Novel fluoroalkylation modified small molecule adjuvant and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a novel fluoroalkylation modified small molecule adjuvant and application thereof. The small molecule adjuvant is shown as a formula I in the specification. Compared with an aluminum adjuvant, the novel fluoroalkylated small molecule adjuvant disclosed by the invention has the advantages that the humoral immunity is improved, the cellular immunity is also improved, the novel fluoroalkylated small molecule adjuvant shows a better effect on tumor therapeutic vaccines and has a better treatment effect on melanoma, and the tumor can be basically and completely removed in a mouse body. The tumor removal mainly depends on cellular immune response to play a role, the problem that the cellular immune effect of an existing adjuvant is not ideal is solved, and the novel fluoroalkylation modified small molecule adjuvant has the application potential of a universal adjuvant. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a novel fluoroalkylation-modified small molecule adjuvant and application thereof. Background Art

[0002] For infectious diseases and tumors, vaccines are the most economical and effective means of prevention and treatment, so vaccine development is crucial. Antigens and adjuvants, as important components of vaccines, are crucial to the effectiveness of vaccines. Vaccine adjuvants can enhance or change the body's immune response to antigens, and play an important role in the development of vaccines. Many vaccines stagnate because of their poor immunogenicity and inability to induce sufficient humoral and cellular immune responses. For most vaccines, suitable adjuvants can better induce the body's immune response and enable the vaccine to achieve the best protective effect.

[0003] Aluminum adjuvant is the most commonly used traditional adjuvant, but it can only enhance humoral immune response, while other MF59 and AS series adjuvants have more balanced immune enhancement effects (both antibody immunity and cellular immunity can be enhanced). Therefore, the development of new adjuvants is crucial to promote vaccine development. Summary of the invention

[0004] In view of the shortcomings of the existing problems, the purpose of the present invention is to provide a novel fluoroalkylated modified small molecule adjuvant and its application.

[0005] The technical solution adopted by the present invention to solve the technical problem is:

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

[0007]

[0008]

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

[0010] In a more specific embodiment, the method is as follows: the amino group of resiquimod (R848) is subjected to an addition reaction with the epoxy group of 3-(perfluoro-n-hexyl)propylene oxide to obtain.

[0011] Specifically, resiquimod is dissolved in dimethyl sulfoxide, methanol is added, and then 3-(perfluoro-n-hexyl)propylene oxide is slowly added dropwise, reacted at 40±5° C. for 48±8 hours, and purified to obtain the compound of formula I.

[0012] In a second aspect, the present invention protects the use of the above-mentioned compound as an adjuvant in the preparation of a vaccine.

[0013] In a fourth aspect, the present invention protects a vaccine, wherein the vaccine contains the compound described above.

[0014] During preparation, the compound described above is formulated into a solution and then mixed with the antigen.

[0015] In a fifth aspect, the present invention also protects the use of the vaccine described above in the preparation of a drug or reagent, wherein the drug or reagent is used to treat, prevent or diagnose a tumor.

[0016] In specific embodiments, the tumor is melanoma.

[0017] Beneficial Effects

[0018] (1) Compared with aluminum adjuvants, the novel fluoroalkylated modified small molecule adjuvant of the present application improves both humoral immunity and cellular immunity, and shows good effects in therapeutic vaccines for tumors. It has a good therapeutic effect on melanoma and 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 independently designed new fluoroalkylated small molecule adjuvant in melanoma vaccine, solving the problem that the cellular immune effect of existing adjuvants is not ideal. The new fluoroalkylated small molecule adjuvant has the potential for application 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, have a better presentation effect on antigens, and thus can effectively improve the level of T cell immune response.

[0021] (4) The novel fluoroalkylated modified small molecule adjuvant of the present application can be stored at room temperature and transported in a short time, and is a safe, stable and effective vaccine adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0024] Figure 3 This is the LC-MS identification result of the novel fluoroalkylation-modified small molecule adjuvant of the present invention.

[0025] Figure 4 The change in tumor size over time.

[0026] Figure 5This novel fluoroalkylated small molecule adjuvant can effectively enhance the vaccine-induced T cell immune response in mice. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0028] Types and sources of raw materials: Resiquimod (R848) was purchased from MCE, 3-(perfluoro-n-hexyl)propylene oxide was purchased from Exploration Platform, DMSO was purchased from Solebao, 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 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-(perfluoro-n-hexyl) propylene oxide (23.92 mg, 0.0636 mmol) was slowly added dropwise, and the mixture was reacted at 40°C for 48 hours. The novel fluoroalkylated small molecule adjuvant was purified by HPLC, and the novel fluoroalkylated small molecule adjuvant was identified by LC-MS in positive ion mode. The Exact Mass of the novel fluoroalkylated small molecule adjuvant is 690.19, and the chemical structure is C 26 H 27 F 13 N4O3. LC-MS results are as follows Figure 3 As shown, the new fluoroalkylated small molecule adjuvant [M+H] + The mass-to-nuclear ratio was 691.19, indicating that the new fluoroalkylation-modified small molecule adjuvant was successfully synthesized.

[0031] Example 2 Vaccine preparation and immunization

[0032] SPF C57BL / 6 mice, 6-8 weeks old, were raised in a sterile environment. The treatment group included 5 groups, with 7 mice in each group. AT, blank control group (Blank); BT, OVA group (O); CT, OVA+R848 group (O+R); DT, OVA+3-(perfluorohexyl)propylene oxide group (O+F); ET, OVA+R848@3-(perfluorohexyl)propylene oxide group (O+RF).

[0033] Specific method: OVA immunization dose was 50 μg / mouse, R848 immunization dose was 10 μg / mouse, 3-(perfluoro-n-hexyl)propylene oxide immunization dose was 11 μg / mouse, OVA+R848@3-(perfluoro-n-hexyl)propylene oxide immunization dose was 21.9 μg / mouse, and the tumor-bearing concentration of B16F10-OVA cells was 10 6 cells / mouse, and the immunization method was subcutaneous injection. The immunization program of the treatment group was as follows: on day 0, cells were loaded with tumors, and the tumor size was measured at intervals of seven days on days 4, 11, and 18.

[0034] Example 3 Tumor size determination

[0035] After SPF grade C57BL / 6 mice were immunized with tumor vaccine, the size of tumor was measured with vernier calipers at different days. The formula for calculating tumor volume is V = (A × B 2 ) / 2. V represents the tumor volume (mm 3 ), A and B represent length and width respectively. When the tumor volume is 1500mm 3 Kill the mice.

[0036] The results are as follows Figure 4 As shown, vaccination showed good therapeutic effect on melanoma in the treated melanoma model and could effectively treat the growth of melanoma.

[0037] Example 4 Tumor flow cytometry results

[0038] Extract tumor tissue cells 1×10 6 cells / ml. Add 100μL stimulator and protein transport blocker, and incubate at 37℃, 5% CO2 for 5 hours. Incubate with anti-mouse surface marker antibodies CD3, CD4, and CD8a at 4℃ in the dark for 30 minutes, and wash twice with PBS. Add 100ul Fix / Perm solution, mix well, incubate at 4℃ in the dark for 20 minutes, centrifuge to remove the supernatant, and wash twice with 1xPerm / Wash. Then stain with anti-mouse intracellular cytokine antibodies TNF-α and IFN-γ, and incubate at 4℃ for 30 minutes. Wash twice with PBS, resuspend the cells and immediately detect them on the Beckman Coulter Cyto FLEX flow cytometer. Data analysis uses flow Jo software, and at least 10 samples are acquired for each sample. 5 The cells were gated on the scatter plot.

[0039] Through the detection of cytokines, the applicant found that the novel fluoroalkylated small molecule adjuvant of the present invention can significantly enhance the secretion level of TNF-α and IFN-γ and thus enhance the T cell response in mice, and has a good effect on the treatment of melanoma. Enhancing the cellular immune response can also optimize the shortcomings of existing adjuvants such as aluminum adjuvants that can only enhance humoral immune response.

[0040] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. The compound represented by formula I:

2. The method for preparing the 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)propylene oxide is slowly added dropwise, reacted at 40±5° C. for 48±8 hours, and purified to obtain the compound of formula I.

3. Use of the compound according to claim 1 as an adjuvant in the preparation of vaccines.

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

5. Use of the vaccine according to claim 4 in the preparation of medicines or reagents, characterized in that: The medicine or agent is used for treating, preventing or diagnosing tumors.

6. The use according to claim 5, characterized in that: The tumor is melanoma.

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