Nanodisk containing racimod prodrug compound, nanovaccine and preparation method and application of nanodisk and nanovaccine

Abstract

The invention relates to a nano-disk containing a racimod prodrug compound, a nano-vaccine and a preparation method and application of the nano-disk and the nano-vaccine. In particular, the present invention relates to nanodisks and nanovaccines comprising a specific racimod prodrug compound, a specific polyethylene glycol fatty acid coupling carrier, and a styrene-maleic anhydride copolymer. The nano-disk can significantly improve the solubility and targeting property of the drug, significantly improve the anti-tumor curative effect of the rasimod, effectively inhibit tumor recurrence and lung metastasis, effectively activate the inherent immunity of the body, and has good safety. The nano vaccine can effectively prevent tumor growth, cause tumor antigen specific immune response, and improve the level of tumor infiltration cytotoxic T lymphocytes.

Description

Technical Field

[0001] The present invention relates to the fields of chemistry and biomedicine, and in particular to nanodiscs and nanovaccines containing resiquimod prodrug compounds, preparation methods thereof, and uses thereof for treating or preventing diseases or conditions for which TLR7 / 8 agonists are applicable. Background Art

[0002] Toll-like receptors (TLRs) are a crucial class of pattern recognition receptors in innate immunity, widely expressed in immune cells such as dendritic cells, macrophages, and NK cells, as well as non-immune cells such as epithelial cells. They recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), regulating innate immunity and promoting adaptive immune responses. TLR activation triggers a series of signaling pathways within the cell, leading to the production of proinflammatory cytokines, chemokines, and antimicrobial molecules, ultimately enhancing the body's resistance to pathogens and exerting anti-tumor and anti-infection effects.

[0003] Currently, a total of 11 TLR family members have been discovered, of which TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 are located on the cell surface, while TLR3, TLR7, TLR8, and TLR9 are located in the endosome / lysosome. In recent years, the development of TLR agonist drugs has made great progress, and many drugs have entered clinical research. In preclinical studies, scientists have observed that the most effective agonists targeting TLRs are TLR7 and TLR8 agonists. Therefore, the research progress of TLR7 / 8 agonists currently in the clinical stage is relatively rapid. As a potential innate immune modulator, small molecule TLR7 / 8 agonists can enhance the tumor infiltration of cytotoxic T lymphocytes through the activation pathway of dendritic cells, thereby exerting anti-tumor effects. At present, the research and development of TLR7 / 8 agonists mainly focuses on imidazoquinoline derivatives. Resiquimod (R848) is a small molecule imidazoquinoline compound that activates immune cells through the TLR7 / 8 signaling pathway. It is currently mainly used in clinical practice to treat tumors and viral infectious diseases.

[0004] Resiquimod has shown potential in enhancing immune responses against tumors, but its clinical application faces several challenges. For example, its poor water solubility allows it to rapidly diffuse into the bloodstream after systemic administration, resulting in insufficient accumulation at target sites, which can trigger cytokine storms or autoimmune diseases. Therefore, there is an urgent need to develop more effective TLR7 / 8 agonists or formulations to improve the drug's solubility, enhance its targeted delivery capabilities, and enhance its efficacy, thereby broadening its scope of application. Summary of the Invention

[0005] This invention introduces different long-chain unsaturated fatty acids into the resiquimod molecule to construct a series of resiquimod prodrug compounds. The nanodiscs derived from these prodrug compounds can significantly increase the drug's solubility, significantly enhance the anti-tumor efficacy of resiquimod, effectively inhibit tumor recurrence and lung metastasis, effectively activate the body's innate immunity, and have a good safety profile. The nanovaccines derived from these prodrug compounds can produce excellent anti-tumor preventive efficacy, effectively preventing tumor development and effectively controlling tumor growth, eliciting tumor antigen-specific immune responses, and increasing the levels of tumor-infiltrating cytotoxic T lymphocytes (CTLs).

[0006] The structural formula of the resiquimod molecule is as follows:

[0007]

[0008] [Nanodiscs containing resiquimod prodrug compounds]

[0009] The present invention provides a nanodisc comprising a resiquimod prodrug compound, a polyethylene glycol fatty acid coupling carrier and a styrene-maleic anhydride copolymer.

[0010] Wherein, the resiquimod prodrug compound is a compound represented by formula (Ia) or formula (Ib), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0011]

[0012] The polyethylene glycol fatty acid coupled carrier is a polyethylene glycol fatty acid coupled carrier represented by formula (IIa) or formula (IIb):

[0013]

[0014] Where each R is independently C 8-30 Alkenyl -C(=O)-, n is selected from integers of 30 to 60.

[0015] In some embodiments, each R is independently C 10-26 Alkenyl-C(=O)-.

[0016] In some embodiments, each R is independently C 14-22 Alkenyl-C(=O)-.

[0017] In some embodiments, each R is independently oleoyl, linoleoyl, docosahexanoyl, eicosapentaenoyl, or docosapentaenoyl.

[0018] In some embodiments, each R is independently

[0019] In some embodiments, the resiquimod prodrug compound is the following compound, a pharmaceutically acceptable salt or stereoisomer thereof:

[0020]

[0021]

[0022] In some embodiments, in Formula (IIa) or Formula (IIb), n is selected from an integer of 35 to 55; or, n is selected from an integer of 40 to 50; or, n is selected from 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

[0023] In some embodiments, the polyethylene glycol fatty acid coupled carrier is a polyethylene glycol fatty acid coupled carrier represented by formula (IIa-1) or formula (IIb-1):

[0024]

[0025] Wherein, R is as described above.

[0026] In some embodiments, the polyethylene glycol fatty acid coupled carrier is the following polyethylene glycol fatty acid coupled carrier:

[0027]

[0028] In some embodiments, the molecular weight of the styrene-maleic anhydride copolymer is 5000-20000 Da; alternatively, the molecular weight of the styrene-maleic anhydride copolymer is 6000-15000 Da; alternatively, the molecular weight of the styrene-maleic anhydride copolymer is 7000-12000 Da; alternatively, the molecular weight of the styrene-maleic anhydride copolymer is 8000-11000 Da.

[0029] In some embodiments, the molecular weight of the styrene-maleic anhydride copolymer is 9500 Da.

[0030] [Method for preparing nanodiscs containing resiquimod prodrug compounds]

[0031] The present invention provides a method for preparing nanodiscs containing a resiquimod prodrug compound, comprising the following steps:

[0032] (1) dissolving the resiquimod prodrug compound and the polyethylene glycol fatty acid coupling carrier in dimethyl sulfoxide (DMSO);

[0033] (2) dissolving styrene-maleic anhydride copolymer (SMA) in deionized water;

[0034] (3) Slowly and uniformly injecting DMSO containing the prodrug compound and the polyethylene glycol fatty acid coupling carrier into the deionized water containing SMA during ultrasonic oscillation;

[0035] (4) The residual dimethyl sulfoxide was removed by dialysis to obtain uniformly dispersed nanodisks.

[0036] In some embodiments, the frequency of ultrasonic oscillation is 200 to 500 kHz.

[0037] In some embodiments, the volume ratio of dimethyl sulfoxide to deionized water is (0.5-2):(8-9.5).

[0038] In some embodiments, the volume ratio of dimethyl sulfoxide to deionized water is 1:9.

[0039] In some embodiments, the molar ratio of the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier and the styrene-maleic anhydride copolymer is (50-200):(1-50):(1-50); or, the molar ratio is (50-150):(1-30):(1-30); or, the molar ratio is (50-100):(1-20):(1-20); or, the molar ratio is (50-80):(1-10):(1-10).

[0040] In some embodiments, the molar ratio of the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier, and the styrene-maleic anhydride copolymer is 65:5:2, 65:5:6, 50:4:5, or 45:4:6.

[0041] [Nanovaccines containing resiquimod prodrug compounds]

[0042] The present invention provides a nano vaccine comprising a resiquimod prodrug compound, a polyethylene glycol fatty acid coupling carrier, an unsaturated fatty acyl modified antigen and a styrene-maleic anhydride copolymer.

[0043] Wherein, the resiquimod prodrug compound is a compound represented by the above formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof; the polyethylene glycol fatty acid coupling carrier is a polyethylene glycol fatty acid coupling carrier represented by the above formula (IIa) or formula (IIb); the unsaturated fatty acyl group is selected from C 8-30 Alkenyl-C(=O)-.

[0044] In some embodiments, the unsaturated fatty acyl group is selected from C 10-26 Alkenyl-C(=O)-.

[0045] In some embodiments, the unsaturated fatty acyl group is selected from C 14-22Alkenyl-C(=O)-.

[0046] In some embodiments, the unsaturated fatty acyl group is selected from oleoyl, linoleoyl, docosahexanoyl, eicosapentaenoyl, or docosapentaenoyl.

[0047] In some embodiments, the unsaturated fatty acyl group is selected from

[0048] In some embodiments, the antigen is selected from chicken ovalbumin (OVA), bovine serum albumin (BSA), human serum albumin (HSA), thyroglobulin (TG), or keyhole limpet hemocyanin (KLH).

[0049] In some embodiments, the molecular weight of the styrene-maleic anhydride copolymer is 5000-20000 Da; alternatively, the molecular weight of the styrene-maleic anhydride copolymer is 6000-15000 Da; alternatively, the molecular weight of the styrene-maleic anhydride copolymer is 7000-12000 Da; alternatively, the molecular weight of the styrene-maleic anhydride copolymer is 8000-11000 Da.

[0050] In some embodiments, the molecular weight of the styrene-maleic anhydride copolymer is 9500 Da.

[0051] In some embodiments, the molar ratio of the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier, the unsaturated fatty acyl-modified antigen and the styrene-maleic anhydride copolymer is (50-200):(1-50):(1-20):(1-50); or, the molar ratio is (50-200):(1-20):(1-20):(1-20); or, the molar ratio is (50-150):(1-20):(1-10):(1-10); or, the molar ratio is (50-100):(1-20):(1-10):(1-10).

[0052] In some embodiments, the molar ratio of the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier, the unsaturated fatty acyl-modified antigen, and the styrene-maleic anhydride copolymer is about 65:5:2:2, 80:10:5:5, or 100:12:8:8.

[0053] [Method for preparing nanovaccines containing resiquimod prodrug compounds]

[0054] The present invention provides a method for preparing a nano vaccine containing a resiquimod prodrug compound, comprising the following steps:

[0055] (1) dissolving the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier, and the unsaturated fatty acyl-modified antigen in dimethyl sulfoxide (DMSO);

[0056] (2) dissolving styrene-maleic anhydride copolymer (SMA) in deionized water;

[0057] (3) Under ultrasonic oscillation, DMSO containing the prodrug, carrier, and antigen is slowly and uniformly injected into deionized water containing SMA to obtain a nanovaccine.

[0058] In some embodiments, the frequency of ultrasonic oscillation is 200 to 500 kHz.

[0059] In some embodiments, the volume ratio of dimethyl sulfoxide to deionized water is (0.5-2):(8-9.5).

[0060] In some embodiments, the volume ratio of dimethyl sulfoxide to deionized water is 1:9.

[0061] [Uses and treatment methods]

[0062] The present invention provides use of the nanodisc or nanovaccine in preparing a medicament for treating or preventing a disease or condition for which a TLR7 / 8 agonist is suitable.

[0063] The present invention provides use of the combination of the above-mentioned nanodiscs or nanovaccines and a PD-1 inhibitor in the preparation of a medicament for treating or preventing diseases or conditions for which TLR7 / 8 agonists are applicable.

[0064] The present invention provides the use of a combination of a resiquimod prodrug compound represented by the above formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof, and a PD-1 inhibitor in the preparation of a medicament for treating or preventing diseases or conditions for which TLR7 / 8 agonists are applicable.

[0065] The present invention provides the above-mentioned nanodiscs or nanovaccines for use in treating or preventing diseases or conditions for which TLR7 / 8 agonists are suitable.

[0066] The present invention provides a combination of the above-mentioned nanodisc or nanovaccine and a PD-1 inhibitor for use in treating or preventing diseases or conditions for which TLR7 / 8 agonists are indicated.

[0067] The present invention provides a combination of a resiquimod prodrug compound represented by the above formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof, and a PD-1 inhibitor, which is used to treat or prevent diseases or conditions suitable for TLR7 / 8 agonists.

[0068] The present invention provides a method for treating or preventing a disease or condition for which a TLR7 / 8 agonist is applicable, comprising administering an effective amount of the above-mentioned nanodisc or nanovaccine to a subject in need thereof.

[0069] The present invention provides a method for treating or preventing a disease or condition for which a TLR7 / 8 agonist is suitable, comprising administering to a subject in need thereof an effective amount of a combination of the above-mentioned nanodisc or nanovaccine and a PD-1 inhibitor.

[0070] The present invention provides a method for treating or preventing diseases or conditions for which TLR7 / 8 agonists are suitable, comprising administering to a subject in need thereof an effective amount of a resiquimod prodrug compound represented by the above-mentioned formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof, and a combination of a PD-1 inhibitor.

[0071] The present invention provides a pharmaceutical preparation comprising the nanodisc or nanovaccine and at least one pharmaceutically acceptable excipient.

[0072] The present invention provides a combination comprising a resiquimod prodrug compound represented by the above formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof, and a PD-1 inhibitor.

[0073] The present invention provides a combination comprising the above-mentioned nanodisc or nanovaccine and a PD-1 inhibitor.

[0074] In some embodiments, the disease or condition for which a TLR7 / 8 agonist is indicated is selected from an inflammatory disease, an infectious disease, cancer, or a precancerous syndrome.

[0075] In some embodiments, the inflammatory disease is selected from arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, or rheumatoid arthritis.

[0076] In some embodiments, the infectious disease is selected from a retroviral infection, a hepatitis virus infection, a COVID-19 novel coronavirus infection, a Zika virus infection, or a dengue virus infection.

[0077] In some embodiments, the cancer is selected from breast cancer, melanoma, colon cancer, lung cancer, pancreatic cancer, kidney cancer, head and neck cancer, lymphoma, skin cancer, urothelial carcinoma, gastric cancer, or hepatocellular carcinoma.

[0078] In some embodiments, the precancerous syndrome is selected from chronic atrophic gastritis, gastric ulcer, ulcerative colitis, cirrhosis, colorectal adenoma, or familial polyposis.

[0079] In some embodiments, the PD-1 inhibitor is a small molecule compound, a polypeptide, or an antibody.

[0080] In some embodiments, the PD-1 inhibitor is selected from Pembrolizumab, Nivolumab, Cemiplimab, Tislelizumab, Toripalimab, Sintilimab, Adebrelimab, Serplulimab, Penpulimab, or Geptanolimab.

[0081] In some embodiments, the PD-1 inhibitor is selected from BioXCell's anti-mouse PD-1 inhibitors, which are clones RMP1-14, 29F.1A12, and J43. In some embodiments, the PD-1 inhibitor is InVivoMAb anti-mouse PD-1.

[0082] In some embodiments, the resiquimod prodrug compound represented by the above formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof and a PD-1 inhibitor are administered simultaneously, formulated independently and co-administered, or formulated independently and administered sequentially.

[0083] In some embodiments, the above-described nanodiscs or nanovaccines and the PD-1 inhibitor are administered simultaneously, formulated independently and co-administered, or formulated independently and administered sequentially.

[0084] In some embodiments, the resiquimod prodrug compound represented by the above formula (Ia) or formula (Ib), a pharmaceutically acceptable salt or stereoisomer thereof is administered once a day, once every two days, once every three days, once a week or once every two weeks.

[0085] In some embodiments, the nanodiscs or nanovaccines are administered once a day, once every two days, once every three days, once a week, or once every two weeks.

[0086] In some embodiments, the PD-1 inhibitor is administered once a day, once every two days, once every three days, once a week, or once every two weeks.

[0087] In some embodiments, the dosage of the resiquimod prodrug compound represented by the above formula (Ia) or formula (Ib), its pharmaceutically acceptable salt or stereoisomer is 0.001 to 100 mg / kg per administration, for example, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg.

[0088] In some embodiments, the dosage of the above-mentioned PD-1 inhibitor is 0.0001 to 100 mg / kg per administration, for example, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg.

[0089] In some embodiments, the above-mentioned nanodiscs or nanovaccines and the PD-1 inhibitor can be administered by the same route of administration or different routes of administration.

[0090] In some embodiments, the administration route of the resiquimod prodrug compound represented by Formula (Ia) or Formula (Ib), its pharmaceutically acceptable salt or stereoisomer is subcutaneous injection; the administration route of the PD-1 inhibitor is intraperitoneal injection.

[0091] In some embodiments, the administration route of the nanodisc or nanovaccine is subcutaneous injection; the administration route of the PD-1 inhibitor is intraperitoneal injection.

[0092] The nanodiscs or nanovaccines described above can act systemically and / or locally. For this purpose, they can be administered by a suitable route, for example, parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, intramuscularly or as an inhalant.

[0093] The above-mentioned administration route can be achieved through a suitable dosage form. The dosage forms that can be used in the present invention include, but are not limited to, tablets, capsules, lozenges, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, syrups, etc.

[0094] The dosage regimen can be adjusted to provide the optimal desired response. For example, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as required for the treatment situation. It should be noted that the dosage value can vary with the type and severity of the condition to be alleviated, and can include single or multiple doses. In general, the dosage for treatment varies, depending on considerations such as: the age, sex, and general health of the patient to be treated; the frequency and desired effect of treatment; the extent of tissue damage; the duration of symptoms; and other variables that can be adjusted by the physician. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the drug. The dosage and administration regimen of the pharmaceutical composition can be determined by a person of ordinary skill in the clinical field.

[0095] [Beneficial Effects]

[0096] The present invention utilizes long-chain unsaturated fatty acids to modify the TLR7 / 8 agonist resiquimod, and synthesizes a series of fatty acid-modified resiquimod prodrug compounds. The nanodiscs obtained from these prodrug compounds can significantly increase the solubility of the drug, significantly improve the anti-tumor efficacy of resiquimod, effectively inhibit tumor recurrence and lung metastasis, effectively activate the body's innate immunity, and have good safety. The nanovaccines obtained from these prodrug compounds can produce excellent anti-tumor prevention efficacy, effectively prevent tumor occurrence, and can also effectively control tumor growth, induce tumor antigen-specific immune response, and increase the level of tumor-infiltrating cytotoxic T lymphocytes (CTLs). At the same time, the unsaturated fatty acids used to modify the drug are nutrients required by the human body and have good biocompatibility. The preparation method of the prodrug compound of the present invention has high yield, low cost, and simple preparation, and the obtained prodrug compound has good clinical application value and industrial production prospects.

[0097] [Related definitions]

[0098] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0099] The terms "include," "comprising," "having," or "containing" and their variations herein are intended to be inclusive or open-ended collective concepts and do not exclude other unrecited elements or method steps. Those skilled in the art will appreciate that the above terms, such as "comprising," encompass the meaning of "consisting of."

[0100] When the lower and upper limits of a numerical range are disclosed, any value or sub-range falling within the range is specifically disclosed. In particular, each numerical range of a parameter disclosed herein (e.g., in the form of "about a to b," or equivalently, "above a and below b") should be understood to encompass every value and sub-range therein. For example, "C 1-4 " should be understood to include any sub-ranges and every point value therein, such as C 2-4 、C 3-4 、C 1-2 、C 1-3 、C 1-4 etc., as well as C1, C2, C3, C4, etc.

[0101] The term "effective amount" refers to a dose that can induce a biological or medical response in cells, tissues, organs or organisms (eg, individuals) and is sufficient to achieve the desired preventive and / or therapeutic effect.

[0102] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is substantially non-toxic to living organisms. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed by reacting a compound of the present invention with a pharmaceutically acceptable inorganic / organic acid or inorganic / organic base, which are also referred to as acid addition salts or base addition salts. For a review of suitable salts, see, for example, Jusiak, Soczewinski, et al., Remington's Pharmaceutical Sciences [M], Mack Publishing Company, 2005 and Stahl, Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use [M], Wiley-VCH, 2002. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0103] The term "pharmaceutically acceptable excipient" refers to an auxiliary material that is administered with a therapeutic agent and is suitable, within the scope of sound medical judgment, for contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. Examples include, but are not limited to, carriers, diluents, binders, absorbents, colorants, adjuvants, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, and the like.

[0104] The terms "treat," "treat," "treat," and "treating" mean to reverse, alleviate, ameliorate the progression of the disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition.

[0105] The term "prevention" includes suppressing and delaying the onset of a disease, and includes not only prevention before the development of a disease but also prevention of recurrence of a disease after treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 1 is a transmission electron micrograph of the nanodisc containing the resiquimod prodrug compound in Example 13 and a particle size distribution diagram.

[0107] Figure 2 The effect of the nanodiscs containing the resiquimod prodrug compound in Example 14 on drug tissue distribution.

[0108] Figure 3 Graph showing the in vivo antitumor activity analysis of the nanodiscs containing the resiquimod prodrug compound in Example 15.

[0109] Figure 4 Graph showing the in vivo immune activation analysis of the nanodiscs containing the resiquimod prodrug compound in Example 16.

[0110] Figure 5 This is a diagram showing the reaction process of the double-chain docosahexaenoic acid-antigen polypeptide in Example 18.

[0111] Figure 6 1 is a transmission electron micrograph and particle size distribution diagram of the nanovaccine containing the resiquimod prodrug compound in Example 19.

[0112] Figure 7 This is a graph evaluating the ability of the nanovaccine containing the resiquimod prodrug compound in Example 20 to prevent tumorigenesis.

[0113] Figure 8 This is a graph evaluating the ability of the nanovaccine containing the resiquimod prodrug compound in Example 21 to control tumor growth and immune activation. DETAILED DESCRIPTION

[0114] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0115] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0116] Example 1 Synthesis of Resiquimod-Oleic Acid Conjugate Prodrug (R848-OA)

[0117]

[0118] Resiquimod (30 mg, 0.095 mmol, 1.0 eq), oleic acid (OA, 30.0 mg, 0.105 mmol, 1.1 eq), and 4-dimethylaminopyridine (DMAP, 12.8 mg, 0.105 mmol, 1.1 eq) were added sequentially to a reaction flask and dissolved in 2 ml of dichloromethane (DCM, ultra-dry reagent). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 16.3 mg, 0.105 mmol, 1.1 eq) was then rapidly added dropwise. The reaction solution was stirred under reflux at 45°C under nitrogen for 24 hours. The reaction endpoint was determined by thin-layer chromatography (TLC). After the reaction, the mixture was washed with 5% citric acid, saturated sodium bicarbonate and saturated brine three times each. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product was separated and purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 30:1) to obtain product 1 (R848-OA) as a white solid product with a yield of 39.9 mg and a yield of 72.3%.

[0119] 1 H NMR (400MHz, DMSO) δ8.55-8.53(d,J=4.0Hz,1H),7.94-7.92(d,J=4.0Hz,1H),7. 64-7.60(t,J=8.0Hz,1H),7.56-7.52(t,J=8.0Hz,1H),5.36-5.28(m,2H),5.00(s ,2H),4.76(s,2H),3.56-3.52(q,J=8.0Hz,2H),2.68(s,2H),2.34(s,2H),1.99- 1.95(m,4H),1.68-1.60(m,2H),1.31-1.12(m,29H),0.84-0.82(t,J=4.0Hz,3H).

[0120] Example 2 Synthesis of Resiquimod-Linoleic Acid Conjugate Prodrug (R848-LA)

[0121]

[0122] Resiquimod (30 mg, 0.095 mmol, 1.0 eq), linoleic acid (LA, 29.4 mg, 0.105 mmol, 1.1 eq) and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) were added to the reaction flask in sequence and dissolved in 2 ml of ultra-dry DCM. EDC (16.3 mg, 0.105 mmol, 1.1 eq) was then added dropwise rapidly. The reaction solution was stirred under reflux at 45 ° C for 24 h under nitrogen protection, and the reaction endpoint was determined by TLC. After the reaction was completed, the product was washed with 5% citric acid, saturated sodium bicarbonate and saturated salt water three times each. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product 2 (R848-LA) was separated and purified by silica gel column chromatography (DCM: MeOH = 30: 1) to obtain a light yellow semi-solid product with a yield of 43.5 mg and a yield of 76.7%.

[0123] 1 H NMR(400MHz, DMSO))δ8.55-8.53(d,J=4.0Hz,1H),7.95-7.93(d,J=4.0Hz,1H),7.64-7.60(t,J=8 .0Hz,1H),7.56-7.52(t,J=8.0Hz,1H),5.36-5.28(t,J=16.0Hz,4H),5.00(s,2H),4.75(s,2H),3 .56-3.52(q,J=8.0Hz,2H),2.74-2.72(d,J=4.0Hz,2H),2.68(s,2H),2.55-2.51(t,J=8.0Hz,2H) ,2.00-1.98(q,J=4.0Hz,4H),1.67-1.60(m,2H),1.32-1.12(m,23H),0.86-0.82(t,J=8.0Hz,3H).

[0124] Example 3 Synthesis of Resiquimod-Docosahexaenoic Acid Conjugate Prodrug (R848-DHA)

[0125]

[0126] Resiquimod (30 mg, 0.095 mmol, 1.0 eq), docosahexaenoic acid (DHA, 34.5 mg, 0.105 mmol, 1.1 eq) and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) were added to the reaction flask in sequence and dissolved in 2 ml of ultra-dry DCM. EDC (16.3 mg, 0.105 mmol, 1.1 eq) was then added dropwise. The reaction solution was stirred at reflux at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the product was washed with 5% citric acid, saturated sodium bicarbonate and saturated salt water three times each. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to dryness. The product 3 (R848-DHA) was separated and purified by silica gel column chromatography (DCM: MeOH = 30: 1) to obtain a light yellow semi-solid product with a yield of 51.2 mg and a yield of 85.8%.

[0127] 1 H NMR (400MHz, DMSO) δ8.54-8.52(d,J=4.0Hz,1H),7.93-7.91(d,J=4.0Hz,1H),7.64-7.60(t ,J=8.0Hz,1H),7.56-7.62(t,J=8.0Hz,1H),5.50-5.24(m,12H),4.99(s,2H),4.76(s,2H),3 .56-3.52(q,J=8.0Hz,2H),2.86-2.74(m,12H),2.55-2.51(t,J=8.0Hz,2H),2.44-2.40(q, J=8.0Hz, 2H), 2.03-1.97 (q, J=12.0Hz, 2H), 1.23-1.12 (m, 9H), 0.91-0.87 (t, J=8.0Hz, 3H).

[0128] Example 4 Synthesis of Resiquimod-Double-chain Oleic Acid Conjugate Prodrug (R848-diOA)

[0129] Step 1:

[0130]

[0131] First, oleic acid was activated with N-hydroxysuccinimide (NHS): oleic acid (169.5 mg, 0.60 mmol, 1.0 eq), NHS (103.6 mg, 0.09 mmol, 1.5 eq), and DMAP (73.2 mg, 0.60 mmol, 1.0 eq) were added sequentially to a reaction flask and dissolved in 2 ml of ultra-dry DCM. EDC (93.2 mg, 0.60 mmol, 1.0 eq) was then added dropwise. The reaction mixture was stirred at reflux at 45°C under nitrogen for 4 h, and the reaction endpoint was determined by TLC. After the reaction was basically completed, the mixture was washed with 5% citric acid, saturated sodium bicarbonate and saturated brine in sequence, the organic layer was dried over anhydrous sodium sulfate and distilled under reduced pressure to dryness, and then purified by silica gel column chromatography (dichloromethane 100%) to obtain product 4 (NHS-activated form of oleic acid) as a white solid product with a yield of 203.1 mg and a yield of 89.2%.

[0132] 1 H NMR (400MHz, CDCl3) δ5.40-5.29(m,2H),2.84(s,4H),2.62-2.58(t,J=8.0Hz,2H),2. 08-1.94(m,4H),1.78-1.70(m,2H),1.44-1.24(m,20H),0.90-0.86(t,J=8.0Hz,3H).

[0133] Step 2:

[0134]

[0135] Synthesis of a double-chain oleic acid intermediate from lysine: Product 4 (178.3 mg, 0.42 mmol, 2.1 eq), L-lysine methyl ester dihydrochloride (46.6 mg, 0.20 mmol, 1.0 eq), and N,N-diisopropylethylamine (DIEA, 54.3 mg, 0.42 mmol, 2.1 eq) were added sequentially to a reaction flask and dissolved in 2 ml of N,N-dimethylformamide (DMF, an ultra-dry reagent). The mixture was stirred under reflux at 45°C under nitrogen for 6 h. The reaction endpoint was determined by TLC. After the reaction was substantially complete, the mixture was washed sequentially with 5% citric acid, saturated sodium bicarbonate, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product 5 was separated and purified by silica gel column chromatography (DCM:MeOH = 50:1) to obtain product 5 as a white solid in a yield of 130.4 mg (92.3%).

[0136] 1H NMR (400MHz, CDCl3) δ6.18-6.16 (d, J=4.0Hz, 1H), 5.67-5.65 (t, J=4.0Hz, 1H), 5.38- 5.29(m,4H),4.60-4.56(q,J=8.0Hz,1H),3.74(s,3H),3.26-3.22(q,J=8.0Hz,2H),2. 25-2.21(t,J=8.0Hz,2H),2.17-2.13(t,J=8.0Hz,2H),2.04-1.98(m,8H),1.90-1.67 (m,2H),1.66-1.58(m,4H),1.56-1.48(m,2H),1.38-1.23(m,42H),0.93-0.83(m,6H).

[0137] Step 3:

[0138]

[0139] Synthesis of dioleic acid: Prepare a 1M sodium hydroxide solution and mix it with methanol in a volume ratio of 1:1 to obtain a mixed solvent. Add product 5 (0.15 mmol) and 3 ml of the mixed solvent to the reaction flask, stir and react at room temperature for 2 hours under nitrogen protection, and determine the reaction endpoint by TLC. After the hydrolysis is substantially completed, use the prepared 1M hydrochloric acid to neutralize the reaction solution to a weak acidity, add DCM and wash with saturated salt water, dry the organic layer with anhydrous sodium sulfate and distill under reduced pressure to dryness, and separate and purify it by silica gel column chromatography (DCM:MeOH=20:1) to obtain product 6 as a white solid product with a yield of 95.5 mg and a yield of 94.3%.

[0140] 1 H NMR (400MHz, CDCl3) δ6.83-6.81(d,J=4.0Hz,1H),5.78-5.76(d,J=4.0Hz,1H),5.38- 5.30(m,4H),4.51-4.47(q,J=8.0Hz,1H),3.37-3.19(m,2H),2.29-2.25(t,J=8.0Hz, 2H),2.20-2.16(t,J=8.0Hz,2H),2.00-1.98(q,J=4.0Hz,8H),1.90-1.82(q,J=16.0H z,2H),1.65-1.59(m,4H),1.56-1.50(m,2H),1.30-1.26(m,42H),0.90-0.86(m,6H).

[0141] Step 4:

[0142]

[0143] Subsequently, resiquimod (R848, 30 mg, 0.095 mmol, 1.0 eq), product 6 (70.8 mg, 0.105 mmol, 1.1 eq), and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) were added to the reaction flask in sequence and dissolved in 2 ml of ultra-dry DCM. EDC (16.3 mg, 0.105 mmol, 1.1 eq) was then added dropwise. The reaction solution was stirred at reflux at 45 ° C under nitrogen protection for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the mixture was washed with 5% citric acid, saturated sodium bicarbonate, and saturated salt water three times each. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product was separated and purified by silica gel column chromatography (DCM: MeOH = 30: 1) to obtain product 7 (R848-diOA) as a white solid product with a yield of 66.5 mg and a yield of 72.1%.

[0144] 1 H NMR (400MHz, DMSO) δ8.54-8.52(d,J=4.0Hz,1H),8.23-8.21(d,J=4.0Hz,1H),7.94-7.92(d,J=4.0Hz,1H),7.86-7 .82(t,J=8.0Hz,1H),7.64-7.60(t,J=8.0Hz,1H),7.56-7.52(t,J=8.0Hz,1H),5.32-5.28(m,4H),4.99(s,2H),4. 77(s,2H),3.56-3.52(q,J=8.0Hz,2H),3.03(s,2H),2.69-2.67(q,J=4.0Hz,1H),2.20-2.16(q,J=8.0Hz,2H),2.0 6-1.90(m,12H),1.87-1.67(m,2H),1.54-1.50(m,2H),1.46-1.42(m,4H),1.28-1.13(m,51H),0.86-0.82(m,6H).

[0145] Example 5 Synthesis of Resiquimod-Double-chain Linoleic Acid Conjugate Prodrug (R848-diLA)

[0146] Step 1:

[0147]

[0148] First, linoleic acid was activated with N-hydroxysuccinimide (NHS): linoleic acid (168.3 mg, 0.60 mmol, 1.0 eq), NHS (103.6 mg, 0.09 mmol, 1.5 eq), and DMAP (73.2 mg, 0.60 mmol, 1.0 eq) were added sequentially to a reaction flask and dissolved in 2 ml of ultra-dry DCM. EDC (93.2 mg, 0.60 mmol, 1.0 eq) was then added dropwise. The reaction mixture was stirred at reflux at 45°C under nitrogen for 4 h, and the reaction endpoint was determined by TLC. After the reaction was basically completed, the mixture was washed with 5% citric acid, saturated sodium bicarbonate and saturated brine in sequence, the organic layer was dried over anhydrous sodium sulfate and distilled under reduced pressure to dryness, and then separated and purified by silica gel column chromatography (dichloromethane 100%) to obtain product 8 (NHS-activated form of linoleic acid) as a slightly yellow oily product with a yield of 198.5 mg and a yield of 87.6%.

[0149] 1 H NMR (400MHz, CDCl3) δ5.48-5.27(m,4H),2.84(s,4H),2.80-2.76(t,J=8Hz,2H),2.62-2.58(t,J=8Hz ,2H),2.07-2.03(q,J=8Hz,4H),1.78-1.72(m,2H),1.43-1.25(m,14H),0.91-0.87(t,J=8.0Hz,3H).

[0150] Step 2:

[0151]

[0152] Synthesis of a double-chain linoleic acid intermediate from lysine: Product 8 (177.5 mg, 0.42 mmol, 2.1 eq), L-lysine methyl ester dihydrochloride (46.6 mg, 0.20 mmol, 1.0 eq), and N,N-diisopropylethylamine (DIEA, 54.3 mg, 0.42 mmol, 2.1 eq) were added sequentially to a reaction flask, dissolved in 2 ml of N,N-dimethylformamide (DMF, ultra-dry reagent), and stirred under reflux at 45°C for 6 h under nitrogen. The reaction endpoint was determined by TLC. After the reaction was substantially complete, the product was washed sequentially with 5% citric acid, saturated sodium bicarbonate, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. It was then purified by silica gel column chromatography (DCM:MeOH = 50:1) to obtain product 9 as a slightly yellow semi-solid product with a yield of 128.4 mg and a yield of 91.2%.

[0153] 1H NMR(400MHz, CDCl3) δ6.16-6.14(d,J=4.0Hz,1H),5.63-5.61(t,J=4.0Hz,1H),5.43-5.28(m,8 H),4.60-4.56(q,J=8.0Hz,1H),3.74(s,3H),3.26-3.22(q,J=8.0Hz,2H),2.79-2.75(t,J=8.0 Hz,4H),2.25-2.21(t,J=8.0Hz,2H),2.17-2.13(t,J=8Hz,2H),2.07-2.03(q,J=8.0Hz,8H),1. 80-1.70(m,2H),1.66-1.62(m,4H),1.56-1.50(m,2H),1.37-1.26(m,30H),0.91-0.87(m,6H).

[0154] Step 3:

[0155]

[0156] Synthesis of di-chain linoleic acid: Prepare a 1M sodium hydroxide solution and mix it with methanol in a volume ratio of 1:1 to obtain a mixed solvent. Add product 9 (0.15 mmol) and 3 ml of the mixed solvent to a reaction flask. Stir the reaction at room temperature under nitrogen for 2 h. The reaction endpoint is determined by TLC. After the hydrolysis is substantially complete, the reaction solution is neutralized to a weak acidity using the prepared 1M hydrochloric acid. DCM is added and washed with saturated brine. The organic layer is dried over anhydrous sodium sulfate and evaporated under reduced pressure to dryness. Purification is performed by silica gel column chromatography (DCM:MeOH = 20:1) to obtain product 10 as a slightly yellow semi-solid product with a yield of 93.0 mg and a yield of 92.1%.

[0157] 1 H NMR (400MHz, CDCl3) δ6.80-6.78 (d, J=4.0Hz, 1H), 6.03-5.99 (t, J=8.0Hz, 1H), 5.40-5.30 (m ,8H),4.57-4.53(q,J=8.0Hz,1H),3.31-3.21(q,J=20.0Hz,2H),2.78-2.76(t,J=4.0Hz,4H), 2.27-2.23(t,J=12.0Hz,4H),2.21-2.17(t,J=12.0Hz,4H),2.07-2.03(q,J=8.0Hz,8H),1.9 0-1.80(m,2H),1.68-1.56(m,4H),1.55-1.51(m,2H),1.38-1.23(m,30H),0.91-0.87(m,6H).

[0158] Step 4:

[0159]

[0160] Subsequently, resiquimod (R848, 30 mg, 0.095 mmol, 1.0 eq), product 10 (70.4 mg, 0.105 mmol, 1.1 eq), and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) were added to the reaction flask in sequence and dissolved in 2 ml of ultra-dry DCM. EDC (16.3 mg, 0.105 mmol, 1.1 eq) was then added dropwise. The reaction solution was stirred at reflux at 45 ° C under nitrogen protection for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the solution was washed with 5% citric acid, saturated sodium bicarbonate, and saturated salt water three times each. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product was separated and purified by silica gel column chromatography (DCM:MeOH=30:1) to obtain product 11 (R848-diLA) as a slightly yellow semi-solid product with a yield of 64.1 mg and a yield of 69.8%.

[0161] 1 H NMR (400MHz, DMSO) δ8.54-8.52(d,J=4.0Hz,1H),8.23-8.21(d,J=4.0Hz,1H),7.94-7.92(d,J=4.0Hz,1H),7.86-7.82( t,J=8.0Hz,1H),7.64-7.60(t,J=8.0Hz,1H),7.56-7.52(t,J=8.0Hz,1H),5.36-5.26(m,8H),4.99(s,1H),4.77(s,2H) ,3.56-3.52(q,J=8.0Hz,2H),3.17-3.15(q,J=4.0Hz,1H),3.03(s,2H),2.76-2.69(m,4H),2.20-2.16(q,J=8.0Hz,2H) ,2.04-1.98(m,12H),1.87-1.77(m,2H),1.53-1.49(m,2H),1.46-1.42(m,4H),1.33-1.11(m,39H),0.86-0.82(m,6H).

[0162] Example 6 Synthesis of Resiquimod-Double-Chained Docosahexaenoic Acid Conjugate Prodrug (R848-diDHA)

[0163] Step 1:

[0164]

[0165] First, Docosahexaenoic acid was activated with N-hydroxysuccinimide (NHS): Docosahexaenoic acid (197.1 mg, 0.60 mmol, 1.0 eq), NHS (103.6 mg, 0.09 mmol, 1.5 eq), and DMAP (73.2 mg, 0.60 mmol, 1.0 eq) were added sequentially to a reaction flask and dissolved in 2 ml of ultra-dry DCM. EDC (93.2 mg, 0.60 mmol, 1.0 eq) was then added dropwise. The reaction mixture was stirred at reflux at 45°C under nitrogen for 4 h, and the reaction endpoint was determined by TLC. After the reaction was substantially completed, the mixture was washed sequentially with 5% citric acid, saturated sodium bicarbonate, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and distilled under reduced pressure to dryness. The mixture was separated and purified by silica gel column chromatography (dichloromethane 100%) to obtain product 12 (NHS-activated form of docosahexaenoic acid) as a slightly yellow oily product with a yield of 230.6 mg and a yield of 90.3%.

[0166] 1 H NMR (400MHz, CDCl3) δ5.52-5.28(m,12H),2.88-2.80(m,14H),2.69-2.67(t,J=4.0Hz,2H) ,2.54-2.48(q,J=12.0Hz,2H), 2.10-2.04(q,J=12.0Hz,2H), 0.98-0.96(t,J=4.0Hz,3H).

[0167] Step 2:

[0168]

[0169] Synthesis of a double-chain docosahexaenoic acid intermediate from lysine: Product 12 (197.6 mg, 0.42 mmol, 2.1 eq), L-lysine methyl ester dihydrochloride (46.6 mg, 0.20 mmol, 1.0 eq), and N,N-diisopropylethylamine (DIEA, 54.3 mg, 0.42 mmol, 2.1 eq) were added sequentially to a reaction flask and dissolved in 2 ml of N,N-dimethylformamide (DMF, ultra-dry reagent). The mixture was stirred under reflux at 45°C under nitrogen for 6 h. The reaction endpoint was determined by TLC. After the reaction was substantially completed, the mixture was washed sequentially with 5% citric acid, saturated sodium bicarbonate, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product 13 was separated and purified by silica gel column chromatography (DCM:MeOH = 50:1) to obtain product 13 as a slightly yellow semi-solid product with a yield of 144.5 mg and a yield of 90.5%.

[0170] 1H NMR (400MHz, CDCl3) δ6.16-6.14(d,J=4.0Hz,1H),5.63-5.61(t,J=4.0Hz,1H),5.45-5.31(m,2 4H),4.60-4.56(q,J=8.0Hz,1H),3.74(s,3H),3.25-3.21(q,J=8.0Hz,2H),2.88-2.80(t,J=16H z,20H),2.44-2.38(q,J=12.0Hz,4H),2.31-2.27(t,J=8.0Hz,2H),2.24-2.20(t,J=8.0Hz,2H), 2.10-2.04(m,4H),1.89-1.69(m,2H),1.55-1.48(m,2H),1.37-1.30(m,2H),1.00-0.96(m,6H).

[0171] Step 3:

[0172]

[0173] Synthesis of double-chain docosahexaenoic acid: Prepare a 1M sodium hydroxide solution and mix it with methanol in a 1:1 volume ratio to obtain a mixed solvent. Add product 13 (0.15 mmol) and 3 ml of the mixed solvent to a reaction flask. Stir the reaction at room temperature under nitrogen for 2 h. The reaction endpoint is determined by TLC. After hydrolysis is substantially complete, the reaction solution is neutralized to a weakly acidic state using 1M hydrochloric acid. DCM is added and washed with saturated saline. The organic layer is dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. Purification is performed by silica gel column chromatography (DCM:MeOH = 20:1) to obtain product 14 as a slightly yellow semi-solid product in a yield of 108.4 mg (94.2%).

[0174] 1 H NMR (400MHz, CDCl3) δ6.64-6.62 (d, J=4.0Hz, 1H), 5.79-5.77 (t, J=4.0Hz, 1H), 5.43-5.28 (m,24H),4.54-4.50(q,J=8.0Hz,1H),3.36-3.24(q,J=24.0Hz,2H),2.88-2.80(m,20H),2. 45-2.37(q,J=16.0Hz,4H),2.34-2.30(t,J=8.0Hz,2H),2.26-2.22(t,J=8.0Hz,2H),2.10 -2.04(m,4H),1.89-1.79(m,2H),1.56-1.50(m,2H),1.40-1.34(m,2H),0.99-0.95(m,6H).

[0175] Step 4:

[0176]

[0177] Subsequently, resiquimod (R848, 30 mg, 0.095 mmol, 1.0 eq), product 14 (80.1 mg, 0.105 mmol, 1.1 eq), and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) were added to the reaction flask in sequence and dissolved in 2 ml of ultra-dry DCM. EDC (16.3 mg, 0.105 mmol, 1.1 eq) was then added dropwise. The reaction solution was stirred at reflux at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the mixture was washed with 5% citric acid, saturated sodium bicarbonate, and saturated salt water three times each. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The product was separated and purified by silica gel column chromatography (DCM:MeOH=30:1) to obtain product 15 (R848-diDHA) as a slightly yellow semi-solid product with a yield of 71.2 mg and a yield of 70.5%.

[0178] 1 H NMR (400MHz, DMSO) δ8.55-8.53(d,J=4.0Hz,1H),8.29-8.27(d,J=4.0Hz,1H),7.94-7.92(d,J=4.0Hz,1H),7.89-7.87( t,J=4.0Hz,1H),7.64-7.60(t,J=8.0Hz,1H),7.56-7.52(t,J=8.0Hz,1H),5.36-5.24(m,24H),4.98(s,2H),4.78(s,2H) ,3.56-3.52(q,J=8.0Hz,2H),3.03(s,2H),2.86-2.74(m,20H),2.69(s,1H),2.33-2.21(m,6H),2.10-2.06(t,J=8.0Hz, 2H), 2.04-2.00(q,J=8.0Hz,4H),1.87-1.77(m,2H),1.48-1.39(m,4H),1.24-1.12(m,9H),0.92-0.88(t,J=8.0Hz,6H).

[0179] Example 7 Synthesis of polyethylene glycol-oleic acid coupled carrier (PEG-OA).

[0180]

[0181] Oleic acid (OA, 30 mg, 0.105 mmol, 1.1 eq), polyethylene glycol (PEG 2K -NH2, 190 mg, 0.095 mmol, 1.0 eq) was dissolved in 2 ml of ultra-dry DCM, and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) and EDC (16.3 mg, 0.105 mmol, 1.1 eq) were quickly added. The reaction solution was refluxed and stirred at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the reaction solution was cooled and spin-dried, and precipitated with glacial ether three times to remove small molecule raw materials. After collecting the precipitate, it was separated and purified by silica gel column chromatography (DCM: MeOH = 15: 1) to obtain product 16 (PEG-OA) as a white solid product with a yield of 135.5 mg and a yield of 62.5%.

[0182] 1 H NMR(400MHz, CDCl3)) δ6.20-6.18(t,J=4.0Hz,1H),5.38-5.31(m,2H),3.68-3.62(m,174H),3.56-3.54(t,J=4.0Hz,2H),3.47-3.44(q,J=6 .0Hz,2H),3.38(s,3H),2.19-2.15(t,J=8.0Hz,2H),2.01-1.98(m,4H),1.65-1.61(m,2H),1.30-1.26(m,20H),0.90-0.86(t,J=8.0Hz,3H).

[0183] Example 8 Synthesis of polyethylene glycol-linoleic acid coupled carrier (PEG-LA).

[0184]

[0185] Linoleic acid (LA, 29.4 mg, 0.105 mmol, 1.1 eq), polyethylene glycol (PEG 2K-NH2, 190 mg, 0.095 mmol, 1.0 eq) was dissolved in 2 ml of ultra-dry DCM, and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) and EDC (16.3 mg, 0.105 mmol, 1.1 eq) were quickly added. The reaction solution was refluxed and stirred at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the reaction solution was cooled and dried, and precipitated with glacial ether three times to remove small molecule raw materials. The precipitate was collected and purified by silica gel column chromatography (DCM: MeOH = 15: 1) to obtain product 17 (PEG-LA) as a slightly yellow solid product with a yield of 130.2 mg and a yield of 60.1%.

[0186] 1 H NMR (400MHz, CDCl3) δ6.30-6.26(m,1H),5.44-5.28(m,4H),3.68-3.62(m, 162H),3.56-3.54(t,J=4.0Hz,2H),3.47-3.45(t,J=4.0Hz,2H),3.38(s,3 H),2.78-2.75(t,J=6.0Hz,2H),2.19-2.15(t,J=8.0Hz,2H),2.07-2.02(m ,4H),1.64-1.61(m,2H),1.34-1.27(m,14H),0.91-0.87(t,J=8.0Hz,3H).

[0187] Example 9 Synthesis of polyethylene glycol-docosahexaenoic acid coupled carrier (PEG-DHA).

[0188]

[0189] Docosahexaenoic acid (DHA, 34.5 mg, 0.105 mmol, 1.1 eq), polyethylene glycol (PEG 2K -NH2, 190 mg, 0.095 mmol, 1.0 eq) was dissolved in 2 ml of ultra-dry DCM, and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) and EDC (16.3 mg, 0.105 mmol, 1.1 eq) were quickly added. The reaction solution was refluxed and stirred at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the reaction solution was cooled and dried, and precipitated with glacial ether three times to remove small molecule raw materials. The precipitate was collected and purified by silica gel column chromatography (DCM: MeOH = 15: 1) to obtain product 18 (PEG-DHA) as a slightly yellow solid product with a yield of 139.6 mg and a yield of 63.1%.

[0190] 1 H NMR (400MHz, CDCl3) δ5.44-5.33(m,12H),3.67-3.63(m,156H),3.56-3.54(t,J=4.0Hz,2H),3.47-3.43(q,J=8.0Hz,2H),3.38(s, 3H), 2.85-2.79 (m, 10H), 2.43-2.39 (q, J = 8.0Hz, 2H), 2.25-2.21 (t, J = 8.0Hz, 2H), 2.11-2.07 (m, 2H), 0.99-0.95 (t, J = 8.0Hz, 3H).

[0191] Example 10 Synthesis of polyethylene glycol-double-chain oleic acid coupled carrier (PEG-diOA).

[0192]

[0193] Product 6 (70.8 mg, 0.105 mmol, 1.1 eq), polyethylene glycol (PEG 2K -NH2, 190 mg, 0.095 mmol, 1.0 eq) was dissolved in 2 ml of ultra-dry DCM, and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) and EDC (16.3 mg, 0.105 mmol, 1.1 eq) were quickly added. The reaction solution was refluxed and stirred at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the reaction solution was cooled and dried, and precipitated with glacial ether three times to remove small molecule raw materials. After collecting the precipitate, it was separated and purified by silica gel column chromatography (DCM: MeOH = 15: 1) to obtain product 19 (PEG-diOA) as a white solid product with a yield of 150.2 mg and a yield of 59.4%.

[0194] 1H NMR (400MHz, CDCl3) δ6.84-6.82(t,J=4.0Hz,1H),6.40-6.38(d,J=4.0Hz,1H),5.86-5.83(t,J=6.0Hz,1H),5.4 0-5.29(m,4H),4.41-4.36(q,J=12Hz,1H),3.66-3.63(m,183H),3.57-3.54(d,J=6.0Hz,2H),3.47-3.43(q,J=8 Hz,2H),3.38(s,3H),3.27-3.19(q,J=16Hz,2H),2.23-2.19(t,J=8.0Hz,2H),2.17-2.13(t,J=8.0Hz,2H),2.01 -1.99(m,8H),1.86-1.66(m,2H),1.65-1.58(m,4H),1.56-1.58(m,2H),1.36-1.27(m,42H),0.90-0.86(m,6H).

[0195] Example 11 Synthesis of polyethylene glycol-double-chain linoleic acid coupled carrier (PEG-diLA).

[0196]

[0197] The product 10 (70.4 mg, 0.105 mmol, 1.1 eq), polyethylene glycol (PEG 2K -NH2, 190 mg, 0.095 mmol, 1.0 eq) was dissolved in 2 ml of ultra-dry DCM, and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) and EDC (16.3 mg, 0.105 mmol, 1.1 eq) were quickly added. The reaction solution was refluxed and stirred at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the reaction solution was cooled and dried, and precipitated with glacial ether three times to remove small molecule raw materials. The precipitate was collected and purified by silica gel column chromatography (DCM: MeOH = 15: 1) to obtain product 20 (PEG-diLA) as a slightly yellow solid product with a yield of 159.3 mg and a yield of 63.1%.

[0198] 1H NMR (400MHz, CDCl3) δ6.84-6.82(t,J=4.0Hz,1H),6.42-6.40(d,J=4.0Hz,1H),5.86-5.83(t,J=6.0Hz,1H ),5.43-5.28(m,8H),4.40-4.38(q,J=4.0Hz,1H),3.66-3.64(m,175H),3.56-3.54(t,J=4.0Hz,2H),3.47 -3.43(q,J=8.0Hz,2H),3.38(s,3H),3.25-3.22(q,J=6.0Hz,2H),2.78-7.75(t,J=6.0Hz,4H),2.23-2.19(t,J=8.0Hz,2H),2.17-2.13 (t,J=8.0Hz,2H),2.07-2.02(m,8H),1.98-1.89(m,4H),1.85-1.66(m,2H),1.55-1.50(m,2H),1.37-1.25(m,33H),0.91-0.87(m,6H).

[0199] Example 12 Synthesis of polyethylene glycol-double-chain docosahexaenoic acid coupled carrier (PEG-diDHA).

[0200]

[0201] The product 14 (80.1 mg, 0.105 mmol, 1.1 eq), polyethylene glycol (PEG 2K -NH2, 190 mg, 0.095 mmol, 1.0 eq) was dissolved in 2 ml of ultra-dry DCM, and DMAP (12.8 mg, 0.105 mmol, 1.1 eq) and EDC (16.3 mg, 0.105 mmol, 1.1 eq) were quickly added. The reaction solution was refluxed and stirred at 45 ° C under nitrogen for 24 h, and the reaction endpoint was determined by TLC. After the reaction was completed, the reaction solution was cooled and dried, and precipitated with glacial ether three times to remove small molecule raw materials. After collecting the precipitate, it was separated and purified by silica gel column chromatography (DCM: MeOH = 15: 1) to obtain product 21 (PEG-diDHA) as a slightly yellow solid product with a yield of 150.1 mg and a yield of 57.1%.

[0202] 1H NMR (400MHz, CDCl3) δ6.96-6.94(t,J=4.0Hz,1H),6.47-6.45(d,J=4.0Hz,1H),5.92-5.89(t,J=6.0Hz,1H ),5.45-5.27(m,24H),4.40-4.38(q,J=4.0Hz,1H),3.66-3.62(m,194H),3.56-3.54(t,J=4.0Hz,2H),3.47 -3.43(q,J=8.0Hz,2H),3.38(s,3H),3.24-3.20(q,J=8.0Hz,2H),2.89-2.78(m,20H),2.43-2.38(m,4H),2.29-2.26(t,J=6.0Hz,2H), 2.23-2.19(t,J=8.0Hz,2H),2.11-2.04(m,4H),1.79-1.69(m,2H),1.57-1.47(m,2H),1.38-1.31(m,2H),0.99-0.95(t,J=8.0Hz,6H).

[0203] Example 13 Preparation of Nanodiscs Containing Resiquimod Prodrug Compounds

[0204] Nanodiscs containing resiquimod prodrug compounds were prepared according to a molar ratio of 65:5:2 of resiquimod prodrug compound: polyethylene glycol fatty acid coupling carrier: styrene-maleic anhydride copolymer (SMA, a new polymer derived from styrene and maleic anhydride, brand: Merck, CAS: 26762-29-8, product number: L9170, molecular weight: 9500 Da). A resiquimod prodrug compound (prepared in Examples 1-6: R848-OA 7.4 mg, R848-LA 7.3 mg, R848-DHA 7.9 mg, R848-diOA 12.3 mg, R848-diLA 12.3 mg, or R848-diDHA 13.5 mg) and a corresponding polyethylene glycol fatty acid coupled carrier (prepared in Examples 7-12: PEG-OA 2.3 mg, PEG-LA 2.3 mg, PEG-DHA 2.3 mg, PEG-diOA 2.6 mg, PEG-diLA 2.6 mg, or PEG-diDHA 2.7 mg) were dissolved in 1 mL of dimethyl sulfoxide (DMSO). Subsequently, 3.72 mg of SMA was dissolved in 9 mL of deionized water. The DMSO containing the prodrug and carrier was slowly and uniformly injected into the deionized water containing SMA under ultrasonic oscillation (frequency 200 kHz). The residual dimethyl sulfoxide was removed by dialysis (molecular weight cut-off 3500) to obtain uniformly dispersed nanodiscs (named after the resiquimod prodrug compound, for example, the nanodiscs prepared from R848-diDHA were named R848-diDHA nanodiscs).

[0205] The results are as follows Figure 1 As shown, all resiquimod prodrug compounds self-assembled into nanodiscs in water, with a single-peak particle size distribution, indicating a uniform size distribution of the nanodiscs. Transmission electron microscopy visually demonstrated a regular morphology, uniform particle size, and high dispersibility.

[0206] The solubility of the original drug of Resiquimod in water is about 0.1 mg / ml. The nanodiscs formed by the self-assembly of the prodrug compound of Resiquimod in water can significantly improve the solubility in water (more than 20 times compared with the original drug).

[0207] Example 14 Drug Tissue Distribution Analysis of Nanodiscs Containing Resiquimod Prodrug Compounds

[0208] In order to compare the tissue distribution of nanodiscs containing resiquimod prodrug compounds and resiquimod original drug in vivo, we determined the actual drug content in major organs of the body after administration of different drugs by high performance liquid chromatography (HPLC). First, we established a 4T1 mouse orthotopic breast cancer model. When the tumor volume grew to 100mm 3Mice were divided equally into two groups of four based on tumor volume. R848 stock drug and R848-diDHA nanodiscs (resiquimod equivalent dose of 0.75 mg / kg) were injected subcutaneously into the tail pad. Twenty-four hours after subcutaneous injection, mice were euthanized and lymph nodes, tumors, heart, liver, spleen, lungs, and kidneys were collected. Tissues were ground into homogenates and processed for testing: 200 μL of tissue homogenate was aspirated, 200 μL of acetonitrile was added to precipitate protein, and the cells were centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and analyzed for drug content using high-performance liquid chromatography (HPLC).

[0209] The HPLC methodology was as follows: the chromatographic analysis column was a C18 ODS reverse phase column (YMC, 5.0 μm, 250 mm × 4.6 mm), the column temperature was 10°C, the injection volume was 100 μL, the UV detection wavelength was 256 nm, and the mobile phase was 10-100% acetonitrile / water with a gradient elution of 30 min, followed by elution with 100% acetonitrile for 5 min at a flow rate of 1.0 mL / min.

[0210] The results are as follows Figure 2 As shown, compared to the original resiquimod, nanodiscs containing the resiquimod prodrug compound can accumulate efficiently and specifically in tumors after subcutaneous injection, with less accumulation in major organs, thereby improving targeting while reducing adverse reactions. R848-OA nanodiscs, R848-LA nanodiscs, R848-DHA nanodiscs, R848-diOA nanodiscs, and R848-diLA nanodiscs were tested using the same method. Compared to the original R848, after subcutaneous injection, tumor accumulation increased by 1.01-fold for R848-OA nanodiscs, 0.97-fold for R848-LA nanodiscs, 1.12-fold for R848-DHA nanodiscs, 1.65-fold for R848-diOA nanodiscs, and 1.71-fold for R848-diLA nanodiscs.

[0211] Example 15 In vivo antitumor activity analysis of nanodiscs containing resiquimod prodrug compounds

[0212] To compare the in vivo antitumor effects of nanodiscs containing resiquimod prodrug compounds with resiquimod original drug and further explore their potential in combination with commonly used immune checkpoint inhibitors in clinical practice, we established a 4T1 mouse orthotopic breast cancer model to evaluate their ability to inhibit tumor growth and inhibit tumor recurrence and metastasis.

[0213] We established a 4T1 mouse orthotopic breast cancer model and waited for the tumor to grow to 50 mm. 3The mice were divided into five groups based on tumor volume, with 10 mice in each group. These groups included normal saline, R848 stock drug, PD-1 inhibitor (InVivoMAb anti-mouse PD-1 (CD279) BioXcell Catalog No. BE0273), R848-diDHA nanodiscs, and R848-diDHA nanodiscs combined with PD-1 inhibitor. The drugs were injected intraperitoneally on days 0, 3, and 6 (the PD-1 inhibitor was injected intraperitoneally, and the remaining drugs were injected subcutaneously via the tail pad). The doses were 4.0 mg / kg resiquimod equivalent and 100 μg PD-1 inhibitor. Tumor volume and body weight were continuously monitored for each mouse, and tumor volume was calculated using the following formula: Volume = (longest diameter of tumor × short axis of tumor × short axis of tumor) / 2.

[0214] The results are as follows Figure 3 As shown in AB, the nanodisc group containing resiquimod prodrug compounds and the nanodisc group combined with PD-1 inhibitors both showed excellent ability to inhibit tumor growth. Figure 3 A shows the tumor inhibition effect of each group. The tumor in the normal saline group grew rapidly. The R848 original drug group and the PD-1 inhibitor alone group produced moderate therapeutic effects. This may be because the R848 original drug is easily metabolized rapidly in the body and is difficult to effectively accumulate in the target site. The R848-diDHA nanodisc group showed a strong ability to inhibit tumor growth, which is due to its ability to efficiently accumulate in the tumor site and exert a more potent anti-tumor activity. In addition, the body weight of the mice remained stable during the treatment ( Figure 3 B), which indicates that the drug is well tolerated and has a good safety profile.

[0215] The same method was used to test R848-OA nanodiscs, R848-LA nanodiscs, R848-DHA nanodiscs, R848-diOA nanodiscs, and R848-diLA nanodiscs. Compared with the original R848 drug, the tumor inhibition effect of the R848-OA nanodisc group increased by 4.05 times, the R848-LA nanodisc group increased by 3.76 times, the R848-DHA nanodisc group increased by 4.63 times, the R848-diOA nanodisc group increased by 6.18 times, and the R848-diLA nanodisc group increased by 5.71 times.

[0216] Triple-negative breast cancer has a poor prognosis and low survival rate, which is mainly related to the easy recurrence and metastasis of tumors after surgery. In order to explore the ability of nanodiscs containing resiquimod prodrug compounds and their combined immune checkpoint inhibitors to inhibit tumor recurrence and metastasis, the experiment was evaluated by simulating a clinical postoperative model. Specifically, when the average tumor volume of the saline group grew to 600mm 3 At 18 days, the tumors in each group were surgically removed, and the tumor recurrence was observed and the survival status of the mice was recorded. Figure 3As shown in Figure C, both the R848 original drug group and the PD-1 inhibitor group alone failed to achieve a good therapeutic effect, with no mice surviving by day 80. The R848-diDHA nanodisc group demonstrated even better results, with two of the five mice still achieving long-term survival by the end of observation (120 days). The R848-diDHA nanodisc combined with the PD-1 inhibitor group achieved the best survival benefit, with all mice surviving until the end of observation (120 days).

[0217] To observe tumor metastasis, three representative mice in each group were sacrificed on day 40 of the experiment, and tumor-draining lymph nodes and lungs were collected for photography and histopathological analysis. Figure 3 As shown in Figures DE, significant tumor-draining lymph nodes and lung metastasis occurred in the saline group, the R848 original drug group, and the PD-1 inhibitor alone group. However, the R848-diDHA nanodisc group effectively inhibited tumor recurrence and lung metastasis. Furthermore, the tumor-draining lymph nodes in the R848-diDHA nanodisc plus PD-1 inhibitor group were similar in size to those in healthy mice. Therefore, these experimental results demonstrate that nanodiscs containing resiquimod prodrug compounds, as well as the combination of nanodiscs and immune checkpoint inhibitors, can effectively inhibit tumor recurrence and lung metastasis, with a good safety profile.

[0218] Example 16 In vivo immune activation analysis of nanodiscs containing resiquimod prodrug compounds

[0219] The activation level of immune cells can specifically reflect the body's anti-tumor immune response. In order to compare the in vivo immune activation effects of nanodiscs containing resiquimod prodrug compounds and resiquimod original drugs, we established a 4T1 mouse orthotopic breast cancer model and assessed the anti-tumor immune level by detecting the activation of target immune cells in the peripheral blood and tumors of treated mice. Specifically, a 4T1 orthotopic breast cancer model was constructed and the tumor was grown to 200mm. 3 Mice were divided equally into a saline group, an R848 stock drug group, and an R848-diDHA nanodisc group, with 5 mice in each group. The drugs (4.0 mg / kg R848 equivalent) were injected subcutaneously via the tail pad on days 0, 3, and 6. Peripheral blood and tumor tissue were collected on day 7 and prepared into single-cell suspensions for subsequent staining and measurement.

[0220] ① Peripheral blood: Take out peripheral blood and add an equal volume of PBS, and add twice the volume of Ficoll cell separation solution to the centrifuge tube. Tilt the peripheral blood PBS mixture and gently drip it into the Ficoll cell separation solution. Centrifuge settings: acceleration 5, deceleration 2, 800g centrifugation for 20 minutes. After the centrifugation, aspirate the middle white cell layer, transfer it to a new centrifuge tube, and fill it with sterile PBS. Centrifuge it at acceleration 9, deceleration 9, and 600g centrifugation for 10 minutes. Discard the supernatant, add red blood cell lysis solution and lyse on ice for 5 minutes. Stop the lysis with 5 times the volume of PBS. After centrifugation at 600g for 10 minutes, the white precipitate is the peripheral blood mononuclear cell (PBMC), and then use Zombie Aqua TM -BV510 was used to stain dead cells at room temperature in the dark for 15 minutes. After washing with PBS twice, anti-mouse CD16 / 32 antibody was used to block on ice for 10 minutes, and then pre-configured fluorescent antibodies were added: APC anti-mouse CD45, FITC anti-mouse CD3, PE anti-mouse CD4, BUV496 anti-mouse CD8 and PE-CY7 anti-mouse CD25. The final volume was controlled to be 100 μL. After incubation on ice in the dark for 20 minutes, PBS was washed twice and intracellular staining was performed. BD Pharmingen TM Transcription Factor Buffer Set, configured for 1× working solution. Add 500 μL of Fix / Perm to the cell pellet after cell surface marker labeling. Incubate at 4°C in the dark for 30 minutes. Then, add two volumes of Perm / Wash buffer and centrifuge at 600g for 5 minutes, washing twice. Add 100 μL of PE-CY5 anti-mouse FOXP3 antibody prepared in Perm / Wash buffer and incubate at 4°C in the dark for 30 minutes. Finally, wash twice with Perm / Wash buffer before analysis.

[0221] ② Tumor: Prepare RPMI culture medium containing collagenase IV (1 mg / mL) and DNase I (0.01 U / μL), mince the removed tumor tissue in the culture medium, and digest it at 37°C for 30 minutes. Under the premise of avoiding cell damage, carefully and thoroughly grind the tissue with PBS buffer and filter it with a 70μm cell strainer to obtain a single cell suspension. Centrifuge at 650g for 5 minutes to collect the cells and use Zombie Aqua TMDead cell staining was performed with BV510 for 15 minutes at room temperature in the dark. After washing twice with PBS, the cells were blocked with anti-mouse CD16 / 32 antibodies for 10 minutes on ice. Pre-assembled fluorescent antibodies were then added: APC anti-mouse CD45, FITC anti-mouse CD3, PE anti-mouse CD4, BUV496 anti-mouse CD8, and PE-CF594 anti-mouse NK1.1. The final volume was adjusted to 100 μL. After incubation on ice in the dark for 20 minutes, the cells were washed twice with PBS and analyzed by flow cytometry.

[0222] The results are as follows Figure 4 As shown in A, in peripheral blood, compared with the saline group and R848 original drug group, the immunosuppressive regulatory T cells (Tregs, Foxp3 + CD25 + ) levels were low (about 2.2%), CTLs (CD8 + ) levels were high (about 34.7%), CD8 + / CD4 + The ratio was elevated (about 1.0%), which confirmed systemic immune activation.

[0223] The immunosuppressive tumor microenvironment (TME) of “cold tumors” hinders the efficacy of immunotherapy, so we further investigated the ability of R848-diDHA nanodiscs to reshape the TME. Figure 4 As shown in B, treatment with R848-diDHA nanodiscs increased the expression of tumor-infiltrating CTLs (CD8a + ) increased significantly from 28.9% (normal saline group) to 51.5%. Compared with the other groups, after treatment with R848-diDHA nanodiscs, CD8a + / CD4 + The proportion of tumor-infiltrating natural killer (NK1.1) cells also increased significantly. Furthermore, a significant difference was observed in the number of natural killer (NK1.1) cells infiltrating the tumor, demonstrating that R848-diDHA nanodiscs can strongly activate the body's innate immunity. Similar results were obtained when R848-OA nanodiscs, R848-LA nanodiscs, R848-DHA nanodiscs, R848-diOA nanodiscs, and R848-diLA nanodiscs were tested using the same method, indicating that nanodiscs containing resiquimod prodrug compounds can effectively enhance the anti-tumor immune activation ability of resiquimod.

[0224] The above data indicate that nanodiscs containing resiquimod prodrug compounds have good in vivo immune activation effects, can effectively activate innate immunity and adaptive immunity, and promote the body's anti-tumor immune effect.

[0225] Example 17 Synthesis of double-chain docosahexaenoic acid maleimidized (diDHA-Mal)

[0226]

[0227] Product 14 (80.1 mg, 0.105 mmol, 1.0 eq), DIEA (46.5 mg, 0.315 mmol, 3.0 eq), and 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU, 45.7 mg, 0.105 mmol, 1.0 eq) were added to a reaction flask in sequence, dissolved in 2 ml of ultra-dry DMF, and stirred at room temperature for 10 min. Subsequently, N-(2-aminoethyl)maleimide (17.4 mg, 0.116 mmol, 1.1 eq) was added and allowed to react for 12 h. The reaction endpoint was determined by TLC. After completion of the reaction, the product was extracted and purified by silica gel column chromatography (DCM:MeOH = 30:1) to obtain product 25 (diDHA-Mal) as a pale yellow solid in a yield of 55.1 mg (59.2%).

[0228] 1 H NMR (400MHz, CDCl3) δ6.72(s,2H),6.67(t,J=6.0Hz,1H),6.25(d,J=8.0Hz,1H),5.76(t,J=6.0Hz,1H ),5.43-5.34(m,25H),4.30(td,J=8.0,6.0Hz,1H),3.68(t,J=5.0Hz,2H),3.53-3.39(m,2H),3.24(p, J=7.0Hz,2H),2.86-2.80(m,21H),2.40(dd,J=7.0,4.0Hz,4H),2.34-2.26(m,2H),2.21(d,J=8.0Hz, 2H), 2.08 (q, J = 7.0Hz, 4H), 1.65-1.56 (m, 2H), 1.53-1.48 (m, 2H), 1.33 (s, 2H), 0.97 (t, J = 8.0Hz, 6H).

[0229] Example 18 Preparation of Double-chain Docosahexaenoic Acid-Antigen Peptide (OVA-diDHA)

[0230] To modify the antigen on the nanodisc surface, a thiol-modified OVA antigen (GenScript Biotech, Product No. RP20398) was conjugated to diDHA containing a maleimide group, resulting in polyunsaturated fatty acidization of the antigen. Specifically, equimolar amounts of thiol-modified OVA and diDHA-Mal were weighed and dissolved in DMSO. The mixture was shaken at room temperature for 3 hours, and the reaction progress was monitored by liquid chromatography.

[0231] The HPLC methodology was as follows: the chromatographic analysis column was a C18 ODS reverse phase column (YMC, 5.0 μm, 250 mm × 4.6 mm), the column temperature was 10°C, the injection volume was 100 μL, the UV detection wavelength was 220 nm, and the mobile phase was 5-65% acetonitrile / water with a gradient elution of 25 min, followed by elution with 95% acetonitrile for 2 min at a flow rate of 1.0 mL / min.

[0232] The results are as follows Figure 5 As shown in FIG, after 12 h of shaking reaction at room temperature, the free OVA polypeptide peak detected by HPLC disappeared, indicating that the reaction was complete.

[0233] Example 19 Construction of Nanovaccines Containing Resiquimod Prodrug Compounds

[0234] 13.5 mg of R848-diDHA (Product 15, containing 4 mg of R848), 2.7 mg of PEG-diDHA (Product 21), and 0.5 mg of OVA-diDHA were weighed and dissolved in 1 mL of dimethyl sulfoxide (DMSO). 3.72 mg of SMA (Mw = 9500) was dissolved in 9 mL of deionized water. Under ultrasonic oscillation (200 kHz), the DMSO containing the prodrug compound, carrier, and modified OVA antigen was slowly and uniformly injected into the deionized water containing SMA to produce the R848-diDHA nanovaccine.

[0235] like Figure 6 As shown, the R848-diDHA nanovaccine exhibited a monodisperse, spherical-like morphology with an ultrasmall particle size (~20.1±1.4 nm). The same method was used to test the R848-OA nanovaccine, R848-LA nanovaccine, R848-DHA nanovaccine, R848-diOA nanovaccine, and R848-diLA nanovaccine, all of which could be prepared to form monodisperse, spherical-like morphologies with ultrasmall particle sizes (20-50 nm).

[0236] Example 20 Evaluation of the ability of nanovaccines containing resiquimod prodrug compounds to prevent tumor growth

[0237] Healthy C57BL / 6 mice of the same age were divided into two groups, 16 in each. On days 0, 7, and 14, they were subcutaneously injected with saline or the R848-diDHA nanovaccine (R848 equivalent dose: 4 mg / kg, OVA equivalent dose: 0.5 mg / kg) to establish immune recognition of tumor-associated antigens. On day 30, the saline group and the R848-diDHA nanovaccine group were each divided into two groups. One group of mice was inoculated with OVA-expressing B16F10 melanoma tumors (referred to as B16OVA, inoculated subcutaneously on the left flank) and B16F10 melanoma tumors (referred to as B16, inoculated subcutaneously on the right flank), while the other group of mice was inoculated with MC38 tumors (inoculated subcutaneously on the left flank) and LLC tumors (inoculated subcutaneously on the right flank). Tumor volume was continuously monitored.

[0238] The results are as follows Figure 7 As shown in the results, R848-diDHA nanovaccine can significantly inhibit the growth and progression of various tumors, showing its great potential in preventing and treating various tumors ( Figure 7 A). Notably, in the same mice, the R848-diDHA nanovaccine demonstrated a 62.5% (5 / 8) anti-tumor efficacy against B16F10 melanoma cells harboring OVA (B16OVA), effectively preventing tumor development. However, it only slowed tumor growth in B16F10 melanoma cells, a similar inhibitory effect observed for MC38 and LLC tumors, which also lack OVA antigens. This result demonstrates that the inclusion of specific antigens is crucial for constructing preventive nanovaccines, as they help generate memory T cells, enabling a rapid and robust response to challenge with the same antigen.

[0239] Memory T lymphocyte detection: This experiment evaluates the long-term anti-tumor immunity level by detecting the activation of related T lymphocytes in the peripheral blood of mice after treatment. Specifically, PBMCs were extracted from peripheral blood, and the cell pellet was obtained and then Zombie Aqua TM Dead cells were stained with BV510 at room temperature in the dark for 15 minutes. After washing twice with PBS, the cells were blocked with anti-mouse CD16 / 32 antibodies on ice for 10 minutes. Pre-configured fluorescent antibodies were then added: APC anti-mouse CD45, FITC anti-mouse CD3, PE anti-mouse CD4, BUV496 anti-mouse CD8, PE-CY7 anti-mouse CD44, and BV421 anti-mouse CD62L. The final volume was controlled to 100 μL. After incubation on ice in the dark for 20 minutes, the cells were washed twice with PBS and analyzed by flow cytometry.

[0240] After vaccination stimulation, unsensitized T cells differentiated into effector memory T cells (TEM), which migrated to peripheral immune organs (such as the spleen) and mediated anti-tumor cytotoxic T lymphocytes to exert immune effects. To study whether vaccination with R848-diDHA nanovaccine can stimulate immune memory in mice against OVA-expressing tumors, mice were vaccinated with R848-diDHA nanovaccine once every 7 days for a total of 3 times, and B16F10 tumor cells or B16F10-OVA tumor cells were subcutaneously inoculated in the right flank of the mice on day 30. On day 31, the mice were euthanized, and peripheral blood was obtained and TEM was analyzed by flow cytometry. Encouragingly, CD4 + T cells and CD8 + The TEM ratio of T cells increased significantly, verifying the immune stimulation effect ( Figure 7 B) Similar results were obtained when the R848-OA, R848-LA, R848-DHA, R848-diOA, and R848-diLA nanovaccines were tested using the same method, indicating that the nanovaccines containing the resiquimod prodrug compound have excellent ability to prevent tumorigenesis.

[0241] Example 21 Evaluation of the ability of nanovaccines containing resiquimod fatty acid-conjugated prodrugs to control tumor growth and activate immunity

[0242] The B16F10 melanoma tumor carrying OVA (B16F10-OVA) was inoculated subcutaneously on the right flank of mice to establish an orthotopic melanoma model. 3 Mice were divided equally into a saline group, an R848-diDHA nanodisc group, and an R848-diDHA nanovaccine group, with 9 mice in each group. The drug (equivalent to 4.0 mg / kg R848) was subcutaneously injected via the tail pad on days 0, 3, and 6. Tumor proliferation and mouse survival were then measured, and tumor volume was calculated using the following formula: volume = (longest diameter of tumor × short axis of tumor × short axis of tumor) / 2.

[0243] The results are as follows Figure 8 As shown in AB, compared with the R848-diDHA nanodisc group, the R848-diDHA nanovaccine group had a significantly better tumor inhibition effect on B16F10-OVA and a significantly prolonged median survival time.

[0244] OVA-specific T lymphocyte detection: Construct an orthotopic melanoma model (B16F10-OVA and B16F10) and wait until the tumor grows to 90 mm. 3At the same time, the mice were divided into normal saline group, R848-diDHA nanodisc group, and R848-diDHA nanovaccine group, with 6 mice in each group, and the above drugs (4.0 mg / kg R848 equivalent dose) were injected subcutaneously through the tail pad on days 0, 3, and 6, respectively. The number and function of immune cells in the tumor were then detected by flow cytometry. This experiment evaluated the level of specific anti-tumor immunity by detecting the immune cell activation of related T lymphocytes in the mouse tumor after treatment. The tumor tissue was extracted to make a single cell suspension, and the cells were stimulated with Cell Activation Cocktail (with Brefeldin A) in a 37°C cell culture incubator for 6 hours. Using Zombie Aqua TM -BV510 was used to stain dead cells at room temperature in the dark for 15 minutes. After washing with PBS twice, anti-mouse CD16 / 32 antibody was used to block on ice for 10 minutes, and then pre-prepared fluorescent antibodies were added: APC anti-mouse CD45, FITC anti-mouse CD3, BUV496 anti-mouse CD8 and PE anti-mouse T-Select H-2K b OVA tetramer-SIINFEKL-PE (MBL LifeScience, cat. no. TS-5001-1C) was added to the final volume of 100 μL. The cells were incubated on ice for 20 min in the dark, then washed twice with PBS for subsequent intracellular staining. FluoroFix was used. TM Buffer, corresponding to 1× working solution. Add 500 μL FluoroFix TM Buffer was added and incubated at room temperature in the dark for 1 hour. Then, two volumes of Perm / Wash buffer were added and the cells were centrifuged at 600g for 5 minutes, washing twice. 100 μL of PE-CY7 anti-mouse INF-γ antibody prepared in Perm / Wash buffer was added and incubated at room temperature in the dark for 20 minutes. Finally, the cells were washed twice with Perm / Wash buffer before analysis.

[0245] To investigate whether the immune response elicited by the R848-diDHA nanovaccine was tumor-specific, we analyzed OVA on the day after the third administration. 257-264 (SIINFEKL)-specific tumor-infiltrating CTLs, and a group of saline-treated B16F10 melanoma mice served as negative controls.

[0246] The results are as follows Figure 8 As shown in C, both R848-diDHA nanodisc and R848-diDHA nanovaccine groups significantly increased the number of tumor-infiltrating CTLs (CD8 +), indicating that the R848-diDHA nanodisc platform has a significant TME remodeling effect and has the potential to turn "cold" tumors into "hot". Figure 8 As shown in D, R848-diDHA nanovaccine significantly increased H-2K in B16F10-OVA tumors. b OVA tetramer + Tumor-infiltrating CTLs (CD8 + OVA + ) level (about 61.6%), which was significantly higher than that of the R848-diDHA nanodisc group (about 41.0%) and the saline group, while the level of OVA-specific CTLs detected in the B16F10 saline group was negligible. This result strongly proves that the R848-diDHA nanovaccine can effectively activate CTLs, enabling them to recognize and carry specific tags, causing the body's specific immunity to further kill tumor cells. Interferon gamma (IFN-γ) can be secreted by CTLs and can directly or indirectly induce tumor cell apoptosis. Figure 8 As shown in Figures EF, R848-diDHA nanovaccine can significantly increase the proportion of CTLs secreting IFN-γ.

[0247] These results indicate that the R848-diDHA nanovaccine can effectively control tumor growth, induce tumor antigen-specific immune responses, and increase the level of tumor-infiltrating CTLs. Similar results were obtained when the R848-OA nanovaccine, R848-LA nanovaccine, R848-DHA nanovaccine, R848-diOA nanovaccine, and R848-diLA nanovaccine were tested using the same method, indicating that the nanovaccine containing the resiquimod prodrug compound has a better ability to control tumor growth and activate the immune system.

Claims

1. A nanodisc comprising a resiquimod prodrug compound, a polyethylene glycol fatty acid coupled carrier and a styrene-maleic anhydride copolymer, in, The resiquimod prodrug compound is a compound represented by formula (Ia) or formula (Ib), or a pharmaceutically acceptable salt or stereoisomer thereof: The polyethylene glycol fatty acid coupled carrier is a polyethylene glycol fatty acid coupled carrier represented by formula (IIa) or formula (IIb): Where each R is independently C 8-30 Alkenyl -C(=O)-, n is selected from integers of 30 to 60.

2. A nano vaccine comprising a resiquimod prodrug compound, a polyethylene glycol fatty acid coupling carrier, an unsaturated fatty acyl modified antigen and a styrene-maleic anhydride copolymer, in, The resiquimod prodrug compound is a compound represented by formula (Ia) or formula (Ib), or a pharmaceutically acceptable salt or stereoisomer thereof: The polyethylene glycol fatty acid coupled carrier is a polyethylene glycol fatty acid coupled carrier represented by formula (IIa) or formula (IIb): Each R is independently C 8-30 Alkenyl -C(=O)-, n is an integer selected from 30 to 60; The unsaturated fatty acyl group is selected from C 8-30 Alkenyl-C(=O)-.

3. The nanodisc of claim 1 or the nanovaccine of claim 2, wherein: Each R is independently C 10-26 alkenyl-C(=O)-; Alternatively, each R is independently C 14-22 alkenyl-C(=O)-; Alternatively, each R is independently oleoyl, linoleoyl, docosahexanoyl, eicosapentaenoyl, or docosapentaenoyl; Alternatively, each R is independently 4. The nanodisc of claim 1 or the nanovaccine of claim 2, wherein the resiquimod prodrug compound is selected from the group consisting of:

5. The nanodisc according to claim 1 or the nanovaccine according to claim 2, wherein In formula (IIa) or formula (IIb), n is selected from an integer of 35 to 55; or, n is selected from an integer of 40 to 50; or, n is 45; Alternatively, the polyethylene glycol fatty acid coupled carrier is the following polyethylene glycol fatty acid coupled carrier:

6. The nanodisc according to claim 1 or the nanovaccine according to claim 2, wherein The molecular weight of the styrene-maleic anhydride copolymer is 5000-20000 Da; or, the molecular weight of the styrene-maleic anhydride copolymer is 6000-15000 Da; or, the molecular weight of the styrene-maleic anhydride copolymer is 8000-11000 Da; or, the molecular weight of the styrene-maleic anhydride copolymer is 9500 Da.

7. The nanovaccine according to claim 2, wherein The unsaturated fatty acyl group is selected from C 10-26 Alkenyl-C(=O)-; or, the unsaturated fatty acyl group is selected from C 14-22 Alkenyl-C(=O)-; or, the unsaturated fatty acyl group is selected from oleoyl, linoleoyl, docosahexaenoyl, eicosapentaenoyl or docosapentaenoyl; Alternatively, the unsaturated fatty acyl group is selected from 8. The nanovaccine according to claim 2, wherein The antigen is selected from chicken ovalbumin, bovine serum albumin, human serum albumin, thyroglobulin or keyhole limpet hemocyanin.

9. The nanodisk according to claim 1, wherein The molar ratio of the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier and the styrene-maleic anhydride copolymer is (50-200):(1-50):(1-50); or, the molar ratio is (50-150):(1-30):(1-30); or, the molar ratio is (50-100):(1-20):(1-20); or, the molar ratio is (50-80):(1-10):(1-10).

10. The nanovaccine according to claim 2, wherein The molar ratio of the resiquimod prodrug compound, the polyethylene glycol fatty acid coupling carrier, the unsaturated fatty acyl-modified antigen and the styrene-maleic anhydride copolymer is (50-200):(1-50):(1-20):(1-50); or, the molar ratio is (50-200):(1-20):(1-20):(1-20); or, the molar ratio is (50-150):(1-20):(1-10):(1-10); or, the molar ratio is (50-100):(1-20):(1-10):(1-10).

11. A pharmaceutical formulation comprising the nanodisc of claim 1 or the nanovaccine of claim 2 and at least one pharmaceutically acceptable excipient.

12. A combination comprising the nanodisc of claim 1 or the nanovaccine of claim 2 and a PD-1 inhibitor.

13. Use of the nanodisc according to claim 1 or the nanovaccine according to claim 2 in the preparation of a medicament for treating or preventing a disease or condition for which a TLR7 / 8 agonist is indicated.

14. Use of the nanodisc according to claim 1 or the nanovaccine according to claim 2 in combination with a PD-1 inhibitor in the preparation of a medicament for treating or preventing a disease or condition for which a TLR7 / 8 agonist is indicated.

15. Use according to claim 13 or 14, wherein the disease or disorder is selected from inflammatory diseases, infectious diseases, cancer or precancerous syndromes.

16. The use according to claim 15, wherein the inflammatory disease is selected from arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis or rheumatoid arthritis; the infectious disease is selected from retroviral infection, hepatitis virus infection, COVID-19 novel coronavirus infection, Zika virus infection or dengue virus infection; the cancer is selected from breast cancer, melanoma, colon cancer, lung cancer, pancreatic cancer, kidney cancer, head and neck cancer, lymphoma, skin cancer, urothelial carcinoma, gastric cancer or hepatocellular carcinoma; the precancerous syndrome is selected from chronic atrophic gastritis, gastric ulcer, ulcerative colitis, cirrhosis, colorectal adenoma or familial polyposis.

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