A novel fluoroalkylated small molecule adjuvant and its application

Abstract

The application belongs to the field of medicine, and particularly relates to a novel fluoralkyl-modified small molecule adjuvant and application thereof. The small molecule adjuvant is shown as formula I. The novel fluoralkyl-modified small molecule adjuvant of the application can improve humoral immunity and cellular immunity compared with aluminum adjuvant, and shows good effects on therapeutic vaccines of tumors, and has good therapeutic effects on melanoma, and can basically completely clear tumors in mice. The tumor clearance is mainly achieved by cellular immune response, and the problem of unsatisfactory cellular immune effect of existing adjuvant is solved, and the novel fluoralkyl-modified small molecule adjuvant has application potential as a universal adjuvant.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a novel fluoroalkylated modified small molecule adjuvant and its application. Background Technology

[0002] For diseases such as infectious diseases and cancer, vaccines are the most economical and effective means of prevention and treatment, making vaccine development crucial. Antigens and adjuvants, as essential components of vaccines, are vital to their effectiveness. Vaccine adjuvants can enhance or alter the body's immune response to antigens, playing a significant role in vaccine development. Many vaccines have stalled due to poor immunogenicity, failing to elicit sufficient humoral and cellular immune responses. For most vaccines, appropriate adjuvants can better elicit the body's immune response, allowing the vaccine to achieve its optimal protective effect.

[0003] Aluminum adjuvants are the most widely used traditional adjuvants, but they only enhance humoral immune responses. In contrast, other adjuvants like MF59 and AS series offer more balanced immune enhancement (enhancing both antibody and cellular immunity). Therefore, developing novel adjuvants is crucial for advancing vaccine development. Summary of the Invention

[0004] To address the shortcomings of existing methods, the purpose of this invention is to provide a novel fluoroalkylated modified small molecule adjuvant and its application.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] In a first aspect, the present invention protects a novel fluoroalkylated modified small molecule adjuvant, said adjuvant being a compound as described in Formula I:

[0007]

[0008]

[0009] In a specific implementation scheme, the small molecule adjuvant synthesis route is as follows: Figure 1 As shown.

[0010] In a more specific embodiment, the method is as follows: the amino group of resimote (R848) is reacted with the epoxy group of 3-(perfluorohexyl)propane to obtain the product.

[0011] Specifically, retsimot is dissolved in dimethyl sulfoxide, methanol is added, and then 3-(perfluorohexyl)propane oxide is slowly added dropwise. The reaction is carried out at 40±5℃ for 48±8 hours, and the compound of formula I is obtained after purification.

[0012] Secondly, this invention protects the use of the compounds described above as adjuvants in the preparation of vaccines.

[0013] Fourthly, the present invention protects a vaccine containing the compounds described above.

[0014] In preparation, the aforementioned compound is prepared into a solution and then mixed with the antigen.

[0015] Fifthly, the present invention also protects the use of the aforementioned vaccine in the preparation of a drug or reagent for the treatment, prevention or diagnosis of tumors.

[0016] In a specific implementation plan, the tumor is a melanoma.

[0017] Beneficial effects

[0018] (1) The novel fluoroalkylated modified small molecule adjuvant of this application enhances both humoral and cellular immunity compared to aluminum adjuvants. It has shown good efficacy in therapeutic vaccines for tumors and has a good therapeutic effect on melanoma. It can basically completely clear tumors in mice.

[0019] (2) Tumor clearance mainly relies on cellular immune response. Therefore, our research results also provide good evidence for the application of our self-designed novel fluoroalkylated modified small molecule adjuvant in melanoma vaccine. This solves the problem of unsatisfactory cellular immune effect of existing adjuvants. The novel fluoroalkylated modified small molecule adjuvant has the potential to be used as a universal adjuvant.

[0020] (3) The novel fluoroalkylated modified small molecule adjuvant of the present invention can effectively promote the maturation of macrophages and the secretion of cytokines, and has a better antigen presentation effect, thereby effectively improving the level of T cell immune response.

[0021] (4) The novel fluoroalkylated modified small molecule adjuvant of this application can be stored at room temperature and transported in a short time. It is a safe, stable and effective vaccine adjuvant. Attached Figure Description

[0022] Figure 1 This is a synthetic route for novel fluoroalkylated modified small molecule adjuvants.

[0023] Figure 2 The results are from the purity test after HPLC purification.

[0024] Figure 3 The results of LC-MS identification of the novel fluoroalkylated modified small molecule adjuvant of the present invention are shown.

[0025] Figure 4 This represents the change in tumor size over time.

[0026] Figure 5This novel fluoroalkylated small molecule adjuvant can effectively enhance vaccine-induced T-cell immune responses in mice. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0028] Raw material types and sources: Resimod (R848) was purchased from MCE, 3-(perfluorohexyl)propane oxide was purchased from the Exploration Platform, DMSO was purchased from Solarbio, methanol was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., and OVA protein was purchased from Sigma.

[0029] Example 1: Synthesis and Identification of Novel Fluoroalkylated Modified Small Molecule Adjuvants

[0030] Resimod (R848) (10 mg, 0.0318 mmol) was dissolved in DMSO (100 μl), 2 mL of methanol was added, and then 3-(perfluorohexyl)propane oxide (23.92 mg, 0.0636 mmol) was slowly added dropwise. The reaction was carried out at 40 °C for 48 hours. A novel fluoroalkylated small molecule adjuvant was obtained by HPLC purification and identified by LC-MS in positive ion mode. The exact mass of the novel fluoroalkylated small molecule adjuvant was 690.19, and its chemical structure was C0. 26 H 27 F 13 N4O3. LC-MS results are as follows: Figure 3 As shown, a novel fluoroalkylated modified small molecule adjuvant [M+H] + The mass-to-nucleus ratio was 691.19, indicating that the novel fluoroalkylated modified small molecule adjuvant was successfully synthesized.

[0031] Example 2: Vaccine preparation and immunization

[0032] SPF-grade C57BL / 6 mice, 6–8 weeks old, were housed in a sterile environment. The treatment groups consisted of 5 subgroups, with 7 mice in each subgroup: AT (blank control); BT (OVA); CT (OVA+R848); DT (OVA+3-(perfluorohexyl)propylene oxide); and ET (OVA+R848@3-(perfluorohexyl)propylene oxide).

[0033] Specific methods: The OVA immunization dose was 50 μg / mouse, the R848 immunization dose was 10 μg / mouse, the 3-(perfluorohexyl)propoxyethylene immunization dose was 11 μg / mouse, the OVA+R848@3-(perfluorohexyl)propoxyethylene immunization dose was 21.9 μg / mouse, and the tumor-bearing concentration of B16F10-OVA cells was 10. 6 Cells / mouse, immunization route: subcutaneous injection. The immunization schedule for the treatment group was: day 0, tumor bearing; tumor size measured at seven-day intervals on days 4, 11, and 18.

[0034] Example 3 Tumor Size Measurement

[0035] Following SPF-grade C57BL / 6 mouse tumor-bearing immunization, tumor size was measured using calipers at different time points. The formula for calculating tumor volume is V = (A × B) / (A × B) 2 ) / 2. V represents tumor volume (mm). 3 A and B represent length and width, respectively. When the tumor volume is more than 1500 mm... 3 The mice were killed at that time.

[0036] The results are as follows Figure 4 As shown, vaccination demonstrated good therapeutic effects on melanoma in a melanoma treatment model, effectively controlling melanoma growth.

[0037] Example 4: Tumor Flow Cytometry Detection Results

[0038] Extract 1×10 tumor tissue cells 6 Cells / ml. Add 100 μL of stimulant and protein transport inhibitor, and incubate at 37°C and 5% CO2 for 5 hours. Incubate with anti-mouse surface-labeled antibodies CD3, CD4, and CD8a at 4°C in the dark for 30 min, then wash twice with PBS. Add 100 μL of Fix / Perm buffer, mix well, and incubate at 4°C in the dark for 20 min. Centrifuge to remove supernatant, and wash twice with 1xPerm / Wash. Then stain with anti-mouse intracellular cytokine antibodies TNF-α and IFN-γ, and incubate at 4°C for 30 min. Wash twice with PBS, resuspend cells, and immediately analyze using a Beckman Coulter Cyto FLEX flow cytometer. Data analysis was performed using FlowJo software; at least 10 cells were obtained from each sample. 5 A scatter plot of cells was generated and gating was performed.

[0039] Through cytokine analysis, the applicant discovered that the novel fluoroalkylated modified small molecule adjuvant of this invention can significantly enhance the secretion levels of TNF-α and IFN-γ, thereby enhancing the T-cell response in mice and showing good therapeutic effects on melanoma. Furthermore, enhancing cellular immune responses can overcome the limitation of existing adjuvants, such as aluminum adjuvants, which only enhance humoral immune responses.

[0040] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A compound of formula I: ###0001### I Ⅰ。 2. Process for the preparation of a compound of formula I according to claim 1, characterized in that The method is as follows: resiquimod is dissolved in dimethyl sulfoxide, methanol is added, and then 3-(perfluoro-n-hexyl)oxetane is slowly added dropwise, and the reaction is carried out at 40±5°C for 48±8 hours, and then purification is carried out to obtain the compound of formula I.

3. Use of the compound of claim 1 as an adjuvant in the preparation of a vaccine.

4. A vaccine, characterized in that, The vaccine contains the compound of claim 1.

5. Use of the vaccine according to claim 4 for the preparation of a medicament or reagent, characterized in that, The drug or agent is used for treating, preventing or diagnosing a tumor, and the tumor is melanoma.

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