A compound for inducing apoptosis and immunogenic death of cancer cells, and a preparation method and application thereof

Abstract

The application discloses a kind of compound based on fatty acid, mitochondrial targeting, inducing cancer cell apoptosis and immunogenic death and preparation method and application thereof, belong to the field of medicinal chemistry and preparation.The structural formula of the drug precursor is as shown in formula (I) and / or (II).The series of compounds can form relatively stable self-assembly in aqueous solution, show high cytotoxicity which simple triphenylphosphine and fatty acid do not have, and induce obvious ICD phenomenon of tumor cells.The drug can be obtained by simple synthesis, with high yield, low preparation cost, high stability, good safety, meet the requirements of clinical medication, meet the requirements of large-scale industrial production, have good market prospect and clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and formulation, specifically relating to a series of fatty acid-based mitochondrial-targeted compounds that induce apoptosis and immunogenic death in cancer cells, their preparation methods, and their anti-tumor applications. Background Technology

[0002] Immunogenic cell death (ICD) refers to the release of tumor-associated antigens (TAAs) and damage-associated pattern molecules (DAMPs) during the death of tumor cells after treatment with chemotherapy drugs or photodynamic therapy. These TAMPs recruit and activate antigen-presenting cells (APCs), further activating tumor-specific T lymphocytes and thus promoting anti-tumor immunity (1. Krysko, DV; Garg, AD; Kaczmarek, A.; Krysko, O.; Agostinis, P.; Vandenabeele, P., Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer 2012, 12(12), 860-875. 2. Kroemer, G.; Galassi, C.; Zitvogel, L.; Galluzzi, L., Immunogenic cell stress and death. Nat Immunol 2022, 23(4), 487-500.). However, despite significant progress in the field of ICD in cancer treatment, the number of ICD inducers currently available for clinical application is relatively limited. Only a small number of drugs have been proven to induce ICD, such as the anthracycline drugs doxorubicin and oxaliplatin. In addition, fully inducing ICD in tumor cells requires high doses of chemotherapy drugs, which are difficult to achieve clinically due to severe toxic side effects (Zhou, JY; Wang, GY; Chen, YZ; Wang, HX; Hua, YQ; Cai, ZD, Immunogenic cell death in cancer therapy: Present and emerging inducers. J Cell Mol Med 2019, 23(8), 4854-4865.).Therefore, there is an urgent need to explore more efficient and precise new ICD-inducing drugs (1. Chen, C.; Ni, X.; Jia, SR; Liang, Y.; Wu, XL; Kong, DL; Ding, D., Massively Evoking Immunogenic Cell Death by Focused Mitochondrial OxidativeStress using an AIE Luminogen with a Twisted Molecular Structure. Adv Mater 2019, 31(52), e1904914. 2. Zheng, P.; Ding, BB; Jiang, ZY; Xu, WG; Li, G.; Ding, JX; Chen, XS, Ultrasound-Augmented Mitochondrial Calcium Ion Overload by Calcium Nanomodulator to Induce Immunogenic Cell Death. Nano Lett 2021, 21(5), 2088-2093.).

[0003] Studies have shown that the generation of reactive oxygen species (ROS) and endoplasmic reticulum stress are closely related to cellular immunogenic cell death. Drugs that directly induce endoplasmic reticulum stress based on ROS can exert ICD effects more efficiently than drugs that indirectly induce endoplasmic reticulum stress, such as doxorubicin (Krysko, DV; Garg, AD; Kaczmarek, A.; Krysko, O.; Agostinis, P.; Vandenabeele, P., Immunogenic cell death and DAMPs in cancer therapy. Nat RevCancer 2012, 12(12), 860-875.). Mitochondria are the main source of ROS in tumor cells. They can interact with the endoplasmic reticulum through mitochondrial-associated membranes (MAMs) and induce endoplasmic reticulum stress. (1. Zorov, DB; Juhaszova, M.; Sollott, SJ, Mitochoondrial Reactive Oxygen Species (Ros) and Ros Induced Ros Release. Physiological Reviews 2014, 94(3), 909-950. 2. Wu, HX; Carvalho, P.; Voeltz, GK, Here, there, and everywhere: The importance of ER membrane contactsites. Science 2018, 361(6401), eaan5835. 3. Csordas, G.; Weaver, D.; Hajnoczky, G., Endoplasmic Reticulum-Mitochondrial Contactology: Structure and Signaling Functions. Trends Cell Biol.) 2018, 28(7), 523-540.4, Senft, D.; Ronai, ZA, UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci 2015, 40(3), 141-148.). Therefore, targeting mitochondria and inducing a large amount of ROS to trigger mitochondrial and endoplasmic reticulum stress may provide insights for developing more efficient ICD inducers. Summary of the Invention

[0004] Fatty acids are essential nutrients for human life. They exhibit low toxicity in vivo, with significant cytotoxicity against tumor cells only at high concentrations (e.g., >100 μM). The applicant synthesized mitochondrial-targeting fatty acid compounds by chemically coupling and modifying fatty acids with amino- or hydroxylated triphenylphosphine. Unexpectedly, it was discovered that these compounds form relatively stable self-assemblies in aqueous solution, specifically damaging mitochondria and generating large amounts of reactive oxygen species (ROS), thus exhibiting high cytotoxicity not found in triphenylphosphine alone or in fatty acids, and inducing significant intracellular drug reaction (ICD) in tumor cells.

[0005] The purpose of this invention is to provide a novel fatty acid-based mitochondrial-targeting ICD inducer that activates specific anti-tumor immunity by inducing ICD in tumor cells. To achieve the above objective, this invention employs the following technical solution:

[0006] This invention provides a fatty acid-based mitochondrial-targeted cancer cell apoptosis and immunogenic death inducer, with the structural formula shown in formula (I) or (II):

[0007]

[0008] Where m = 1 to 8; R comes from different fatty acids.

[0009] Furthermore, R is:

[0010] Where n is an integer from 3 to 30; one or more CH2-CH2 in R can be further replaced by CH=CH. Furthermore, R contains 0 to 8 CH=CH.

[0011] Furthermore, m is an integer from 1 to 8; furthermore, m is an integer from 2 to 8; specifically, m is 2, 3, 4, 5, 6, 7, 8.

[0012] Furthermore, the R structure is as follows:

[0013]

[0014] More specifically, the compound is characterized in that it is one of the following compounds:

[0015]

[0016] In this invention, various fatty acids were modified using the mitochondrial-targeting group triphenylphosphonium (TPP) to synthesize a series of mitochondrial-targeting fatty acid compounds. Specifically, the triphenylphosphonium group provides the compounds with mitochondrial-targeting properties, and these compounds can self-assemble into nanoparticle structures in aqueous solution.

[0017] The results of this invention show that the nanoparticles assembled from the above compounds have significantly better antitumor activity than fatty acids and triphenylphosphine, and can induce immunogenic cell death in tumor cells.

[0018] The present invention also provides a method for preparing the compound described in any of the above technical solutions, comprising: (1) reacting R precursor fatty acid ROH with N-hydroxysuccinimide under the action of a base or a base / catalyst to obtain an intermediate product; (2) reacting the intermediate product with a triphenylphosphine amino- or hydroxylated derivative under the action of a base or a base / catalyst, and performing post-treatment after the reaction to obtain a drug with the structural formula shown in formula (I) or (II).

[0019] Furthermore, a method for synthesizing the aforementioned fatty acid-based mitochondrial-targeted immunogenic death inducer includes the following steps:

[0020] (1) Under the action of alkali or alkali / catalyst, fatty acids react with N-hydroxysuccinimide to obtain intermediate products; the molar ratio of fatty acids to N-hydroxysuccinimide in the reaction is 1:1 to 1.5.

[0021] (2) Under the action of alkali or alkali / catalyst, the intermediate product reacts with triphenylphosphine amino or hydroxylated derivatives. After the reaction is completed, the crude product is separated and purified to obtain the drug with the structural formulas shown in formulas (I) and (II).

[0022] Preferably, in steps (1) and (2), the base is selected from, but not limited to, N,N-diisopropylethylamine (DIEA) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); and the catalyst is selected from, but not limited to, 4-dimethylaminopyridine (DMAP).

[0023] Preferably, in steps (1) and (2), the reaction solvent is dichloromethane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixture of two or three of the above solvents.

[0024] Preferably, in step (1), the molar ratio of fatty acid to N-hydroxysuccinimide is 1:1 to 1.5.

[0025] Preferably, in step (1), the reaction temperature is 20–50°C and the reaction time is 1–4 h. More preferably, the reaction temperature is 45°C.

[0026] Preferably, in step (2), the molar ratio of the intermediate product to the triphenylphosphine-amino or hydroxylated derivative is 1:1 to 1.5. The structural formula of the triphenylphosphine-amino or hydroxylated derivative is as follows:

[0027]

[0028] Where m = 1 to 8.

[0029] Preferably, the triphenylphosphine amination derivative is 3-aminopropyl (triphenyl)phosphine bromide, 5-aminopentyl (triphenyl)phosphine bromide, or 9-aminononyl (triphenyl)phosphine bromide. The triphenylphosphine hydroxylation derivative is 3-hydroxypropyl (triphenyl)phosphine bromide, 5-hydroxypentyl (triphenyl)phosphine bromide, or 9-hydroxynonyl (triphenyl)phosphine bromide.

[0030] Preferably, in step (2), the reaction temperature is 20–50°C and the reaction time is 8–24 h. Thin-layer chromatography is used to monitor the reaction progress. More preferably, the reaction temperature is 45°C and the reaction time is 8 h.

[0031] Another object of the present invention is to provide a mitochondrial-targeted fatty acid compound self-assembly formulation. Specifically, the mitochondrial-targeted fatty acid compound is dissolved in an organic solvent such as dimethyl sulfoxide (DMSO), and self-assembled nanoparticles (mtDSN) are prepared by slowly injecting the DMSO into deionized water under ultrasonic water bath. The volume ratio of DMSO to deionized water is fixed at 3-20%.

[0032] Preferably, the volume ratio of DMSO to deionized water is fixed at 5%.

[0033] During respiration and oxidation, mitochondria store the energy generated as electrochemical potential energy in the inner mitochondrial membrane, resulting in a negative potential difference (-180mV to -200mV) across the inner mitochondrial membrane. Driven by this mitochondrial membrane potential, lipophilic cations preferentially accumulate in mitochondria (Murphy, MP, Selective targeting of bioactive compounds to mitochondria. Trends in biotechnology 1997, 15(8), 326-30; Murphy, MP, Targeting lipophilic cations to mitochondria. Biochim. Biophys. Acta. 2008, 1777(7-8), 1028-31). Triphenylphosphonium (TPP) is the most typical and widely used lipophilic cation mitochondrial targeting group. The delocalization of the π electron clouds of the three benzene rings in its structure makes the entire molecule positively charged, which can target mitochondria under the drive of the mitochondrial membrane potential. Therefore, triphenylphosphine salts can achieve mitochondrial targeting by virtue of the lipophilic cationic properties of triphenylphosphine (Zhang Haifeng, An Lu. Application of triphenylphosphine salts in tumor diagnosis and treatment [J]. Journal of Shanghai Normal University (Natural Science Edition, Chinese and English), 2024, 53(01):137-145.). Salts exist in aqueous solutions as free ions, and the drug precursor in this invention also exists in the form of free ions in the solution environment, thus not requiring the participation of anions to exert its targeting effect.

[0034] During preparation, the fatty acid-based mitochondrial-targeted cancer cell apoptosis and immunogenic death inducers mentioned in this invention can form externally neutral compound structures with the corresponding anions. Furthermore, the triphenylphosphine amino- or hydroxylated derivatives used in the preparation process also exist in cationic form in the reaction system, simultaneously forming externally neutral intermediate structures with the aforementioned anions. These anions include, but are not limited to, fluoride ions, bromide ions, iodide ions, or astatine ions.

[0035] The present invention demonstrates that, compared to low-toxicity fatty acids and triphenylphosphine amino or hydroxylated derivatives, the fatty acid-based mitochondrial-targeted immunogenic death inducer provided by the present invention exhibits strong cytotoxic effects in various tumor cells and induces immunogenic cell death in tumor cells.

[0036] The present invention also provides the application of the aforementioned fatty acid-based mitochondrial-targeted immunogenic death inducer in the preparation of antitumor drugs.

[0037] Specifically, the tumors mentioned are pancreatic cancer, lung cancer, cervical cancer, liver cancer, breast cancer, stomach cancer, ovarian cancer, colorectal cancer, bone cancer, melanoma, oral cancer, glioma, prostate cancer, bladder cancer, and kidney cancer.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] (1) This invention synthesizes a mitochondrial-targeted cancer cell apoptosis and immunogenic death inducers based on fatty acids. These inducers can self-assemble into nano-formations in aqueous solution without the need for additional amphiphilic polymer excipients. They exhibit strong anti-tumor activity not found in simple fatty acids and triphenylphosphine amino derivatives, and can induce immunogenic death of tumor cells. This provides a new approach for developing highly efficient ICD inducers.

[0040] (2) The present invention can obtain the drug precursor through simple synthesis, with high yield, low preparation cost, high stability, good safety, and meets the requirements of clinical drug use and large-scale industrial production, and has good market prospects and clinical application value. Attached Figure Description

[0041] Figure 1 This is the synthetic route for fatty acid compound 1 in Example 1.

[0042] Figure 2 This is the synthetic route for fatty acid compound 2 in Example 2.

[0043] Figure 3 This is the synthetic route for fatty acid compound 3 in Example 3.

[0044] Figure 4 This is the synthetic route for fatty acid compound 4 in Example 4.

[0045] Figure 5 This is the synthetic route for fatty acid compound 5 in Example 5.

[0046] Figure 6 This is the synthetic route for fatty acid compound 6 in Example 6.

[0047] Figure 7 This is the synthetic route for fatty acid compound 7 in Example 7.

[0048] Figure 8 This is the synthetic route for fatty acid compound 8 in Example 8.

[0049] Figure 9 The synthetic route for fatty acid compound 9 in Example 9 is shown.

[0050] Figure 10 The synthetic route for fatty acid compound 10 in Example 10 is shown.

[0051] Figure 11 This is the synthetic route for fatty acid compound 11 in Example 11.

[0052] Figure 12 The synthetic route for fatty acid compound 12 in Example 12 is shown.

[0053] Figure 13 The synthetic route for fatty acid compound 13 in Example 13 is shown.

[0054] Figure 14 The synthetic route for fatty acid compound 14 in Example 14 is shown.

[0055] Figure 15 The synthetic route for fatty acid compound 15 in Example 15 is shown.

[0056] Figure 16 The synthetic route for fatty acid compound 16 in Example 16 is shown.

[0057] Figure 17 The synthetic route for fatty acid compound 17 in Example 17 is shown.

[0058] Figure 18 The synthetic route for fatty acid compound 18 in Example 18 is shown.

[0059] Figure 19 The synthetic route for fatty acid compound 19 in Example 19 is shown.

[0060] Figure 20 This is the synthetic route for fatty acid compound 20 in Example 20. Detailed Implementation

[0061] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0063] N-Hydroxysuccinimide, CAS No. 6066-82-6; Triphenylphosphine, CAS No. 603-35-0; 3-Bromopropylamine, CAS No. 5003-71-4; 5-Bromopentylamine, CAS No. 51874-27-2; Heptanoic acid, CAS No. 142-62-1; Lauric acid, CAS No. 143-07-7; Oleic acid, CAS No. 112-80-1; Linoleic acid, CAS No. 60-33-3; DHA, CAS No. 6217-54-5; 9-Aminononyl(triphenyl)phosphine bromide, CAS No. 2248017-20-9, Merck 906107; 3-Hydroxypropyl(triphenyl)phosphine bromide, CAS No. 51860-45-8, Merck S860166.

[0064] Example 1: Synthesis of Fatty Acid Compound 1

[0065] 1. Synthesis of 3-aminopropyl(triphenyl)phosphine bromide

[0066] 3-Bromopropylamine (0.6 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 0.9 g of a white solid product, in 80.4% yield.

[0067] 2. Synthesis of fatty acid compound 1

[0068] Heptanonic acid (Hep, 58.6 mg, 0.45 mmol), N-hydroxysuccinimide (NHS, 62.1 mg, 0.54 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 77.6 mg, 0.50 mmol), and 4-dimethylaminopyridine (DMAP, 61.1 mg, 0.50 mmol) were dissolved in 2 mL of anhydrous dichloromethane (DCM). The reaction mixture was stirred and heated to 45 °C for 2 h to obtain Hep-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous N,N-dimethylformamide (DMF) was added. Then, 3-aminopropyl(triphenyl)phosphine bromide (200.2 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were added, and the mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated using an oil pump and washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 110.5 mg, with a yield of 47.9%. See the synthetic route below. Figure 1 . 1 H NMR (400MHz, CDCl3) δ9.04-9.01 (t, J = 6Hz, 1H), 7.82-7.67 (m, 15H), 3.87-3.80 (m, 2H), 3.50-3.46 (m, 2H), 2 .40-2.36(t,J=8Hz,2H),1.89-1.83(m,2H),1.66-1.59(m,2H),1.36-1.24(m,6H),0.87-0.84(t,J=6Hz,3H). 13 C NMR (100MHz, CDCl3) δ174.8,135.1,135.1,135.1,133.5,133.5,133.5,133.4,133.4,133.4,130.6,130.6,130 .6,130.5,130.5,130.5,118.6,117.8,117.8,38.7,36.4,31.7,29.1,25.9,22.5,21.1,20.6,14.1.HRMS:calcd for[C 28 H 35 NOP] + =432.2451; obsd:432.2442.

[0069] Example 2 Synthesis of fatty acid compound 2

[0070] 1. Synthesis of 3-aminopropyl(triphenyl)phosphine bromide

[0071] 3-Bromopropylamine (0.6 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 0.9 g of a white solid product, in 80.4% yield.

[0072] 2. Synthetic fatty acid compound 2

[0073] Lauric acid (Lau, 90.1 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain Lau-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by 3-aminopropyl(triphenyl)phosphine bromide (200.2 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 105.6 mg, with a yield of 40.3%. The synthetic route is described in [reference needed]. Figure 2 . 1 H NMR (400MHz, CDCl3) δ9.01-8.98 (t, J = 6Hz, 1H), 7.82-7.67 (m, 15H), 3.88-3.81 (m, 2H), 3.50-3.47 (m, 2H), 2. 40-2.36(t,J=8Hz,2H),1.87-1.82(m,2H),1.65-1.58(m,2H),1.31-1.19(m,16H),0.89-0.86(t,J=6Hz,3H). 13 C NMR (100MHz, CDCl3) δ174.8,135.1,135.1,135.1,133.5,133.5,133.5,133.4,133.4,133.4,130.6,130.6,130.6,130.5,130. 5,130.5,118.7,117.8,117.8,38.7,36.4,31.9,29.6,29.6,29.6,29.4,29.3,26.0,22.7,22.6,21.0,20.5,14.1.HRMS:calcd for[C 33 H 45 NOP] + =502.3233; obsd:502.3227.

[0074] Example 3: Synthesis of fatty acid compound 3

[0075] 1. Synthesis of 3-aminopropyl(triphenyl)phosphine bromide

[0076] 3-Bromopropylamine (0.6 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 0.9 g of a white solid product, in 80.4% yield.

[0077] 2. Synthetic fatty acid compounds 3

[0078] Oleic acid (OA, 127.1 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain OA-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by 3-aminopropyl(triphenyl)phosphine bromide (200.2 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 101.4 mg, with a yield of 33.9%. The synthetic route is described in [reference needed]. Figure 3 . 1 H NMR (400MHz, CDCl3) δ8.99-8.96 (t, J = 6Hz, 1H), 7.81-7.67 (m, 15H), 5.37-5.29 (m, 2H), 3.86-3.79 (m, 2H), 3.48 (s, 2H), 2. 39-2.35(t,J=8Hz,2H),2.01-1.98(t,J=6Hz,4H),1.85(s,2H),1.62-1.60(m,2H),1.27(s,20H),0.89-0.86(t,J=6Hz,3H). 13C NMR (100MHz, CDCl3) δ174.8,135.1,135.1135.1,133.5,133.5,133.5,133.4,133.4,133.4,130.6,130.6,130.6,130.5,130.5,130.5,129.9,12 9.8,118.7,117.8,117.8,38.8,36.4,31.9,29.8,29.8,29.5,29.5,29.5 ,29.4,29.3,27.2,25.9,22.7,22.7,22.6,20.9,20.4,14.1.HRMS:calcd for[C 39 H 55 NOP] + =584.4016; obsd:584.4006.

[0079] Example 4: Synthesis of fatty acid compound 4

[0080] 1. Synthesis of 3-aminopropyl(triphenyl)phosphine bromide

[0081] 3-Bromopropylamine (0.6 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 0.9 g of a white solid product, in 80.4% yield.

[0082] 2. Synthetic fatty acid compounds 4

[0083] Linoleic acid (LA, 126.2 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain LA-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by the addition of 3-aminopropyl(triphenyl)phosphine bromide (200.2 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 145.8 mg, with a yield of 48.9%. The synthetic route is described in [reference needed]. Figure 4 . 1 H NMR (400MHz, CDCl3) δ9.11-9.08(t,J=6Hz,1H),7.82-7.61(m,15H),5.41-5.28(m,4H),3.88-3.81(dd,J=16,12Hz,2H),3.49-3.47(m,2H),2.78-2. 75(t,J=6Hz,2H),2.41-2.37(t,J=8Hz,2H),2.07-2.00(m,4H),1.88-1.79 (m,2H),1.64-1.59(m,2H),1.39-1.22(m,14H),0.90-0.87(t,J=6Hz,3H). 13 C NMR (100MHz, CDCl3) δ174.8,135.1,135.1,135.1,133.5,133.5,133.5,1 33.4,133.4,133.4,130.6,130.6,130.6,130.5,130.5,130.5,130.2,130 .2,127.9,127.9,118.7,117.8,117.8,38.7,36.4,31.5,29.7,29.5,29. 4,29.3,27.3,27.2,25.9,25.6,22.7,22.6,21.1,20.6,14.1.HRMS:calcd for[C 39 H 53 NOP] + =582.3859; obsd:582.3860.

[0084] Example 5: Synthesis of Fatty Acid Compound 5

[0085] 1. Synthesis of 3-aminopropyl(triphenyl)phosphine bromide

[0086] 3-Bromopropylamine (0.6 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 0.9 g of a white solid product, in 80.4% yield.

[0087] 2. Synthetic fatty acid compounds 5

[0088] Weigh out DHA (147.8 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) and dissolve them in 2 mL of anhydrous DCM. Stir and heat the reaction mixture to 45 °C for 2 h to obtain DHA-NHS. Rotate the DCM to dryness, add 4 mL of anhydrous DMF, then add 3-aminopropyl(triphenyl)phosphine bromide (200.2 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol), and stir and heat at 45 °C for 8 h. Monitor the reaction process using thin-layer chromatography. After the reaction is complete, rotate the DMF to dryness using an oil pump, wash successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, and then remove water with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 135.9 mg, with a yield of 42.5%. The synthetic route is described in [reference needed]. Figure 5 . 1 H NMR(400MHz, CDCl3)δ9.07-9.05(t,J=4Hz,1H),7.81-7.67(m,15H),5.47-5.25(m,12H),3.87-3.79(dd,J=16,12Hz,2H),3 .49-3.48(d,J=4Hz,2H),2.84-2.82(m,10H),2.49-2.37(m,4H),2.11-2.04(m,2H),1.86(s,2H),0.99-0.95(t,J=8Hz,3H). 13 C NMR (100MHz, CDCl3) δ173.9,135.1,135.1,135.1,133.6,133.6,133.6,133.5,13 3.5,133.5,132.0,130.6,130.6,130.6,130.5,130.5,130.5,129.1,128.6,128.5 ,128.3,128.2,128.2,128.1,127.9,127.9,127.0,127.0,118.7,117.8,117.8,3 8.8,35.8,25.6,25.6,25.6,25.5,23.5,22.6,21.0,20.5,20.5,14.3.HRMS:calcd for[C 43 H 53 NOP] + =630.3859; obsd:630.3858.

[0089] Example 6: Synthesis of Fatty Acid Compound 6

[0090] 1. Synthesis of 5-aminopentyl(triphenyl)phosphine bromide

[0091] 5-Bromopentylamine (0.7 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 1 g of a white solid product, in 83.3% yield.

[0092] 2. Synthetic fatty acid compounds 6

[0093] Heptanonic acid (Hep, 58.6 mg, 0.45 mmol), N-hydroxysuccinimide (NHS, 62.1 mg, 0.54 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 77.6 mg, 0.50 mmol), and 4-dimethylaminopyridine (DMAP, 61.1 mg, 0.50 mmol) were dissolved in 2 mL of anhydrous dichloromethane (DCM). The reaction mixture was stirred and heated to 45 °C for 2 h to obtain Hep-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous N,N-dimethylformamide (DMF) was added. Then, 5-aminopentyl(triphenyl)phosphine bromide (214.1 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were added, and the mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated using an oil pump and washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was purified by silica gel column chromatography and dried under vacuum to obtain a white solid. The yield was 143.8 mg, with a yield of 59.1%. See the synthetic route below. Figure 6 .

[0094] Example 7 Synthesis of fatty acid compound 7

[0095] 1. Synthesis of 5-aminopentyl(triphenyl)phosphine bromide

[0096] 5-Bromopentylamine (0.7 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 1 g of a white solid product, in 83.3% yield.

[0097] 2. Synthetic fatty acid compounds 7

[0098] Lauric acid (Lau, 90.1 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain Lau-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by the addition of 5-aminopentyl(triphenyl)phosphine bromide (214.1 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 132.6 mg, with a yield of 48.3%. The synthetic route is described in [reference needed]. Figure 7 .

[0099] Example 8: Synthesis of Fatty Acid Compound 8

[0100] 1. Synthesis of 5-aminopentyl(triphenyl)phosphine bromide

[0101] 5-Bromopentylamine (0.7 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 1 g of a white solid product, in 83.3% yield.

[0102] 2. Synthetic fatty acid compounds 8

[0103] Oleic acid (OA, 127.1 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain OA-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by 5-aminopentyl(triphenyl)phosphine bromide (214.1 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 122.5 mg, with a yield of 39.3%. The synthetic route is described in [reference needed]. Figure 8 .

[0104] Example 9: Synthesis of fatty acid compound 9

[0105] 1. Synthesis of 5-aminopentyl(triphenyl)phosphine bromide

[0106] 5-Bromopentylamine (0.7 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 1 g of a white solid product, in 83.3% yield.

[0107] 2. Synthetic fatty acid compounds 9

[0108] Linoleic acid (LA, 126.2 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain LA-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by the addition of 5-aminopentyl(triphenyl)phosphine bromide (214.1 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 147.4 mg, with a yield of 47.4%. The synthetic route is described in [reference needed]. Figure 9 .

[0109] Example 10 Synthesis of fatty acid compound 10

[0110] 1. Synthesis of 5-aminopentyl(triphenyl)phosphine bromide

[0111] 5-Bromopentylamine (0.7 g, 2.8 mmol) and triphenylphosphine (1.4 g, 5.3 mmol) were refluxed in butanol (6 mL) at 120 °C for 6 h, then cooled to room temperature. Benzene and diethyl ether were added to the reaction mixture, and the precipitate was washed with diethyl ether until non-sticky. The solid was then redissolved in ethanol, followed by the addition of diethyl ether. Purification by silica gel column chromatography yielded 1 g of a white solid product, in 83.3% yield.

[0112] 2. Synthetic fatty acid compounds 10

[0113] Weigh out DHA (147.8 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) and dissolve them in 2 mL of anhydrous DCM. Stir and heat the reaction mixture to 45 °C for 2 h to obtain DHA-NHS. Rotate the DCM to dryness, add 4 mL of anhydrous DMF, then add 5-aminopentyl(triphenyl)phosphine bromide (214.1 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol), and stir and heat at 45 °C for 8 h. Monitor the reaction process using thin-layer chromatography. After the reaction is complete, rotate the DMF to dryness using an oil pump, wash successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, and then remove water with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 149.7 mg, with a yield of 45.0%. The synthetic route is described in [reference needed]. Figure 10 .

[0114] Example 11 Synthesis of fatty acid compound 11

[0115] 1. Synthetic fatty acid compounds 11

[0116] Heptanonic acid (Hep, 58.6 mg, 0.45 mmol), N-hydroxysuccinimide (NHS, 62.1 mg, 0.54 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 77.6 mg, 0.50 mmol), and 4-dimethylaminopyridine (DMAP, 61.1 mg, 0.50 mmol) were dissolved in 2 mL of anhydrous dichloromethane (DCM). The reaction mixture was stirred and heated to 45 °C for 2 h to obtain Hep-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous N,N-dimethylformamide (DMF) was added. Then, 9-aminononyl(triphenyl)phosphine bromide (242.3 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were added, and the mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated using an oil pump and washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was purified by silica gel column chromatography and dried under vacuum to obtain a white solid. The yield was 153.2 mg. See the synthetic route below. Figure 11 .

[0117] Example 12 Synthesis of fatty acid compound 12

[0118] 1. Synthetic fatty acid compounds 12

[0119] Lauric acid (Lau, 90.1 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain Lau-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by 9-aminononyl(triphenyl)phosphine bromide (242.3 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 150.5 mg. The synthetic route is described in [reference needed]. Figure 12 .

[0120] Example 13 Synthesis of fatty acid compound 13

[0121] 1. Synthetic fatty acid compounds 13

[0122] Oleic acid (OA, 127.1 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain OA-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by 9-aminononyl(triphenyl)phosphine bromide (242.3 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 159.3 mg. The synthetic route is described in [reference needed]. Figure 13 .

[0123] Example 14 Synthesis of fatty acid compound 14

[0124] 1. Synthetic fatty acid compounds 14

[0125] Linoleic acid (LA, 126.2 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were weighed and dissolved in 2 mL of anhydrous DCM. The reaction mixture was stirred and heated to 45 °C for 2 h to obtain LA-NHS. The DCM was evaporated to dryness, and 4 mL of anhydrous DMF was added, followed by 9-aminononyl(triphenyl)phosphine bromide (242.3 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated to 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the DMF was evaporated to dryness using an oil pump, and the mixture was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 156.1 mg. The synthetic route is described in [reference needed]. Figure 14 .

[0126] Example 15 Synthesis of fatty acid compound 15

[0127] 1. Synthetic fatty acid compounds 15

[0128] Weigh out DHA (147.8 mg, 0.45 mmol), NHS (62.1 mg, 0.54 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) and dissolve them in 2 mL of anhydrous DCM. Stir and heat the reaction mixture to 45 °C for 2 h to obtain DHA-NHS. Rotate the DCM to dryness, add 4 mL of anhydrous DMF, then add 9-aminononyl(triphenyl)phosphine bromide (242.3 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol), and stir and heat at 45 °C for 8 h. Monitor the reaction process using thin-layer chromatography. After the reaction is complete, rotate the DMF to dryness using an oil pump, wash successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, and then remove water with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 159.2 mg. The synthetic route is described in [reference needed]. Figure 15 .

[0129] Example 16 Synthesis of fatty acid compound 16

[0130] 1. Synthetic fatty acid compounds 16

[0131] Heptanoic acid (Hep, 58.6 mg, 0.45 mmol) was weighed and added to 4 mL of anhydrous N,N-dimethylformamide (DMF). Then, 3-hydroxypropyl(triphenyl)phosphine bromide (200.6 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were added, and the mixture was stirred and heated at 45 °C for 8 h. The reaction was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness using an oil pump, and the product was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 143.9 mg. The synthetic route is described in [reference needed]. Figure 16 .

[0132] Example 17 Synthesis of fatty acid compound 17

[0133] 1. Synthetic fatty acid compounds 17

[0134] Lauric acid (90.1 mg, 0.45 mmol) was weighed and added to 4 mL of anhydrous N,N-dimethylformamide (DMF). Then, 3-hydroxypropyl(triphenyl)phosphine bromide (200.6 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were added, and the mixture was stirred and heated at 45 °C for 8 h. The reaction was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness using an oil pump, and the product was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 147.2 mg. The synthetic route is described in [reference needed]. Figure 17 .

[0135] Example 18 Synthesis of fatty acid compound 18

[0136] 1. Synthetic fatty acid compounds 18

[0137] Oleic acid (OA, 127.1 mg, 0.45 mmol) was weighed and added to 4 mL of anhydrous N,N-dimethylformamide (DMF). Then, 3-hydroxypropyl(triphenyl)phosphine bromide (200.6 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol) were added, and the mixture was stirred and heated at 45 °C for 8 h. The reaction was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness using an oil pump, and the product was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 158.3 mg. The synthetic route is described in [reference needed]. Figure 18 .

[0138] Example 19 Synthesis of fatty acid compound 19

[0139] 1. Synthetic fatty acid compounds 19

[0140] Linoleic acid (LA, 126.2 mg, 0.45 mmol) was weighed and added to 4 mL of anhydrous N,N-dimethylformamide (DMF), followed by 3-hydroxypropyl(triphenyl)phosphine bromide (200.6 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol). The mixture was stirred and heated at 45 °C for 8 h. The reaction was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness using an oil pump, and the product was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was purified by silica gel column chromatography and dried under vacuum to obtain a white solid. The yield was 157.6 mg. The synthetic route is described in [reference needed]. Figure 19 .

[0141] Example 20 Synthesis of fatty acid compound 20

[0142] 1. Synthetic fatty acid compounds 20

[0143] Weigh out DHA (147.8 mg, 0.45 mmol), add 4 mL of anhydrous N,N-dimethylformamide (DMF), then add 3-hydroxypropyl(triphenyl)phosphine bromide (200.6 mg, 0.50 mmol), EDC (77.6 mg, 0.50 mmol), and DMAP (61.1 mg, 0.50 mmol), and stir and heat at 45 °C for 8 h. The reaction process was monitored by thin-layer chromatography. After the reaction, the DMF was evaporated to dryness using an oil pump, and the product was washed successively with 5% citric acid, saturated NaHCO3, and saturated NaCl, followed by dehydration with anhydrous Na2SO4. The primary product was separated and purified by silica gel column chromatography, and dried under vacuum to obtain a white solid. The yield was 163.4 mg. See the synthetic route for details. Figure 20 .

[0144] Example 21: Preparation and Testing of mtDSN Nanoparticle Formulation

[0145] The fatty acid compounds prepared in Examples 1-10 were dissolved in DMSO and slowly injected into deionized water under ultrasonic water bath to prepare self-assembled nanoparticles (mtDSN). The volume ratio of DMSO to deionized water was fixed at 5%. Uniformly dispersed nanoparticles were obtained. The nanoparticle prepared from fatty acid compound 1 was named mtDSN-1, and so on, the corresponding nanoparticles prepared from fatty acid compounds 2-5 were named mtDSN-2-5.

[0146] Example 22: Toxicity test of mtDSN on tumor cells

[0147] The toxicity of the nanoparticles mtDSN-1-5 prepared from fatty acid compounds 1-5 to various tumor cells was investigated in the examples, and the specific methods are as follows:

[0148] Logarithmic growth phase cells were seeded into 96-well plates (2000-5000 cells / well). After incubation at 37°C for 24 hours, mtDSN-1-5 were added, with 3-aminopropyl(triphenyl)phosphine bromide (TPP-NH2) and fatty acids (dissolved in DMSO) as control drugs. Four replicates were performed for each drug and concentration. After drug addition, the 96-well plates were placed in a cell culture incubator. After 72 hours of incubation, 30 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for another 4 hours. The culture medium was discarded, and 100 μL of DMSO was added to each well. After thorough shaking, the absorbance was measured at 490 nm using a microplate reader. The relative cell viability was calculated using the following formula, and a survival curve was plotted to obtain the IC50 of the drug for cell growth. 50 (Half-maximal inhibitory concentration).

[0149] Cell viability (%)=(At-Ab) / (Acon-Ab)×100%.

[0150] At, Acon, and Ab represent the absorbance of the experimental group (Treatment), control group (no fatty acid compounds or control drugs added, only cells added), and blank well (no drugs or cells added), respectively. The in vitro cytotoxicity results of mtDSN-1 to mtDSN-5 against tumor cells are shown in Table 1.

[0151] Table 1. Cytotoxicity assays (IC50) of mtDSN-1 to 5 in A549 lung cancer, HeLa cervical cancer, Panc2 pancreatic cancer, LM3 liver cancer, 4T1 breast cancer, 7901 gastric cancer, A2780 ovarian cancer, MC38 colorectal cancer, U2OS osteosarcoma, B16F10 melanoma, DU145 prostate cancer, HSC-3 oral cancer, U251 glioma, MBT-2 bladder cancer, and TK10 renal cell carcinoma tumor cells. 50 ±SD, μM)

[0152]

[0153] Table 2. Cytotoxicity assays (IC50) of fatty acids and TPP-NH2 in A549 lung cancer, HeLa cervical cancer, Panc2 pancreatic cancer, LM3 liver cancer, 4T1 breast cancer, 7901 gastric cancer, A2780 ovarian cancer, MC38 colorectal cancer, U2OS osteosarcoma, B16F10 melanoma, DU145 prostate cancer, HSC-3 oral cancer, U251 glioma, MBT-2 bladder cancer, and TK10 renal cell carcinoma tumor cells. 50 ±SD,

[0154] μM)

[0155]

[0156] Note: TPP+Hep, TPP+Lau, TPP+OA, TPP+LA, and TPP+DHA are the fatty acids and TPP-NH2 corresponding to nanoparticles mtDSN-1, mtDSN-2, mtDSN-3, mtDSN-4, and mtDSN-5, respectively.

[0157] The results in Tables 1 and 2 show that after co-culturing with various tumor cells for 72 h, the nanoparticles mtDSN-1-5 prepared from fatty acid compounds 1-5 exhibited significantly enhanced antitumor activity compared to fatty acids and TPP-NH2, realizing the transformation of fatty acids from "low-toxicity to tumor" to "high-toxicity antitumor".

[0158] Example 23: Detection of mtDSN-induced apoptosis in cancer cells

[0159] The apoptosis-inducing ability of nanoparticles mtDSN-1-5, prepared from fatty acid compounds 1-5, to triple-negative breast cancer cells 4T1 was investigated in the examples. The specific methods are as follows:

[0160] 4T1 cells were administered at a rate of 1×10⁻⁶. 5 Cells were seeded per well in six-well plates and incubated overnight for adhesion. Then, nanoparticles mtDSN-1–5 (10 μM), prepared from fatty acid compounds 1–5, were added to each well and incubated for another 48 hours. At the experimental endpoint, cells were digested with trypsin, washed with PBS, and centrifuged to obtain cell pellets. Annexin V-FITC and PI were then added according to the kit instructions, and the cells were stained at room temperature in the dark for 15 minutes. Each sample was then filtered through a cell sieve and analyzed by flow cytometry.

[0161] Table 3. Percentage of apoptosis induced by mtDSN-1 to 5 in 4T1 cells

[0162]

[0163] As shown in Table 3, after incubation at a concentration of 10 μM for 48 hours, the apoptosis rate of the mtDSN-2-5 nanoparticles was significantly increased.

[0164] Example 24: Evaluation of mtDSN-1 to 5 inducing ICD in tumor cells and promoting anti-tumor immune activation.

[0165] Primary bone marrow cells were extracted from the femur and tibia of healthy C57BL / 6 mice and cultured for 5 days with colony-stimulating factor and IL-4 to induce differentiation into bone marrow-derived cells (BMDCs). 4T1 cells were seeded in six-well plates and co-cultured with equal concentrations of free drug and nanoparticles for 12 hours. After drug withdrawal, fresh culture medium was added and incubated for another 12 hours. The supernatant was then co-incubated with the induced BMDCs for 24 hours. BMDCs incubated with fresh culture medium served as a blank control. CD80 levels of BMDCs under different treatments were analyzed by flow cytometry. + / CD86 + The expression of MHCII was investigated to explore whether the small molecule self-assembled mtDSN-1-5 could induce antigen-presenting cell maturation by promoting ICD in breast cancer cells.

[0166] Table 4. Maturation of BMDC induced by mtDSN-1 to 5

[0167]

[0168] As shown in Table 4, the small molecule self-assembled mtDSN-2~5 can significantly improve the CD80 of BMDC. + / CD86 +The expression of MHCII was verified to induce immunogenic death of tumor cells. The above embodiments are merely preferred embodiments of the present invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without inventive effort are all within the scope of protection of the present invention.

Claims

1. A nanoparticle, characterized in that, Formed by self-assembly of compounds with the configuration shown in formula (I) or (II): (Ⅰ) or (II) Where m = 1~8; R comes from different fatty acids; and R is: , where n is an integer from 3 to 30; one or more CH2-CH2 in its R can be further replaced by CH=CH; The nanoparticles are prepared by dissolving the compound of formula (Ⅰ) or (ⅠI) in an organic solvent and slowly injecting it into deionized water under an ultrasonic water bath to obtain self-assembled nanoparticles.

2. The nanoparticles according to claim 1, characterized in that, The R structure is as follows: 。 3. The nanoparticles according to claim 1, characterized in that, Composed of one of the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 4. A method for preparing the nanoparticles according to any one of claims 1 to 3, characterized in that: The compound of formula (I) or (II) is dissolved in an organic solvent and slowly injected into deionized water under an ultrasonic water bath to prepare self-assembled nanoparticles.

5. An antitumor drug, characterized in that, The nanoparticles contained in any one of claims 1 to 3 in a therapeutically effective amount.

6. The antitumor drug according to claim 5, characterized in that, The tumors include one or more of the following: pancreatic cancer, lung cancer, cervical cancer, liver cancer, breast cancer, stomach cancer, ovarian cancer, colorectal cancer, osteosarcoma, melanoma, oral cancer, glioma, prostate cancer, bladder cancer, and kidney cancer.

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