhydrous N,N-dimethylformamide and stir the reaction under an inert gas atmosphere at 0±5℃ for 72~96 h to obtain the product.

[0013] Specifically, in step (1), the molar ratio of 3,5-dimethylbenzoic acid, NHS, EDCI and methoxy polyethylene glycol amino 2000 is 1:(1~1.2):(1~1.2):(0.5~0.8).

[0014] Specifically, in step (2), the molar ratio of 3 ((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid, TSTU, DIEA and intermediate 1 is 1:(1~1.2):(1~1.2):(0.35~0.4).

[0015] Specifically, in step (3), the molar ratio of intermediate product 2 to trifluoroacetic acid is 1:(50~100); in step (4), the molar ratio of intermediate product 3, polyunsaturated fatty acid, NHS and EDCI is 1:(2~7):(2~7):(2~7).

[0016] As a preferred embodiment, the present invention provides a method for preparing the above-mentioned polyethylene glycol-disulfide bond-polyunsaturated fatty acid small molecule prodrug (taking methoxy polyethylene glycol amino2000-disulfide bond-docosahexaenoic acid as an example), a reduction-sensitive polyethylene glycol-polyunsaturated fatty acid small molecule prodrug (PEG-SS-DHA2): which includes the following steps: (1) Weigh 0.1~0.3 mmol of 3,5-dihydroxybenzoic acid, 0.1~0.36 mmol of NHS and 0.1~0.36 mmol of EDCI, dissolve them in a three-necked flask containing 5 mL of anhydrous N,N-dimethylformamide, weigh 0.05~0.15 mmol of methoxy polyethylene glycol amino 2000 (mPEG-NH2) and dissolve it in 6 mL of anhydrous N,N-dimethylformamide until it is completely dissolved, then add it dropwise to the three-necked flask containing 3,5-dihydroxybenzoic acid, stir the reaction under nitrogen protection for 48~72 h, dialyze, and freeze dry to obtain intermediate product 1; (2) Weigh 0.07~0.08 mmol of 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid and 0.07~0.096 mmol of TSTU into a 50 mL flask, add 6 mL of anhydrous N,N-dimethylformamide to dissolve, then add 0.07~0.096 mmol of DIEA, stir and activate under N2 protection at 0℃, then add 0.0245~0.028 mmol of intermediate 1 and continue stirring under N2 protection for 72~96 h, dialyze, and freeze dry to obtain intermediate 2; (3) Weigh 0.01~0.03 mmol of intermediate product 2 and dissolve it in 4 mL of dichloromethane. Then add 1~1.2 mmol of trifluoroacetic acid and continue stirring for 4~6 h. Then add saturated sodium bicarbonate solution to adjust the pH to 7 and continue stirring for 1~2 h. Dialyze and freeze dry to obtain intermediate product 3. (4) Weigh 0.007 mmol of intermediate product 3, 0.035~0.045 mmol of PUFA (DHA, EPA, ALA and LA are collectively referred to as PUFA in this content), 0.035~0.045 mmol of NHS and 0.035~0.045 mmol of EDCI and dissolve them in 4 mL of anhydrous N,N-dimethylformamide. Continue stirring under N2 protection for 72~96 h, dialyze, and freeze dry to obtain the final product.

[0017] Preferably, in step (1), the reaction temperature is 25~30℃ and the reaction time is 48~72 h; in step (2), the reaction temperature is -5~5℃ (preferably 0℃) and the reaction time is 72~96 h; in step (3), the reaction temperature is 25~30℃ and the reaction time is 4~6 h; in step (4), the reaction temperature is -5~5℃ (preferably 0℃) and the reaction time is 72~96 h.

[0018] This invention also provides a method for preparing the above-mentioned polyethylene glycol-polyunsaturated fatty acid derivative, wherein when the derivative has the structural formula a, the method includes the following steps: 3,5-Dihydroxybenzoic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in N,N-dimethylformamide for activation, and then N-hydroxysuccinimide was added and the mixture was stirred. Subsequently, methoxy polyethylene glycol amino 2000 was added and the reaction was continued to obtain an intermediate product. Polyunsaturated fatty acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane for activation, and then the intermediate product was added and the reaction was continued to be stirred to obtain the final product.

[0019] As a preferred technical solution, the preparation method of the above-mentioned polyethylene glycol-polyunsaturated fatty acid derivative, when the derivative has the structural formula a, includes the following steps: (a) 3,5-Dihydroxybenzoic acid, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved in anhydrous N,N-dimethylformamide (DMF) to obtain solution A; methoxy polyethylene glycol amino 2000 (mPEG-NH2) was dissolved in anhydrous N,N-dimethylformamide and then added to solution A. The mixture was stirred and reacted at 25-30°C for 48-72 h under an inert gas atmosphere to obtain the intermediate product; (b) Dissolve polyunsaturated fatty acids (PUFAs) in dichloromethane (DCM), then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) and stir to activate under an inert gas atmosphere at 45±5℃; dissolve the intermediate product in dichloromethane (DCM) and add it dropwise to the above reaction system and continue stirring at 45±5℃ for 24~48 h to obtain the final product.

[0020] Furthermore, in step (a), the molar ratio of 3,5-dihydroxybenzoic acid, EDCI, NHS and methoxy polyethylene glycol amino 2000 can be 1:(1~1.2):(1~1.2):(0.5~0.8).

[0021] Furthermore, in step (b), the molar ratio of the intermediate product, EDCI, DMAP and PUFA can be 1:(2~6):(2~6):(2~6).

[0022] As a preferred embodiment, the present invention provides a method for preparing the above-mentioned polyethylene glycol-polyunsaturated fatty acid small molecule prodrug, a reduction-sensitive polyethylene glycol-polyunsaturated fatty acid small molecule prodrug (taking PEG-CC-DHA2 as an example): It includes the following steps: (a) Weigh 0.1-0.3 mmol of 3,5-dihydroxybenzoic acid, 0.1-0.36 mmol of NHS, and 0.1-0.36 mmol of EDCI, and dissolve them in a three-necked flask containing 5 mL of anhydrous N,N-dimethylformamide. Weigh 0.05-0.15 mmol of methoxy polyethylene glycol amino 2000 (mPEG-NH2) and dissolve it in 6 mL of anhydrous N,N-dimethylformamide until it is completely dissolved. Then add it dropwise to the three-necked flask containing 3,5-dihydroxybenzoic acid. Stir the reaction under nitrogen protection for 48-72 h, dialyze, and freeze dry to obtain the intermediate product. (b) Weigh 0.6 mmol of docosahexaenoic acid (DHA) into a 25 mL flask, add 6 mL of dichloromethane (DCM) to dissolve it completely, then add 0.6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 0.6 mmol of 4-dimethylaminopyridine (DMAP), and stir to activate under an inert atmosphere at 45±5℃. Then, dissolve 0.2 mmol of the intermediate product in 2 mL of DCM and quickly add it dropwise to the flask. Continue stirring under nitrogen protection for 24-48 h. After cooling, remove DCM by rotary evaporation, then add DMF to dissolve it completely. Extract with 5% citric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, dialyze, and freeze dry to obtain the final product.

[0023] Preferably, in step (a), the reaction treatment temperature is 25~30℃ and the reaction treatment time is 48~72 h; in step (b), the reaction activation temperature is 45℃ and the activation time is 1 h, the reaction treatment temperature is 40~50℃ and the reaction treatment time is 24~48 h.

[0024] This invention also provides the application of the above-mentioned polyethylene glycol-polyunsaturated fatty acid derivatives in the preparation of antitumor drugs.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects: The synthesis method of the polyethylene glycol-polyunsaturated fatty acid derivatives described in this invention is simple, environmentally friendly, and suitable for industrial production. The polyethylene glycol-polyunsaturated fatty acid derivatives provided by this invention exhibit good resistance to multidrug resistance. Their anti-multidrug resistance effect has been verified through in vitro tumor cell experiments, indicating their promising application prospects in multidrug resistance research. Attached Figure Description

[0026] Figure 1 For intermediate product 1, intermediate product 2, and intermediate product 3 1 H NMR (DMSO-d6, 400 MHz); Figure 2 For PEG-SS-DHA2 1 H NMR (DMSO-d6, 400 MHz); Figure 3 For PEG-SS-EPA2, PEG-SS-ALA2, PEG-SS-LA2 1 H NMR (DMSO-d6, 400 MHz); Figure 4 A complex of PEG-CC-DHA2, 3,5-dihydroxybenzoic acid and mPEG-NH2 1 H NMR (DMSO-d6, 400MHz); Figure 5 For PEG-CC-DHA2 1 H NMR (DMSO-d6, 400 MHz); Figure 6 For PEG-CC-EPA2 1 H NMR (DMSO-d6, 400 MHz); Figure 7 For PEG-CC-ALA2 1 H NMR (DMSO-d6, 400 MHz); Figure 8 For PEG-CC-LA2 1 H NMR (DMSO-d6, 400 MHz); Figure 9 The FT-IR images are of intermediate product 1, intermediate product 2, and intermediate product 3. Figure 10 The FT-IR spectra of PEG-SS-DHA2 (abbreviated as SDHA), DHA, and intermediate product 3 are shown. Figure 11FT-IR plots of PEG-SS-EPA2 (abbreviated as SEPA), EPA, PEG-SS-ALA2 (abbreviated as SALA), ALA, PEG-SS-LA2 (abbreviated as SLA), and LA; Figure 12 FT-IR spectra of PEG-CC-DHA2, mPEG-NH2, 3,5-DHBA, and DHA; Figure 13 FT-IR plots of PEG-CC-EPA2 (abbreviated as CEPA), EPA, PEG-CC-ALA2 (abbreviated as CALA), ALA, PEG-CC-LA2 (abbreviated as CLA), and LA; Figure 14 Cell survival rate of HepG2 cells after 24 hours of treatment with PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, PEG-SS-LA2 and PEG-SS-LA2; Figure 15 Cell survival rate of HepG2 cells after 24 hours of treatment with PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2; Figure 16 Cell survival rate of HepG2 cells after 24 hours of treatment with DHA, EPA, ALA, LA, and other active ingredients; Figure 17 Cell survival rate of MCF-7 cells after 24 h of treatment with PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2; Figure 18 Cell survival rate of MCF-7 cells after 24 hours of treatment with PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2; Figure 19 Cell survival rate of DHA, EPA, ALA, LA and MCF-7 cells after 24 hours of treatment. Detailed Implementation

[0027] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention.

[0028] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field and in accordance with the instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Example 1

[0029] In this embodiment, the synthetic route of the polyethylene glycol-polyunsaturated fatty acid small molecule prodrug (taking PEG-SS-DHA2 as an example) is as follows.

[0030] .

[0031] The synthesis method of the reduction-sensitive polyethylene glycol-polyunsaturated fatty acid small molecule prodrug (taking PEG-SS-DHA2 as an example) provided in this embodiment includes the following steps: (1) Weigh 0.3 mmol of 3,5-dihydroxybenzoic acid, 0.3 mmol of NHS, and 0.3 mmol of EDCI into a 50 mL flask. Add 5 mL of anhydrous N,N-dimethylformamide (DMF) and stir at room temperature (25~30℃) at a speed of 600 r / min. Weigh 0.15 mmol of methoxy polyethylene glycol amino 2000 and dissolve it completely in 6 mL of anhydrous N,N-dimethylformamide. Then add it dropwise to the reaction flask and continue stirring for 48 h under nitrogen protection at room temperature (25~30℃). After the reaction is complete, dialyze using a dialysis bag with a pore size of 3500 Da. The volume of the dialysate (using deionized water as the dialysate) is 100 times the volume of the reaction solution. Dialyze every 2~4 hours. Replace the dialysis solution and dialyze for 2-3 days. Then freeze the dialyzed reaction solution at -20°C. After the reaction solution is completely frozen, freeze-dry it (freeze at -60°C for 24 hours) to obtain intermediate product 1.

[0032] (2) Weigh 0.03 mmol of 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid and 0.036 mmol of TSTU into a 50 mL flask. Add 6 mL of anhydrous N,N-dimethylformamide and dissolve completely. Then add 0.036 mmol of DIEA and stir for 2 h under nitrogen protection at 0 °C at a speed of 600 r / min. Then weigh 0.0105 mmol of intermediate product 1 and add it to the reaction system. Continue stirring under nitrogen protection at 0 °C for 72 h. After the reaction is completed, dialyze using a dialysis bag with a pore size of 3500 Da. The volume of the dialysate (using deionized water as the dialysate) is 100 times the volume of the reaction solution. Change the dialysate every 2-4 h and dialyze for 2-3 days. Then freeze at -20 °C. After the reaction solution is completely frozen, freeze dry (freeze at -60 °C for 24 h) to obtain intermediate product 2.

[0033] (3) Weigh 0.01 mmol of intermediate product 2 into a 25 mL flask, add 4 mL of dichloromethane, then add 1 mmol of trifluoroacetic acid and stir at room temperature for 4 h at a speed of 600 r / min. Then add saturated sodium bicarbonate solution to adjust the pH of the reaction solution to neutral and continue stirring for 2 h. After the reaction is completed, dialyze using a dialysis bag with a pore size of 3500 Da. The volume of the dialysate (using deionized water as the dialysate) is 100 times the volume of the reaction solution. Change the dialysate every 2-4 h and dialyze for 2-3 days. Then freeze at -20℃. After the reaction solution is completely frozen, freeze dry (freeze at -60℃ for 24 h) to obtain intermediate product 3.

[0034] (4) Weigh 0.007 mmol of intermediate product 3, 0.042 mmol of polyunsaturated fatty acid docosahexaenoic acid (DHA), 0.042 mmol of NHS, and 0.042 mmol of EDCI into a 25 mL flask, add 4 mL of anhydrous N,N-dimethylformamide, and stir at 0℃ under nitrogen protection for 72 h at a speed of 600 r / min. After the reaction is complete, dialyze using a dialysis bag with a pore size of 3500 Da. The volume of the dialysate (using deionized water as the dialysate) is 100 times the volume of the reaction solution. Change the dialysate every 2-4 h and dialyze for 2-3 days. Then freeze at -20℃. After the reaction solution is completely frozen, freeze-dry (freeze at -60℃ for 24 h) to obtain the final product polyethylene glycol-polyunsaturated fatty acid, denoted as PEG-SS-DHA2.

[0035] The intermediate products 1, 2, and 3 obtained 1 HNMR (DMSO-d6, 400 MHz) and FT-IR spectra are shown below. Figure 1 and Figure 9 .

[0036] The final product obtained is PEG-SS-DHA2 1 The H NMR (DMSO-d6, 400 MHz) and FT-IR spectra are shown below. Figure 2 and Figure 10 .

[0037] Furthermore, as a preferred option, the DHA in the aforementioned PEG-SS-DHA2 can be replaced by compounds such as eicosapentaenoic acid (EPA), α-linolenic acid (ALA), and linoleic acid (LA). Its synthesis follows the synthesis steps of PEG-SS-DHA2, and its final products are named PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2, respectively.

[0038] The final products obtained are PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2. 1 The HNMR (DMSO-d6, 400 MHz) NMR and FT-IR spectra are shown below. Figure 3 and Figure 11 .

[0039] Experimental results: PEG-SS-DHA2 1 H NMR and infrared spectroscopy are shown below. Figure 2 and Figure 10 .

[0040] The NMR spectra of intermediates 1, 2, 3, PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2 obtained in Example 1 are as follows: Figure 1 , Figure 2 , Figure 3 As shown. Spectrum Figure 1 From bottom to top, they are mPEG-NH2, intermediate 1, intermediate 2, and intermediate 3. Figure 1 Intermediate product 1 showed an increased absorption peak on the phenolic hydroxyl group and a secondary amine absorption peak compared to the raw material mPEG-NH2, indicating that intermediate product 1 was successfully synthesized. Intermediate product 2 showed an increased absorption peak on the Boc group compared to intermediate product 1, indicating that intermediate product 2 was successfully synthesized. Intermediate product 3 showed the disappearance of the Boc group absorption peak and the presence of absorption peaks on other groups compared to intermediate product 2, indicating that intermediate product 3 was successfully synthesized. Figure 2 The absorption peak a is the hydrogen atom on the terminal methoxy group (-OCH3), the absorption peak b is the hydrogen atom on the polyoxyethyl chain, the absorption peak c is the hydrogen atom on the amide bond, the absorption peaks e and d are the aromatic hydrogen atoms on the benzene ring, the absorption peak f is the hydrogen atom on the double bond alkene (CH=CH), and the absorption peak g is the hydrogen atom on the saturated hydrocarbon chain. Compared with intermediate product 3, the spectrum of PEG-SS-DHA2 adds the absorption peak of the double bond in DHA to the spectrum of intermediate product 3. Therefore, it is speculated that DHA and mPEG-NH2 are successfully linked through disulfide bonds. Figure 3 From bottom to top, the sequence is intermediate 3: PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2. Similarly, the characteristic peaks of PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2 also correspond in the 1H NMR spectrum, suggesting that EPA, ALA, and LA are all successfully linked to mPEG-NH2 via disulfide bonds.

[0041] The infrared spectra of intermediates 1, 2, 3, PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2 synthesized in Example 1 are shown below. Figure 9 , Figure 10 , Figure 11 As shown. Figure 9 It can be seen that in mPEG-NH2, 2877 cm⁻¹ -1 The peak at 1108 cm⁻¹ represents the stretching vibration of the methylene C-H bond (-CH₂-). This strong and sharp peak is a characteristic peak of mPEG-NH₂. -1 The peak at 3310 cm⁻¹ represents the stretching vibration of an ether bond (-COC-), which can be identified as a characteristic infrared peak of mPEG-NH₂. Intermediate product 1 also exhibits this characteristic peak, therefore it is inferred that intermediate product 1 was successfully synthesized. -1 The left and right sides represent the NH stretching vibration of the secondary amine (-NH-) in the carbamate structure of 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid, 1700 cm⁻¹. -1 The peaks on the left and right represent the stretching vibrations of the ester carbonyl group (-O-(C=O)-NH-) in the Boc group, which are among the most characteristic peaks of the Boc group. Intermediate product 2 exhibits these characteristic peaks, therefore it is inferred that intermediate product 2 was successfully synthesized. Intermediate product 3 does not have the characteristic peaks of the Boc group, therefore it is inferred that intermediate product 3 was successfully synthesized. Figure 10 It can be seen that DHA contains 3006 cm -1 This is a stretching vibration of the carbon-hydrogen bond (CH) of an unsaturated olefin, 1745 cm⁻¹ -1 The strong absorption peak indicates the stretching vibration of the carboxyl carbon-oxygen double bond (-C=O). PEG-SS-DHA2 exhibits these characteristic peaks, therefore it is inferred that the synthesis of PEG-SS-DHA2 was successful. Similarly, based on... Figure 11 It is speculated that PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2 were also successfully synthesized. Example 2

[0042] In this embodiment, the synthetic route of the polyethylene glycol-polyunsaturated fatty acid small molecule prodrug (taking PEG-CC-DHA2 as an example) is as follows.

[0043] .

[0044] The synthesis method of the reduction-free polyethylene glycol-polyunsaturated fatty acid small molecule prodrug (PEG-CC-DHA2 as an example) provided in this embodiment includes the following steps: (1) Weigh 0.3 mmol of 3,5-dihydroxybenzoic acid, 0.3 mmol of NHS, and 0.3 mmol of EDCI into a 50 mL flask. Add 5 mL of anhydrous N,N-dimethylformamide (DMF) and stir at room temperature (25~30℃) at a speed of 600 r / min. Weigh 0.15 mmol of methoxy polyethylene glycol amino 2000 and dissolve it completely in 6 mL of anhydrous N,N-dimethylformamide. Then add it dropwise to the reaction flask and continue stirring for 48 h under nitrogen protection at room temperature (25~30℃). After the reaction is complete, dialyze using a dialysis bag with a pore size of 3500 Da. The volume of the dialysate (using deionized water as the dialysate) is 100 times the volume of the reaction solution. Dialyze every 2~4 hours. Replace the dialysis fluid and dialyze for 2-3 days. Then freeze the dialyzed reaction solution at -20°C. After the reaction solution is completely frozen, freeze-dry it (freeze at -60°C for 24 hours) to obtain the intermediate product. (2) Weigh 0.6 mmol of docosahexaenoic acid (DHA) into a 25 mL flask, add 6 mL of dichloromethane (DCM) to dissolve it completely, then add 0.6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 0.6 mmol of 4-dimethylaminopyridine (DMAP), and stir to activate for 1 h under N2 atmosphere at 45 °C. Subsequently, 0.2 mmol of the intermediate product was dissolved in 2 mL of DCM and rapidly added dropwise to the flask. The mixture was stirred for 48 h under nitrogen protection. After cooling, the DCM was removed by rotary evaporation. DMF was then added and dissolved completely. The mixture was extracted twice with 5% citric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively. Dialysis was performed using a dialysis bag with a pore size of 3500 Da. The volume of the dialysate (using deionized water as the dialysate) was 100 times the volume of the reaction solution. The dialysate was changed every 2-4 h, and dialyzed for 2-3 days. The dialyzed reaction solution was then frozen at -20 °C. After the reaction solution was completely frozen, it was lyophilized (frozen at -60 °C for 24 h) to obtain the final product, denoted as PEG-CC-DHA2.

[0045] The final product obtained is PEG-CC-DHA2 1 HNMR (DMSO-d6, 400 MHz) and FT-IR spectra are shown below. Figure 4 , Figure 5 and Figure 12 .

[0046] Furthermore, as a preferred option, the DHA in the aforementioned PEG-CC-DHA2 can be replaced by compounds such as eicosapentaenoic acid (EPA), α-linolenic acid (ALA), and linoleic acid (LA). Its synthesis follows the synthesis steps of PEG-CC-DHA2, and its final products are named PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2, respectively.

[0047] The FT-IR spectra of the final products PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 are shown below. Figure 13 The 1H NMR spectra of the final products PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 are shown below. Figure 6 , Figure 7 , Figure 8 .

[0048] The NMR spectrum of the polyethylene glycol-polyunsaturated fatty acid small molecule prodrug PEG-CC-DHA2 synthesized in Example 2 is shown below. Figure 4 , Figure 5 As shown. Figure 4 From bottom to top, they are PEG-CC-DHA2, DHA, 3,5-dihydroxybenzoic acid, and mPEG-NH2. Figure 5 The image shows the 1H NMR spectrum of PEG-CC-DHA2. Absorption peak a represents the hydrogen atom on the terminal methoxy group (-OCH3), absorption peak b represents the hydrogen atom on the polyoxyethyl chain, absorption peak c represents the hydrogen atom on the amide bond, absorption peaks e and d represent aromatic hydrogen atoms on the benzene ring, absorption peak f represents the hydrogen atom on the double-bonded alkene (-C=CH), and absorption peak g represents the hydrogen atom on the saturated hydrocarbon chain. Furthermore, the absorption peak on the phenolic hydroxyl group in the intermediate product completely disappears in the NMR spectrum of PEG-CC-DHA2, indicating that the intermediate product reacted completely with DHA. Therefore, it is inferred that PEG-CC-DHA2 was successfully synthesized. Similarly, based on... Figure 6 , Figure 7 , Figure 8 It is speculated that PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 were also successfully synthesized.

[0049] The infrared spectrum of the small molecule prodrug PEG-CC-DHA2 synthesized in Example 2 is shown below. Figure 12 As shown, the infrared spectra of PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 are as follows: Figure 13 As shown. Figure 12 As can be seen from this, in DHA, 2922 cm -1 and 2853 cm -1The positions represent the asymmetric and symmetric stretching vibrations of saturated carbon-hydrogen bonds (-CH2-, -CH3)d, respectively. 1745 cm⁻¹ -1 The strong absorption peak at 3,5-DHBA is due to the stretching vibration of the carboxyl carbon-oxygen double bond (-C=O). This peak occurs in the 3000–3300 cm⁻¹ range. -1 The region exhibits a typical broad peak for free carboxylic acid (-OH), at 1689 cm⁻¹. -1 There is stretching vibration of the carboxyl group (-C=O) at 1450~1600 cm⁻¹ -1 The region contains vibrations of the benzene ring (-C=C-) skeleton. In mPEG-NH2, at 2877 cm⁻¹... -1 The peak at 1108 cm⁻¹ represents the stretching vibration of the methylene C-H bond (-CH₂-). This strong and sharp peak is a characteristic peak of mPEG-NH₂. -1 The peaks at this point represent the stretching vibration of the ether bond (-COC-). The final product, PEG-CC-DHA2, exhibits these characteristic peaks, thus suggesting successful synthesis of PEG-CC-DHA2. Similarly, in... Figure 13 Based on the above absorption peak positions, it is inferred that PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 were also successfully synthesized. Example 3

[0050] HepG2 cytotoxicity.

[0051] The antitumor effects of PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, PEG-SS-LA2, PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, PEG-CC-LA2, DHA, EPA, ALA, and LA were detected by the MTT assay using tetramethylazobium salts. HepG2 cells in logarithmic growth phase were selected and subjected to a assay of 6 × 10⁻⁶ cells. 3The plates were seeded at a density of 10% fetal bovine serum (FBS) in DMEM medium in 96-well plates. After incubation at 37°C and 5% CO2 for 24 h, different concentrations of PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, PEG-SS-LA2, PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, PEG-CC-LA2, DHA, EPA, ALA, and LA were added, with six replicates for each concentration. After incubation for 24 h, 10 µl of 5 mg / ml MTT was added to each well, and the plates were incubated for another 4 h. The 96-well plates were then removed, the liquid in the wells was shaken off, and 100 µl of DMSO was added to each well. The plates were gently shaken until the crystals at the bottom of the wells were completely dissolved. The absorbance of each well was measured at 570 nm using a microplate reader, and the results were recorded. The experimental results are attached. Figure 14 , Figure 15 and Figure 16 .

[0052] Figure 14 It can be seen that as the concentrations of PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2 (these four prodrug molecules are collectively referred to as PEG-SS-PUFA2) increase, their killing effect on HepG2 cells becomes stronger and more concentration-dependent. Figure 15 It can be seen that as the concentrations of PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 (these four prodrug molecules are collectively referred to as PEG-CC-PUFA2) increase, their killing effect on HepG2 cells becomes stronger and more concentration-dependent. Figure 16 The results show that as the concentrations of DHA, EPA, ALA, and LA (collectively referred to as PUFAs) increase, their cytotoxic effect on HepG2 cells becomes stronger and more concentration-dependent. PUFAs have a certain cytotoxic effect on HepG2 cells; compared to PUFAs, PEG-CC-PUFA2 further enhances the cytotoxic effect on HepG2 cells; compared to PEG-CC-PUFA2, PEG-SS-PUFA2 significantly enhances the cytotoxic effect on HepG2 cells, indicating that this material has a good effect in killing cancer cells. Example 4

[0053] MCF-7 cytotoxicity.

[0054] The antitumor effects of PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, PEG-SS-LA2, PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, PEG-CC-LA2, DHA, EPA, ALA, and LA were detected by the MTT assay using tetramethylazobium salts. Logarithmically growing MCF-7 cells were selected and subjected to a assay at a concentration of 6 × 10⁻⁶ cells. 3 The plates were seeded at a density of 10% fetal bovine serum (FBS) in DMEM medium in 96-well plates. After incubation at 37°C and 5% CO2 for 24 h, different concentrations of PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, PEG-SS-LA2, PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, PEG-CC-LA2, DHA, EPA, ALA, and LA were added, with six replicates for each concentration. After incubation for 24 h, 10 µl of 5 mg / ml MTT was added to each well, and the plates were incubated for another 4 h. The 96-well plates were then removed, the liquid in the wells was shaken off, and 100 µl of DMSO was added to each well. The plates were gently shaken until the crystals at the bottom of the wells were completely dissolved. The absorbance of each well was measured at 570 nm using a microplate reader, and the results were recorded. The experimental results are attached. Figure 17 , Figure 18 and Figure 19 .

[0055] Figure 17 It can be seen that as the concentrations of PEG-SS-DHA2, PEG-SS-EPA2, PEG-SS-ALA2, and PEG-SS-LA2 (these four prodrug molecules are collectively referred to as PEG-SS-PUFA2) increase, their killing effect on MCF-7 cells becomes stronger and more concentration-dependent. Figure 18 It can be seen that as the concentration of PEG-CC-DHA2, PEG-CC-EPA2, PEG-CC-ALA2, and PEG-CC-LA2 (these four prodrug molecules are collectively referred to as PEG-CC-PUFA2) increases, their killing effect on MCF-7 cells becomes stronger and more concentration-dependent. Figure 19The results show that as the concentrations of DHA, EPA, ALA, and LA (collectively referred to as PUFAs) increase, their killing effect on MCF-7 cells becomes stronger and more concentration-dependent. PUFAs have a certain killing effect on MCF-7 cells; compared to PUFAs, PEG-CC-PUFA2 further enhances the killing effect on MCF-7 cells; compared to PEG-CC-PUFA2, PEG-SS-PUFA2 significantly enhances the killing effect on MCF-7 cells, further demonstrating that this material has a good effect in killing cancer cells.

[0056] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A polyethylene glycol-polyunsaturated fatty acid derivative, characterized by, It has any of the following structural formulas: 。 2. The method for producing the polyethylene glycol-polyunsaturated fatty acid derivative according to claim 1, characterized by, Includes the following steps: Methoxy polyethylene glycol amino 2000 and 3,5-dihydroxybenzoic acid were dissolved in anhydrous N,N-dimethylformamide and reacted to obtain intermediate product 1. Intermediate product 1 and 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid were then dissolved in anhydrous N,N-dimethylformamide and reacted to obtain intermediate product 2. Intermediate product 2 was then dissolved in dichloromethane and trifluoroacetic acid was added dropwise to react and obtain intermediate product 3. Intermediate product 3 and polyunsaturated fatty acids were dissolved in anhydrous N,N-dimethylformamide and reacted to obtain polyethylene glycol-polyunsaturated fatty acid derivatives.

3. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative as described in claim 2, characterized in that, Includes the following steps: (1) Dissolve 3,5-dihydroxybenzoic acid, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in anhydrous N,N-dimethylformamide to obtain solution A; dissolve methoxy polyethylene glycol amino 2000 in anhydrous N,N-dimethylformamide and then add it to solution A. Stir and react at 25-30°C for 48-72 h under an inert gas atmosphere to obtain intermediate product 1; (2) Dissolve 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid and N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate TSTU in anhydrous N,N-dimethylformamide, then add N,N'-diisopropylethylamine DIEA, and stir the reaction under an inert gas atmosphere at 0±5℃ for 2~4 h. Then add intermediate product 1 and continue stirring the reaction under an inert gas atmosphere at 0±5℃ for 72~96 h to obtain intermediate product 2; (3) Dissolve intermediate product 2 in dichloromethane, then add trifluoroacetic acid, stir at 25~30℃ for 4~6 h to obtain intermediate product 3; (4) Dissolve intermediate product 3, polyunsaturated fatty acid, NHS and EDCI in anhydrous N,N-dimethylformamide and stir the reaction under an inert gas atmosphere at 0±5℃ for 72~96 h to obtain the product.

4. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative as described in claim 2, characterized in that, In step (1), the molar ratio of 3,5-dimethylbenzoic acid, NHS, EDCI and methoxy polyethylene glycol amino 2000 is 1:(1~1.2):(1~1.2):(0.5~0.8).

5. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative as described in claim 2, characterized in that, In step (2), the molar ratio of 3((2-((tert-butoxycarbonyl)amino)ethyl)dithio)propionic acid, TSTU, DIEA and intermediate 1 is 1:(1~1.2):(1~1.2):(0.35~0.4).

6. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative as described in claim 2, characterized in that, In step (3), the molar ratio of intermediate product 2 to trifluoroacetic acid is 1:(50~100); in step (4), the molar ratio of intermediate product 3, polyunsaturated fatty acid, NHS and EDCI is 1:(2~7):(2~7):(2~7).

7. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative according to claim 1, characterized in that, Includes the following steps: (a) 3,5-Dihydroxybenzoic acid, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved in anhydrous N,N-dimethylformamide to obtain solution A; methoxy polyethylene glycol amino 2000 was dissolved in anhydrous N,N-dimethylformamide and then added to solution A. The mixture was stirred and reacted at 25-30°C for 48-72 h under an inert gas atmosphere to obtain the intermediate product. (b) Dissolve polyunsaturated fatty acid PUFA in dichloromethane, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) and stir to activate under an inert gas atmosphere at 45±5℃; dissolve the intermediate product in dichloromethane and add it dropwise to the above reaction system and continue stirring at 45±5℃ for 24~48 h to obtain the final product.

8. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative as described in claim 7, characterized in that, In step (a), the molar ratio of 3,5-dihydroxybenzoic acid, EDCI, NHS and methoxy polyethylene glycol amino 2000 is 1:(1~1.2):(1~1.2):(0.5~0.8).

9. The method for preparing the polyethylene glycol-polyunsaturated fatty acid derivative as described in claim 7, characterized in that, In step (b), the molar ratio of intermediate product, EDCI, DMAP and PUFA is 1:(2~6):(2~6):(2~6).

10. The use of the polyethylene glycol-polyunsaturated fatty acid derivative of claim 1 in the preparation of antitumor drugs.

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