A curcumin derivative prodrug, preparation method and application thereof

Abstract

The present invention discloses a curcumin derivative prodrug, a preparation method, and applications thereof. The curcumin derivative prodrug is a compound wherein a single sulfide bond or a disulfide bond is located at the α- and β-positions of adjacent carbonyl groups, obtained by connecting a curcumin derivative via 2,2'-thiodiacetic acid, 2,2'-thiodipropionic acid, 3,3'-dithiodiacetic acid, or 2,2'-dithiodipropionic acid with oleyl alcohol. The curcumin derivative prodrug can be prepared using a nanoparticle precipitation method to form non-PEGylated curcumin derivative prodrug self-assembled nanoparticles or PEG-modified curcumin derivative prodrug self-assembled nanoparticles. The curcumin derivative prodrug can be used to treat breast cancer or liver cancer. The resulting self-assembled nanoparticles have a high drug loading capacity, good stability, low toxicity and side effects, and rapid, tumor-site-specific drug release, thereby extending systemic circulation time, increasing bioavailability, and enhancing therapeutic efficacy.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor drug preparation, and in particular to the synthesis of a monosulfide / disulfide bond-linked oleyl alcohol-curcumin derivative C210 prodrug and the preparation of its self-assembled nanoparticles and their application in the preparation of anti-tumor drugs. Background Art

[0002] Curcumin, a diketone polyphenolic compound extracted from the tuber of turmeric, exhibits significant in vitro antitumor activity against leukemia cells and solid tumors, including colon, lung, liver, and breast cancer, demonstrating a broad antitumor spectrum. Curcumin has been extensively studied clinically and has demonstrated efficacy in the treatment of numerous cancers. In addition to its potent antitumor activity, curcumin is also highly safe. Clinical trials have shown no significant toxic side effects, even at doses as high as 8g / day. However, its rapid elimination from the body and poor oral bioavailability limit its application. Based on the fact that the main metabolic pathway of curcumin in the body is the combination of the phenolic hydroxyl group on the benzene ring with glucuronic acid, thus delaying curcumin metabolism without reducing its activity, the introduction of a methoxy group at the phenolic hydroxyl position can enhance the activity of the compound. Based on this, the inventors designed and synthesized a new curcumin derivative C210 (for its chemical structure, see compound No. 4 disclosed in patent CN104030904A). Studies have shown that C210 has a stronger molecular chaperone function that inhibits heat shock protein 90 and stronger in vitro anti-tumor activity than curcumin. However, the pharmacokinetic properties of this compound are not significantly improved compared to curcumin, which has become a bottleneck for its development and application.

[0003] Prodrugs are drugs that have low or no in vitro activity but are metabolized or release their active ingredients in vivo. Chemical modification can be used to target specific tissues for drug release, thereby enhancing drug selectivity. Small molecule prodrugs also offer advantages such as low molecular weight, high drug loading, and the ability to self-assemble into nanoparticles. Consequently, small molecule prodrug nanoparticles have gained increasing attention. Some prodrugs modified with fatty acids and fatty alcohols can prolong drug retention in the systemic circulation. For example, paliperidone palmitate, a currently marketed lipid-modified prodrug, significantly prolongs the duration of effective in vivo concentrations after a single injection, suggesting that lipid-modified drugs may be an effective strategy for improving pharmacokinetic behavior. Tumor tissues have higher redox levels than normal tissues. The intracytoplasmic glutathione content of tumor cells is over 10 times that of normal cells, and the concentration of reactive oxygen species (ROS) in tumor cells is approximately 100 times higher than that of normal cells. Therefore, drugs based on the redox response of tumor cells have been widely used. Summary of the Invention

[0004] To address the development bottleneck of existing curcuminoids, which suffer from poor pharmacokinetic properties, the present invention provides a curcumin derivative C210 prodrug and its self-assembling nanoparticles, as well as their preparation methods and applications, to improve the pharmacokinetic properties of C210 and enhance its selective antitumor activity. The C210 prodrug uses monosulfide or disulfide bonds of varying chain lengths to link the curcumin derivative C210 with oleic acid. The presence of redox-responsive monosulfide or disulfide bonds allows the prodrug to specifically release C210 within tumor cells with high redox levels, enhancing the drug's specific distribution and release within tumor tissue and strengthening its selective antitumor activity. The oleic acid modification enables the prodrug to self-assemble into nanoparticles, thereby extending the drug's average residence time in the systemic circulation and improving its pharmacokinetic properties. The resulting nanoparticles exhibit high stability, excellent pharmacokinetic properties, specific release of C210 within tumor cells, high tumor tissue distribution, and strong in vivo antitumor activity.

[0005] Specifically, the present application uses different redox intermediates to connect unsaturated fatty acid oleic acid and curcumin derivative C210 to prepare a prodrug nano drug delivery system, to improve the drugability of curcumin derivative C210, thereby improving the purpose of its in vivo efficacy. A total of 4 prodrugs were designed and synthesized. These prodrugs are named according to the key bridge containing the central element bond (single sulfur S, disulfide SS) and ester bond (α is ethyl ester, β is propyl ester), and are abbreviated as α-C210-S-OA, α-C210-SS-OA, β-C210-S-OA, β-C210-SS-OA accordingly. The self-assembled nanoparticles of this type of prodrug can significantly improve the systemic circulation time and bioavailability of C210. Based on the background of high redox levels in tumor cells and tumor microenvironment, the designed sulfur-containing bridge can enhance the targeted release ability of the drug, so that the prodrug nanoparticles are specifically distributed in tumor cells or tumor tissues, and based on the high redox level of the tumor, the intelligent release of C210 is triggered, thereby improving the distribution and release of the drug in tumor tissue, enhancing the selective anti-tumor activity, and improving the anti-tumor effect of the drug in vivo.

[0006] To achieve the above objectives, the present invention provides a monosulfide / disulfide-linked oleyl alcohol-curcumin derivative C210 prodrug having the following general structural formula, wherein “monosulfide / disulfide-linked” is referred to herein and hereinafter, and “ / ” represents “or”.

[0007]

[0008] wherein n is an integer of 1-2, and R is a monosulfide bond or a disulfide bond.

[0009] The curcumin derivative C210 is described in CN104030904A and is abbreviated as C210 or C210 prodrug in this application. Its structural formula is:

[0010]

[0011] The prodrug structure of the curcumin derivative C210 having a single sulfide bond and a diethyl ester structure is as follows:

[0012]

[0013] The prodrug structure of the curcumin derivative C210 having a disulfide bond and a diethyl ester structure is:

[0014]

[0015] The prodrug structure of the curcumin derivative C210 having a single sulfide bond and a dipropyl ester structure is:

[0016]

[0017] The prodrug structure of the curcumin derivative C210 having a disulfide bond and a dipropyl ester structure is:

[0018]

[0019] The present invention provides a method for preparing a prodrug of an oleyl alcohol-curcumin derivative C210 containing a monosulfide bond / disulfide bond connection, comprising the following steps:

[0020] (1) Weigh 2,2'-thiodiacetic acid, 2,2'-dithiodiacetic acid, 3,3'-thiodipropionic acid or 3,3'-dithiodipropionic acid, oleyl alcohol, DMAP, and EDCI in a 100 ml round-bottom flask, add 30 ml of dichloromethane as the reaction solvent, and react at room temperature for 24 h under nitrogen protection. Check the reaction progress by thin-layer chromatography. The solvent is removed by rotary evaporation under reduced pressure, and the concentrate is separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) and dried under vacuum to obtain white oily intermediates α-S-OA, α-SS-OA, β-S-OA, and β-SS-OA.

[0021] (2) α-S-OA, α-SS-OA, β-S-OA, β-SS-OA obtained in step (1) and curcumin derivative C210, DMAP, and EDCI were weighed separately in a 100 ml round-bottom flask, 10 ml of dichloromethane was added as the reaction solvent, and the mixture was reacted at room temperature for 24 h under nitrogen protection. The reaction progress was checked by thin layer chromatography. After the reaction solution was concentrated, saturated brine and ethyl acetate were added and extracted three times. After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated. The concentrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1-3:1) and dried under vacuum. The target products α-C210-S-OA, α-C210-SS-OA, β-C210-S-OA, and β-C210-SS-OA were obtained.

[0022] The present invention also provides self-assembled nanoparticles of the monosulfide bond / disulfide bond-linked oleyl alcohol-curcumin derivative C210 prodrug, comprising PEG-modified prodrug self-assembled nanoparticles, and the preparation method is a nanoprecipitation method.

[0023] The method for preparing the self-assembled nanoparticles of the monosulfide / disulfide-linked oleyl alcohol-curcumin derivative C210 prodrug is as follows: each prodrug and DSPE-PEG2000 are completely dissolved in acetone. The mixed solution is then added dropwise to an appropriate amount of physiological saline under magnetic stirring. The acetone is removed by evaporation under reduced pressure to obtain an organic solvent-free nanoparticle solution. DSPE-PEG2000 is not added in the preparation of the non-PEGylated C210 prodrug nanoparticles.

[0024] The present invention also provides use of the self-assembled nanoparticles of the monosulfide bond / disulfide bond-connected oleyl alcohol-curcumin derivative C210 prodrug in preparing a drug delivery system.

[0025] The present invention also provides the use of the self-assembled nanoparticles of the monosulfide bond / disulfide bond-connected oleyl alcohol-curcumin derivative C210 prodrug in the preparation of anti-tumor drugs.

[0026] The present invention also provides the use of the self-assembled nanoparticles of the monosulfide bond / disulfide bond-linked oleyl alcohol-curcumin derivative C210 prodrug in the preparation of an injection, oral administration or local administration system.

[0027] The technical problem solved by the present invention is to introduce monosulfide / disulfide bonds of different chain lengths into oleyl alcohol-curcumin derivative C210 prodrugs, design redox-sensitive C210 prodrugs with different monosulfide / disulfide bridges, and use the C210 prodrugs in the construction of self-assembling nanoparticles, thereby achieving high drug loading, good stability, low toxicity and side effects, and rapid tumor-specific drug release, extending systemic circulation time, increasing bioavailability, and improving therapeutic efficacy. At the same time, the differences in the self-assembly of C210 prodrugs with different monosulfide / disulfide linker chain lengths are examined, as well as their effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the prodrug self-assembled nanoparticles.

[0028] The advantages of the present invention are:

[0029] (1) C210 prodrugs containing monosulfide / disulfide linkage chains of different lengths were designed and synthesized, and the synthesis method was simple and easy.

[0030] (2) C210 prodrug self-assembled nanoparticles with uniform particle size were prepared. The preparation method is simple and easy, and efficient drug encapsulation is achieved, with an ultra-high drug loading of about 50%.

[0031] (3) The differences in self-assembly of monosulfide / disulfide linker chains of different lengths were investigated, as well as their effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the prodrug self-assembled nanoparticles. Based on the comprehensive experimental results, the prodrug containing a monosulfide bond and a diethyl ester structure has a suitable particle size and chemical stability. At the same time, it has the best in vitro redox-responsive release of C2120, significantly improving the bioavailability of C210 and the anti-tumor effect in vivo. The present invention provides new strategies and more options for the development of intelligent responsive drug delivery systems for tumor microenvironment, meeting the urgent clinical demand for highly efficient and selective anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Purity confirmation of C210 prodrug;

[0033] Figure 2 Electron microscopy characterization of C210 prodrug self-assembled nanoparticles;

[0034] Figure 3 Stability results of C210 prodrug self-assembled nanoparticles;

[0035] A: Stability of C210 prodrug nanoparticles in PBS solution containing 10% FBS within 48 h;

[0036] B: Stability of C210 prodrug nanoparticles at 4°C in a dark environment for 3 months;

[0037] Figure 4 In vitro release test of C210 prodrug self-assembled nanoparticles under high redox conditions simulating tumors;

[0038] A: Release of C210 prodrug nanoparticles in PBS (pH = 7.4) medium containing 0 mM H2O2;

[0039] B: Release of C210 prodrug nanoparticles in PBS (pH = 7.4) medium containing 1 mM H2O2;

[0040] C: Release of C210 prodrug nanoparticles in PBS (pH = 7.4) medium containing 10 mM H2O2;

[0041] D: Release of C210 prodrug nanoparticles in PBS (pH = 7.4) medium containing 0 mM DTT;

[0042] E: Release of C210 prodrug nanoparticles in PBS (pH = 7.4) medium containing 1 mM DTT;

[0043] F: Release of C210 prodrug nanoparticles in PBS (pH = 7.4) medium containing 10 mM DTT;

[0044] Figure 5 Plasma CT curve of C210 prodrug self-assembled nanoparticles in rats after intravenous administration;

[0045] Figure 6 The distribution of C210 prodrug self-assembled nanoparticles in tumor tissue;

[0046] A: Distribution of C210 prodrug in tumor tissue at 1 h, 2 h, 4 h, and 12 h, and the released C210;

[0047] B: Tumor accumulation of C210 released from C210 prodrug nanoparticles within 0-12 h;

[0048] Figure 7 In vivo anti-tumor experimental results of C210 prodrug self-assembled nanoparticles;

[0049] A: Tumor volume growth curve of Balb / C tumor-bearing mice after treatment with C210 prodrug nanoparticles;

[0050] B: Photographs of tumors in Balb / C tumor-bearing mice after treatment with C210 prodrug nanoparticles;

[0051] C: Tumor weight of Balb / C tumor-bearing mice after treatment with C210 prodrug nanoparticles;

[0052] D: Body weight changes of Balb / C tumor-bearing mice after treatment with C210 prodrug nanoparticles. DETAILED DESCRIPTION

[0053] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0054] Example 1: Synthesis of C210 prodrug (α-C210-S-OA) containing a single sulfide bond and a diethyl ester structure

[0055] Oleyl alcohol (268 mg, 1 mmol), 2,2'-thiodiacetic acid (150 mg, 1 mmol), EDCI (191 mg, 1 mmol), and DMAP (24 mg, 0.2 mmol) were added to a 10 ml dichloromethane solution. The reaction mixture was stirred at room temperature under nitrogen. The formation of the desired product was monitored by thin-layer chromatography. After the reaction was complete, the dichloromethane was evaporated and saturated brine was added. The aqueous layer was extracted with dichloromethane, washed with a saturated NaCl solution, and dried over anhydrous sodium sulfate. The concentrate was separated by silica gel column chromatography to obtain the intermediate product, (E)-2-[(2-(octadec-9-en-1-yloxy)-2-oxyethyl)thio)acetic acid] (α-S-OA), in a 23% yield.

[0056] C210 (592 mg, 1 mmol), α-S-OA (400 mg, 1 mmol), EDCI (191 mg, 1 mmol), and DMAP (24 mg, 0.2 mmol) were dissolved in 10 ml of aqueous dichloromethane. The reaction mixture was then stirred overnight at room temperature under nitrogen. The formation of the desired product was determined by thin-layer chromatography.

[0057] After completion of the reaction, the reaction solution was extracted with dichloromethane, washed with a saturated NaCl solution, and dried over anhydrous sodium sulfate. The concentrate was separated by silica gel column chromatography to obtain the target product, [(2-{[(10E)-octadec-9-enyl]oxy}-2-oxyylideneethyl)thio]acetate-2-methoxy-4-[(4E)-3-oxyylidene-2-[(2E)-1-oxyylidene-3-(3,4,5-trimethoxyphenyl)prop-2-enyl]-5-(3,4,5-trimethoxyphenyl)pent-4-enyl]phenyl ester (α-C210-S-OA), in a yield of 78%.

[0058] The structure was confirmed by high-resolution mass spectrometry, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. The spectrum analysis results are as follows: [MH] - Peak 973.4774, proved that α-C210-S-OA was successfully synthesized, and the molecular formula was C 55 H 74 O 13 S.

[0059] 1 H NMR(500MHz, CDCl3)δ7.69(d,J=15.3Hz,2H),6.93–6.86(m,2H),6.83(d,J=15.3Hz,2H ),6.67(s,3H),5.35(dqd,J=7.9,4.6,4.0,2.1Hz,2H),4.13(t,J=6.9Hz,2H),3.98(s, 2H),3.93–3.82(m,19H),3.77(s,3H),3.62(d,J=5.6Hz,2H),3.48(s,2H),2.06–1.97( m, 4H), 1.64 (t, J = 7.3Hz, 2H), 1.58 (s, 2H), 1.35–1.22 (m, 24H), 0.88 (t, J = 6.8Hz, 3H).

[0060] 13C NMR (126MHz, CDCl3) δ183.56,169.82,168.05,153.44,151.27,140.40,140.20 ,138.17,130.72,130.01,129.76,122.79,119.92,111.82,108.68,105.92,10 5.40,,65.75,61.00,56.24,56.19,55.90,33.39,33.23,31.91,31.78,29.77,29.72,29.53,29.40,29.32,29.20,28.53,27.22,27.18,25.81,22.69,14.13.

[0061] Example 2: Synthesis of C210 prodrug (α-C210-SS-OA) containing disulfide bonds and diethyl ester structures

[0062] The preparation method of Example 1 was adopted, and 2,2'-thiodiacetic acid was replaced with 2,2'-dithiodiacetic acid to prepare [(2-{[(10E)-octadec-9-enyl]oxy}-2-oxyylideneethyl)disulfide]acetic acid-2-methoxy-4-[(4E)-3-oxyylidene-2-[(2E)-1-oxyylidene-3-(3,4,5-trimethoxyphenyl)prop-2-enyl]-5-(3,4,5-trimethoxyphenyl)pent-4-enyl]phenyl ester (α-C210-SS-OA).

[0063] The structure was confirmed by high-resolution mass spectrometry, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. The spectrum analysis results are as follows: [MH] - Peak 1005.4497, proving that α-C210-SS-OA was successfully synthesized, with the molecular formula C 55 H 74 O 13 S2.

[0064] 1 H NMR(500MHz, CDCl3)δ7.69(d,J=15.3Hz,2H),6.93–6.80(m,4H),6.67(s,3H),5.39–5.30(m,2H),4.13(dd,J=7.3,6.2Hz,2H),3 .98(s,2H),3.91–3.76(m,23H),3.62(s,2H),2.06–1.92(m,4H),1.68–1.60(m,3H),1.36–1.23(m,24H),0.88(t,J=6.9Hz,3H).

[0065] 13C NMR (126MHz, CDCl3) δ183.56,169.38,167.68,153.44,151.33,142.26,140.45,14 0.19,138.18,130.72,130.00,129.77,122.82,119.93,119.89,111.85,108.69,1 05.40,65.90,61.02,61.00,56.19,55.90,55.86,41.44,41.03,31.91,29.77,29.73,29.53,29.41,29.33,29.21,29.18,28.51,27.22,27.19,25.81,22.69,14.13.

[0066] Example 3: Synthesis of C210 prodrug (β-C210-S-OA) containing a monosulfide bond and a dipropyl ester structure

[0067] The preparation method of Example 1 was adopted, and 2,2'-thiodiacetic acid was replaced with 3,3'-thiodipropionic acid to prepare 3-[(3-{[(10E)-19-enyl]oxy}-3-oxyylidenepropyl)thio]propanoic acid-2-methoxy-4-[(4E)-3-oxyylidene-2-[(2E)-1-oxyylidene-3-(3,4,5-trimethoxyphenyl)prop-2-enyl]-5-(3,4,5-trimethoxyphenyl)pent-4-enyl]phenyl ester (β-C210-S-OA).

[0068] The structure was confirmed by high-resolution mass spectrometry, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. The spectrum analysis results are as follows: [MH] - Peak 1001.5082, proving that β-C210-S-OA was successfully synthesized, with the molecular formula C 57 H 78 O 13 S.

[0069] 1H NMR(500MHz, CDCl3)δ7.62(d,J=15.3Hz,2H),6.85–6.73(m,3H),6.60(s,3 H),5.33–5.21(m,3H),4.01(t,J=6.8Hz,2H),3.90(s,2H),3.85–3.77(m,18 H),3.70(d,J=7.1Hz,3H),2.87–2.74(m,6H),2.59–2.52(m,2H),1.94(t,J =6.3Hz,3H),1.59–1.46(m,4H),1.22–1.18(m,24H),0.81(d,J=1.8Hz,3H). 13 C NMR (126MHz, CDCl3) δ182.53,170.86,169.05,168.95,152.44,152.41,150.34,141.19,139.15,139.13 ,137.20,129.71,128.96,128.75,121.92,118.94,118.85,110.75,107.71,104.89,104.37,63.94,59.9 9,59.97,55.21,55.16,54.87,33.78,33.60,30.90,30.88,30.75,30.41,29.16,28.74,28.71,28.68,28.63,28.50,28.39,28.34,28.30,28.25,28.19,27.56,26.19,26.16,26.06,25.96,24.86,21.66,13.10.

[0070] Example 4: Synthesis of C210 prodrug (β-C210-SS-OA) containing disulfide bonds and dipropyl ester structures

[0071] The preparation method of Example 1 was adopted, and 2,2'-thiodiacetic acid was replaced with 3,3'-dithiodipropionic acid to prepare 3-[(3-{[(10E)-octadec-9-enyl]oxy}-3-oxyylidenepropyl)disulfide]propionic acid-2-methoxy-4-[(4E)-3-oxyylidene-2-[(2E)-1-oxyylidene-3-(3,4,5-trimethoxyphenyl)prop-2-enyl]-5-(3,4,5-trimethoxyphenyl)pent-4-enyl]phenyl ester (β-C210-SS-OA).

[0072] The structure was confirmed by high-resolution mass spectrometry, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. The spectrum analysis results are as follows: [MH] -Peak 1035.4805, proving that β-C210-SS-OA was successfully synthesized, with the molecular formula C 57 H 78 O 13 S2.

[0073] 1 H NMR(500MHz, CDCl3)δ7.69(d,J=15.3Hz,2H),6.92–6.78(m,4H),6.67(s,3H),5.40–5 .30(m,2H),4.08(t,J=6.8Hz,2H),3.97(s,1H),3.93–3.84(m,18H),3.76(s,3H),3.06 –2.99(m,4H),2.95(t,J=7.2Hz,2H),2.74(t,J=7.2Hz,2H),2.17(d,J=3.1Hz,2H),2. 01(q,J=6.7Hz,3H),1.63(h,J=6.9Hz,4H),1.30–1.23(m,24H),0.88(d,J=1.9Hz,3H).

[0074] 13 C NMR (126MHz, CDCl3) δ183.56,171.73,169.93,153.47,153.43,151.36,142.23,140.18,138.21,130.89,130.7 3,129.99,129.78,122.91,119.96,119.87,111.78,108.73,105.92,105.39,68.15,65.03,64.62,61.02,61.00 ,56.23,56.19,55.90,38.72,34.14,33.89,33.23,33.02,31.91,31.44,30.94,30.19,29.77,29.73,29.70,29.66,29.52,29.41,29.36,29.32,29.27,29.21,28.92,28.57,27.22,27.19,25.89,23.74,22.99,22.69,14.13.

[0075] Example 5: Purity confirmation of C210 prodrug

[0076] The purity of C210 prodrug was determined by HPLC. Figure 1 As shown, the purity of the four prodrugs was above 98%, which met the requirements of subsequent experiments.

[0077] Example 6: Preparation and Stability Investigation of C210 Prodrug Self-Assembled Nanoparticles

[0078] 4 mg of prodrug (α-C210-S-OA, α-C210-SS-OA, β-C210-S-OA, or β-C210-SS-OA synthesized in Examples 1-4, respectively) and 0.8 mg of DSPE-PEG2000 were weighed and completely dissolved in 1 ml of acetone. The mixed solution was then added dropwise to 4 ml of normal saline under magnetic stirring, and the acetone was evaporated under reduced pressure. The nanoparticles were mixed with PBS containing 10% fetal bovine serum and placed in a shaking incubator at 37°C. Samples were taken at 0, 2, 4, 6, 8, 12, 24, and 48 hours. After dilution, the nanoparticles were measured for particle size. Particle size and PDI were used as indicators of stability to assess the short-term stability of the nanoparticles.

[0079] The nanoparticles were placed at 4°C in a dark environment and samples were taken at 1, 3, 5, 7, 14, 28, 60, and 90 days. The particle size of the nanoparticles was measured after dilution. The particle size and PDI were used as indicators to evaluate the long-term stability. The results are shown in Table 1. Figure 2 As shown in the figure, the DSPE PEG2K modified prodrug nanoparticles are spherical, with a particle size of about 120nm, a uniform particle size distribution, and a surface charge of about 36mV, which proves that the nanoparticles are stable and not easy to aggregate. In addition, the drug loading of each prodrug nanoparticle is about 50%, effectively reducing the use of excipients and the potential risks they bring. Then the stability of the prodrug nanoparticles was investigated, such as Figure 3 As shown, the prodrug nanoparticles exhibited minimal size change within 48 hours at 37°C in PBS (pH 7.4) containing 10% fetal bovine serum. The nanoparticles also showed excellent stability after being stored at 4°C in the dark for three months, demonstrating their excellent stability and potential for long-term storage.

[0080] Table 1 Particle size, particle size distribution, surface charge, encapsulation efficiency and drug loading of C210 prodrug self-assembled nanoparticles

[0081]

[0082]

[0083] Example 7: In vitro simulated tumor high redox responsive release experiment of C210 prodrug self-assembled nanoparticles

[0084] PBS (pH 7.4) containing 5% SDS was used as the release medium to study the release profile of C210 from the prodrug nanoparticles. 1 ml of C210 prodrug nanoparticles was added to 30 ml of release medium containing 0, 1, or 10 mM H2O2 or DTT and incubated at 37°C. At predetermined time points (0, 2, 4, 6, 8, 12, and 24 hours), 100 μl of the solution was removed and replaced with an equal amount of release medium. The amount of released C210 was determined by high-performance liquid chromatography. The oxidative release results are shown in Figure 2. Figure 4 As shown in Figures AC, when each prodrug nanoparticle was incubated in PBS (pH 7.4) at 37°C for 24 h, only a small amount of C210 (less than 10% w / w) was released. However, when incubated in PBS containing 1 mM H2O2 or 10 mM H2O2, the prodrug nanoparticles were able to rapidly release C210, with the release rates in the order of α-C210-S-OA > α-C210-SS-OA > β-C210-S-OA > β-C210-SS-OA. The reduction release results are shown in Figures 4A and 4B. Figure 4 As shown in Figures DF, incubation in PBS containing 1mM / 10mM DTT revealed that prodrugs containing disulfide bonds exhibited a more sensitive reduction reaction, with response rates ranking α-C210-SS-OA > β-C210-SS-OA > β-C210-S-OA > α-C210-S-OA, with α-C210-SS-OA being nearly completely reduced within approximately 2 hours. In contrast, under non-reducing conditions, nanoparticles underwent slow conversion within 24 hours. This result demonstrates that both monosulfide and disulfide bonds exhibit dual redox responsiveness, with monosulfide bonds being more sensitive to oxidative environments and disulfide bonds being more sensitive to reducing environments, resulting in a slower release rate for long-chain prodrugs than for short-chain prodrugs.

[0085] Example 8: Selective cytotoxicity of C210 prodrug self-assembled nanoparticles against tumor cells

[0086] The MTT assay was used to determine the cell viability of MCF-7 cells, MDA-MB-231 cells, 4T1 cells, MCF-10A cells (normal mammary epithelial cells), and HepG2 cells treated with C210 and prodrug nanoparticles. Cancer cells were seeded in 96-well plates at 3,000 cells per well and cultured for 12 hours until the cells were fully adhered. The culture medium was then replaced with medium containing C210 or prodrug nanoparticles at concentrations ranging from 0 to 200 μmol / l. The cells were then incubated for 48 or 72 hours. Afterwards, 15 μl of MTT solution was added to each well, and the cells were incubated at 37°C for an additional 4 hours. After removing the culture medium, the formazan crystals were dissolved in 200 μl of DMSO. The absorbance of the plate was then measured at a wavelength of 570 nm using a microplate reader.

[0087] The results are shown in Table 2. The cytotoxicity of the four prodrug nanoparticles against breast cancer and liver cancer cells showed significant differences. α-C210-S-OANPs and α-C210-SS-OANPs exhibited stronger antitumor activity, while β-C210-S-OA NPs and β-C210-SS-OA NPs exhibited slightly weaker antitumor activity. The cytotoxicity of C210 and the prodrug nanoparticles against breast cancer and liver cancer cells revealed that while the C210 prodrug nanoparticles exhibited lower cytotoxicity than free C210, as shown in Table 3, the prodrug nanoparticles exhibited significant selectivity against normal breast cells (MCF-10A) and breast cancer cells (MCF-7), with relative therapeutic indices higher than those of the original drug.

[0088] Table 2 IC of C210 and various prodrug nanoparticles on tumor cells 50 value

[0089]

[0090] Table 3 IC of C210 and various prodrug nanoparticles on tumor cells and normal cells 50 Value (relative therapeutic index = normal cell IC 50 / tumor cell IC 50 )

[0091]

[0092] Example 9: Pharmacokinetics and tumor tissue-specific distribution of C210 prodrug self-assembled nanoparticles

[0093] Pharmacokinetic studies were conducted using SD rats. The rats were fasted for 12 hours before the experiment and had free access to water. 18 rats were divided into 6 groups and injected intravenously with free C210 or C210 prodrug nanoparticles at an equivalent dose of 20 μmol / kg C210. Blood samples were collected at predetermined time points (0.08, 0.15, 0.5, 1, 1.5, 2, 4, 6, 8, and 12 hours) and plasma was obtained by centrifugation. The concentrations of C210 and corresponding prodrugs were determined by LC-MS. Pharmacokinetic parameters were analyzed using Drugs and Statistics 2.0 (DAS) software. The biodistribution of C210 and prodrug nanoparticles was studied by measuring the concentrations of C210 and prodrug nanoparticles in the major organs of 4T1 tumor-bearing BALB / c mice. 60 mice were randomly divided into 5 groups and injected intravenously with free C210 or C210 prodrug nanoparticles at an equivalent dose of 20 μmol / kg C210. The mice were killed 1, 2, 4, and 12 hours after intravenous administration, and the tumor tissues were collected and the accumulation of C210 in the tumors was determined by LC-MS.

[0094] The results are as follows Figure 5As shown in Table 4, free C210 is rapidly eliminated from the blood. Compared with free C210, the mean residence time (MRT) of C210 released from prodrug nanoparticles in the blood circulation is significantly prolonged, with the MRTs of α-C210-S-OA NPs, α-C210-SS-OA NPs, β-C210-S-OA NPs, and β-C210-SS-OA NPs being 7.3, 6.8, 8.7, and 6.1 times that of the free C210 group, respectively. Compared with the free C210 group, C210 prodrug nanoparticles significantly increased the AUC of C210. The AUCs of α-C210-S-OANPs, α-C210-SS-OA NPs, β-C210-S-OA NPs, and β-C210-SS-OA NPs were 9.9, 9.6, 3.4, and 2.2 times that of the free C210 group, respectively. This indicates that prodrug nanoparticles can effectively improve the pharmacokinetic behavior of C210. Figure 6 As shown in the data, the accumulation of α-C210-S-OA NPs in tumor tissue was much higher than that of the free C210 group. The accumulation of C210 released from α-C210-S-OANPs, α-C210-SS-OA NPs, β-C210-S-OANPs, and β-C210-S-OA NPs in tumor tissue was 3.3, 1.5, 1.2, and 0.8 times that of the free C210 group, respectively, indicating that the nanodelivery system can effectively increase drug accumulation in tumor tissue.

[0095] Table 4 Pharmacokinetic parameters of C210 and prodrug nanoparticles

[0096]

[0097] Example 10: In vivo anti-tumor study of C210 prodrug self-assembled nanoparticles

[0098] Construction of mouse breast cancer 4T1 subcutaneous tumor model: 1×10 6 4T1 cells were injected into the right abdomen of female BALB / c mice to observe the antitumor efficacy of C210 and prodrug nanoparticles. When the tumor volume reached 100 mm3, the tumor-bearing mice were randomly divided into 7 groups (normal saline, CTX, C210 and α-C210-S-OA-NPs, α-C210-SS-OA-NPs, β-C210-S-OA-NPs, β-C210-SS-OA-NPs), with 8 mice in each group. CTX (dose: 30 mg / kg) was injected intraperitoneally once every 3 days. The remaining groups were injected intravenously once a day (equivalent to C210 80 μmol / kg). Treatment was continued for 14 days or when the tumor size reached 2000 mm 3 The mice were killed at 4 hr. Tumor tissues were collected, weighed, and photographed to calculate the tumor inhibition rate.

[0099] The results are as follows Figure 7 As shown in the data, α-C210-S-OANPs has the most significant anti-tumor effect among several prodrug nanoparticles. Its anti-tumor effect is stronger than that of the same dose of free C210 and slightly stronger than that of the positive drug cyclophosphamide 30 mg / kg. Moreover, the anti-tumor effect of α-C210-S-OANPs is stronger than that of β-C210-S-OANPs, that is, the activity of short-chain prodrugs is significantly better than that of long-chain prodrugs.

[0100] In summary, α-C210-S-OANPs have many advantages, including good colloidal stability, rapid prodrug release, efficient cellular uptake, long blood circulation time, and high tumor-specific accumulation.

Claims

1. A curcumin derivative prodrug, characterized in that The prodrug is an oleyl alcohol-curcumin derivative connected by a single sulfur bond or a disulfide bond of different chain lengths, and its structural formula is shown in the general formula (1): wherein n is an integer of 1-2, and R is a monosulfide bond or a disulfide bond; The structural formula of the curcumin derivative is as follows:

2. The curcumin derivative prodrug according to claim 1, wherein It is a compound in which a single sulfide bond or a disulfide bond is located at the α and β positions of the adjacent carbonyl groups, obtained by connecting a curcumin derivative with oleyl alcohol through 2,2'-thiodiacetic acid, 2,2'-thiodipropionic acid, 3,3'-dithiodiacetic acid or 2,2'-dithiodipropionic acid. The structural formula is as follows:

3. The method for preparing the curcumin derivative prodrug according to claim 2, wherein: The steps include: (1) 2,2'-thiodiacetic acid, 2,2'-thiodipropionic acid, 3,3'-dithiodiacetic acid, 2,2'-dithiodipropionic acid, oleyl alcohol, EDCI and DMAP were added to a dichloromethane solution respectively, and the reaction mixture was stirred at room temperature under nitrogen protection. Four intermediate products α-S-OA, α-SS-OA, β-S-OA and β-SS-OA were separated by silica gel column chromatography; (2) The α-S-OA, α-SS-OA, β-S-OA, and β-SS-OA obtained in step (1) were dissolved in dichloromethane with a curcumin derivative, EDCI, and DMAP, respectively. The reaction mixture was stirred at room temperature under nitrogen protection, and the target products α-C210-S-OA, α-C210-SS-OA, β-C210-S-OA, and β-C210-SS-OA were separated by silica gel column chromatography.

4. The self-assembled nanoparticles carrying the curcumin derivative prodrug according to claim 1, characterized in that: The self-assembled nanoparticles are non-PEGylated curcumin derivative prodrug self-assembled nanoparticles or PEG-modified curcumin derivative prodrug self-assembled nanoparticles; the particle size is about 120 nm, the particle size distribution is uniform, and the surface charge is about 36 mV.

5. The self-assembled nanoparticles carrying curcumin derivative prodrug according to claim 4, characterized in that: The nanoparticles are obtained by a nanoparticle precipitation method, wherein the curcumin derivative prodrug and DSPE-PEG2000 are dissolved in acetone solvent, and the resulting solution is slowly added dropwise to water under stirring, so that the curcumin derivative prodrug spontaneously forms uniform nanoparticles. The solvent is removed by evaporation under reduced pressure to obtain a PEG-modified curcumin derivative prodrug self-assembled nanoparticle solution free of organic solvent. Wherein, DSPE-PEG2000 is not added in the preparation method of non-PEGylated C210 prodrug nanoparticles.

6. A pharmaceutical composition, characterized in that The invention relates to a self-assembled nanoparticle comprising the curcumin derivative prodrug according to claim 1 or 2 or the curcumin derivative prodrug-carrying nanoparticle according to claim 4, and a pharmaceutically acceptable excipient.

7. Use of the curcumin derivative prodrug according to claim 1 or 2 for preparing a drug for treating tumors, wherein the curcumin derivative prodrug contains a redox-responsive monosulfide bond or a disulfide bond, so that the prodrug can specifically release the curcumin derivative in tumor cells with high redox levels, thereby improving the specific distribution and release of the drug in tumor tissue and enhancing its selective anti-tumor effect.

8. The use according to claim 7, characterized in that The tumor is breast cancer or liver cancer.

9. Use of the self-assembling nanoparticles carrying a curcumin derivative prodrug according to claim 4 for preparing a drug for treating tumors, wherein the curcumin derivative prodrug is modified with oleic acid to enable the prodrug to self-assemble into nanoparticles, thereby prolonging the average residence time of the drug in the systemic circulation and improving its pharmacokinetic properties.

10. The use according to claim 9, characterized in that The tumor is breast cancer or liver cancer.

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