CN116903479A - Water-soluble pentabiphenyl aromatic hydrocarbon, synthesis method thereof and application thereof in vaccine adjuvant - Google Patents
Water-soluble pentabiphenyl aromatic hydrocarbon, synthesis method thereof and application thereof in vaccine adjuvant Download PDFInfo
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- 239000012646 vaccine adjuvant Substances 0.000 title claims abstract description 23
- 229940124931 vaccine adjuvant Drugs 0.000 title claims abstract description 23
- 238000001308 synthesis method Methods 0.000 title abstract description 5
- -1 arene ammonium salt Chemical class 0.000 claims abstract description 35
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
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Abstract
The invention relates to a water-soluble pentabiphenyl aromatic hydrocarbon, a synthesis method thereof and application thereof as a tumor vaccine adjuvant. The method has the advantages of simple reaction operation, mild and efficient reaction conditions and contribution to industrial production. The pentabiphenyl [3] arene ammonium salt has good water solubility and biocompatibility, and can form strong electrostatic interaction with OVA antigen with net negative charge (the binding constant reaches 9.58 mu M), so that the pentabiphenyl [3] arene ammonium salt is a good tumor vaccine adjuvant. The tumor therapeutic effect is good in a mouse lymphatic tumor vaccine model, and particularly the lymphatic tumor inhibition rate is up to 82%.
Description
Technical Field
The invention belongs to the field of supermolecular chemistry and biological medicine, and relates to synthesis of water-soluble pentabiphenyl aromatic hydrocarbon and application of the water-soluble pentabiphenyl aromatic hydrocarbon as a tumor vaccine adjuvant.
Background
Tumor immunotherapy is the intentional elimination of cancerous cells by activating immune cells in the body. Compared with the traditional modes of chemotherapy, radiotherapy, operation treatment and the like, the treatment method has the advantages of strong specificity, long acting period, small side effect and the like, and is a good choice for replacing the traditional treatment or assisting in improving the curative effect of the traditional treatment. The primary tumor immunotherapy regimen includes immune checkpoint blockade, adoptive cell transfer, and vaccination. Tumor vaccines typically involve the use of a selected tumor antigen in combination with an adjuvant that activates dendritic cells. Conventional antigens cannot induce effective anti-tumor immunity in vivo, and the immunogenicity of the antigens or the specific response of a host to the antigens must be enhanced by means of an adjuvant. The key element for promoting the application progress of tumor vaccines is the research and development of vaccine adjuvants for efficiently activating the immune system. Co-delivery of antigen and adjuvant may improve the stability and retention rate of antigen in vivo, sustained release and/or specific activation of the immune system, enhancing immune response. Therefore, the development of novel vaccine adjuvants that efficiently and consistently activate and maintain immune responses is critical to drive the development and use of tumor vaccines.
Macrocyclic compounds have for decades, by virtue of their good physicochemical properties, such as rigid circulatory structure, geometry (diameter and height), hydrophobic cavities and hydrophilic interfaces, demonstrated great potential in molecular recognition, drug delivery, disease treatment, etc., however their immunological properties, especially in terms of immune activation as vaccine adjuvants for the delivery of antigens, have been neglected. Mainly because traditional macrocyclic compounds such as cyclodextrin, calixarene, cucurbituril and column arene are generally only suitable for complexing small and medium-sized guests and cannot complex huge molecules or structures such as proteins.
Therefore, the invention synthesizes the pentabiphenyl [3] arene with five benzene rings connected with each other for the first time, and aims to increase the size of the cavity by increasing the number of benzene rings of the monomer.
Chicken ovalbumin OVA, a typical representation of a model antigen, is a good vehicle for studying the immunomodulatory effects of adjuvants. In view of its net negative charge, we decided to multi-site modify the side chain of the biphenyl arene macrocycle with a positively charged ammonium salt. Based on the research and analysis, the invention synthesizes the quaternary ammonium salt side chain modified pentabiphenyl [3] arene for the first time. The modification of ammonium salt ions not only solves the problem of water solubility, but also provides enough positive charges for the ammonium salt ions, can form strong electrostatic interaction with the OVA with net negative charges, is very beneficial to improving the antigen retention rate, activating and maintaining immune response, and provides convenience for the ammonium salt ions to become excellent immune adjuvants. By establishing a mouse lymphatic tumor model, the effect of tumor treatment is evaluated, and the tumor inhibition rate is up to about 82%, and the weight of the mouse is not obviously changed.
Disclosure of Invention
In order to achieve the above object, the present invention discloses the following technical contents:
a water-soluble pentabiphenyl [3] arene shown in a formula III is characterized by having the following structural characteristics:
the invention further discloses a preparation method of water-soluble pentabiphenyl [3] arene shown in a formula III, which is characterized by comprising the following steps:
(1) Synthesizing a bromochain pentabiphenyl monomer;
(2) Synthesizing bromochain pentabiphenyl [3] arene;
(3) And (3) carrying out nucleophilic substitution reaction on the compound obtained in the step (2) under the catalysis of Lewis acid to obtain the pentabiphenyl [3] arene ammonium salt.
The Lewis acid catalyst is as follows: boron trifluoride diethyl etherate, p-toluene sulphonic acid or trifluoroacetic acid.
The invention further discloses the tumor treatment effect of the water-soluble pentabiphenyl [3] arene shown in the formula III as a vaccine adjuvant: experimental results show that the water-soluble pentabiphenyl [3] arene shown in the formula III not only has better protein loading and binding capacity in vitro and has a binding constant of 9.58 mu M with a tumor model antigen OVA, but also has good tumor treatment effect in vivo and a tumor inhibition rate of about 82%. The synthesis and preparation of the macrocyclic compound capable of being used as a vaccine adjuvant provide a good choice for replacing the traditional treatment or assisting in improving the curative effect of the traditional treatment, and provide a feasible scheme for constructing an immune activation adjuvant molecule.
The invention is described in more detail below:
(1) The pentabiphenyl full-hydroxy monomer, 1, 3-dibromopropane and cesium carbonate are added into acetonitrile according to the proportion of 1:40:12, N 2 Stirring and refluxing 12 h under the condition of protecting 90 ℃, naturally cooling to room temperature after the reaction is finished, carrying out suction filtration, extracting the obtained solid with dichloromethane and water for 3 times, mixing an organic phase with silica gel, and separating by a column chromatography to obtain a compound shown in a formula I;
(2) Adding a compound shown in a formula I and paraformaldehyde into dichloromethane according to the proportion of 1:1, stirring until the raw materials are completely dissolved, adding a Lewis acid catalyst, reacting for 25 min at room temperature, quenching the reaction by using a saturated sodium bicarbonate solution, washing an organic phase by using the saturated sodium bicarbonate solution and the saturated sodium chloride solution in sequence, stirring the organic phase by using silica gel, and separating the organic phase by using a column chromatography method to obtain the compound shown in the formula II;
wherein: (CH) 2 O) n Representing paraformaldehyde.
(3) Adding a compound of formula II to acetonitrile at N 2 Reflux was performed under heating under protection, and then 30% trimethylamine (20 eq) ethanol solution was added thereto for 7 days. Naturally cooling to room temperature after the completion, spin-drying the reaction solution, and washing the solid with acetonitrile and dichloromethane respectively to obtain water-soluble pentabiphenyl arene shown in a formula III;
the invention has simple reaction operation and higher yield, and simultaneously researches the effect of the pentabiphenyl [3] arene ammonium salt as a vaccine adjuvant in tumor treatment. The invention relates to the technical field of supermolecular biological medicine, in particular to a synthesis method of pentabiphenyl [3] arene ammonium salt and application of the pentabiphenyl [3] arene ammonium salt as a vaccine adjuvant.
The modification of the ammonium salt ion on the side chain not only solves the water solubility problem, but also provides enough positive charges for the ammonium salt ion, can form strong electrostatic interaction with the OVA with net negative charges, and is very beneficial to improving the antigen retention rate, activating and maintaining immune response. The pentabiphenyl [3] arene ammonium salt in the inclusion compound has hydrophobic rigid cavity, and each pentabiphenyl contains five benzene rings, and the pi-pi conjugation between the benzene rings further enhances the bonding capacity to guest molecules.
The technical scheme provided by the embodiment of the invention has the beneficial effects that at least:
the invention designs and synthesizes the water-soluble pentabiphenyl [3] arene modified by the quaternary ammonium salt side chain, has simple reaction operation and mild and high-efficiency reaction conditions, and is beneficial to industrial production; has good water solubility, biocompatibility and tumor treatment effect, and can be used as a novel tumor vaccine adjuvant.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the structural formula and nuclear magnetic resonance hydrogen spectrum of a pentabiphenyl full-hydroxy monomer;
FIG. 2 shows the structural formula and nuclear magnetic resonance spectrum of a bromochain pentabiphenyl monomer;
FIG. 3 shows the structural formula and nuclear magnetic resonance hydrogen spectrum of bromopentabiphenyl [3] arene;
FIG. 4 shows the structural formula and nuclear magnetic resonance hydrogen spectrum of pentabiphenyl [3] arene ammonium salt;
FIG. 5 is a graph of the binding constant of pentabiphenyl [3] arene ammonium salts to tumor pattern antigen OVA;
FIG. 6 shows a graph of antibody titres against pentabiphenyl [3] arene ammonium salts as vaccine adjuvants; wherein:
A. IgG antibody titer determination chart produced by using pentabiphenyl [3] arene ammonium salt as vaccine adjuvant
B. IgG1 antibody titer determination chart produced by using pentabiphenyl [3] arene ammonium salt as vaccine adjuvant
C. IgG2a antibody titer determination chart produced by using pentabiphenyl [3] arene ammonium salt as vaccine adjuvant
D. IgG2b antibody titer determination chart generated by using pentabiphenyl [3] arene ammonium salt as vaccine adjuvant;
FIG. 7 is a graph showing the therapeutic effect of pentabiphenyl [3] arene ammonium salt on mouse lymphoma;
figure 8 statistical plot of the effect of pentabiphenyl [3] arene ammonium salt as vaccine adjuvant on tumor bearing mice body weight.
Detailed Description
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are as follows: the reagents such as pentabiphenyl full-hydroxy monomer, 1, 3-dibromopropane, cesium carbonate, methylene dichloride, trifluoromethanesulfonic acid, paraformaldehyde, trimethylamine ethanol, methylene dichloride, petroleum ether, RMPI 1640 culture medium, fetal bovine serum, endotoxin-free chicken egg albumin (OVA), mouse lymphoma cells (EG 7-OVA) and the like are all sold in the market.
In order to make the technical scheme and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Synthesis method of pentabiphenyl [3] arene ammonium salt
(1) Preparation and characterization of a bromochain pentabiphenyl monomer:
a250 mL round bottom flask was charged with a pentabiphenyl full hydroxy monomer (0.20 g,1 mmol), 1, 3-dibromopropane (12 g,40 mmol) and cesium carbonate (1.80 g,12 mmol), and 100 mL acetonitrile was added thereto as a solvent, stirred until the starting materials were completely dissolved, heated to 90℃and then cooled to N 2 Reflux 12 h was stirred under protection and monitored by thin layer chromatography during the reaction. Naturally cooling to room temperature after the reaction is finished, filtering, washing the obtained solid with dichloromethane (3×50 mL), merging filtrate, distilling under reduced pressure to remove solvent, and purifying the product by a silica gel column chromatography to obtain a white solid, namely the bromochain pentabiphenyl monomer. (eluent composition: from pure petroleum ether to petroleum ether/dichloromethane=1/1,v/vyield 84%). 1 H NMR (400 MHz, CDCl 3 )δ7.75 (s, 4H), 7.69 (d,J= 8.3 Hz, 4H), 7.58 (d,J= 8.1 Hz, 4H), 7.31 (d,J= 9.1 Hz, 2H), 6.62 (d,J= 7.1 Hz, 4H), 4.17 (t,J= 5.8 Hz, 4H), 4.13 (t,J= 5.7 Hz, 4H), 3.64 (t,J= 6.4 Hz, 4H), 3.54 (t,J= 6.3 Hz, 4H), 2.36 (p,J= 6.2 Hz, 4H), 2.27 (p,J= 6.0 Hz, 4H) ppm。
(2) Preparation and characterization of bromochain pentabiphenyl [3] arene:
into a 150 mL round bottom flask was added bromopentabiphenyl monomer (0.20 g,1 mmol), paraformaldehyde (0.01 g, 2 mmol), then 50 mL dichloromethane as solvent, and after stirring until the starting material was completely dissolved, trifluoromethanesulfonic acid (0.01 mL, 0.12 mmol) was added. The reaction is carried out at room temperature, the reaction process is monitored by thin layer chromatography, after the reaction is finished for 30 min, the reaction solution is transferred to a 500 mL separating funnel, the reaction is quenched by saturated sodium bicarbonate solution, the organic phase is washed by saturated sodium bicarbonate solution (3×50 mL) and saturated sodium chloride solution (50 mL) in sequence, finally, the reaction solution is dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation, and the product is purified by silica gel column chromatography (eluent composition: petroleum ether/dichloromethane=2/1,v/v) Finally, the white solid product is obtainedIs bromine chain pentabiphenyl [3]]Aromatic hydrocarbon (0.40, g, 83% yield).
1 H NMR (400 MHz, CDCl 3 )δ7.70 (s, 4H), 7.64 (d,J= 8.3 Hz, 4H), 7.54 (d,J= 8.2 Hz, 4H), 7.05 (s, 2H), 6.61 (s, 2H), 4.15 (dt,J= 11.4,5.6 Hz, 8H), 3.94(s, 2H), 3.51 (dt,J= 13.8,6.4 Hz, 8H), 2.35-2.29 (m, 4H), 2.25 (q,J= 5.9 Hz, 4H) ppm。
(3) Preparation and characterization of pentabiphenyl [3] arene ammonium salts:
adding bromochain pentabiphenyl [3] into 250 mL two-mouth bottle]Aromatic hydrocarbon (0.20. 0.20 g, 0.07 mmol) was dissolved in N by adding 120mL of acetonitrile as a solvent 2 Heating to 90 ℃ under protection, adding 30% trimethylamine ethanol solution 4 mL, stirring and refluxing for 5 days. Naturally cooling to room temperature after the reaction is finished, spin-drying the reaction solution, dispersing the solid in acetonitrile, ultrasonically washing (2X 50 mL), washing (50 mL) with dichloromethane, and centrifuging to obtain a solid product, namely the pentabiphenyl [3]]Aromatic hydrocarbon ammonium salt (0.21 g, 84%), melting point 335 ℃.
1 H NMR (400 MHz, DMSO-d 6 )δ7.83-7.72 (m, 8H), 7.53 (d,J= 8.0 Hz, 4H), 6.97 (s, 2H), 6.88 (s, 2H), 4.16 (d,J= 20.5 Hz, 8H), 3.92 (s, 2H), 3.44 (s, 8H), 3.11 (d,J= 19.7 Hz, 36H), 2.17 (s, 8H) ppm。
Example 2
Determination experiment of binding constant of pentabiphenyl [3] arene ammonium salt and tumor model antigen OVA
Measuring pentabiphenyl [3] by micro-scale thermophoresis method]Binding constant of aromatic ammonium salt to OVA protein was labeled with fluorescent dye NT-647 using single molecule NT protein labeling kit. PBS buffer containing 0.05% Tween-20 (pH 7.4) was used as detection buffer. The concentration of the OVA protein with the fluorescent label was kept unchanged, while pentabiphenyl [3]]The concentration of the aromatic ammonium salt solution was diluted from 1 mM to 0.03 μm in a double ratio to give a series of concentration gradient solutions. The fluorescent protein solution was then mixed with solutions of different concentrations in a 1:1 volume ratio. After 1 minute incubation, the samples were loaded into nt.115 standard glass capillaries using nt.115 monomersThe analysis is performed.K D The values were calculated using the nanosampler software package. The binding constant obtained was 9.58. Mu.M, indicating that pentabiphenyl [3]]The aromatic hydrocarbon ammonium salt has stronger interaction with OVA protein.
Example 3
Experiment of antibody titre of pentabiphenyl [3] arene ammonium salt as vaccine adjuvant
Female C57BL/6 mice were first randomized into 2 groups of 5 mice each. Immunization was performed on day 0 and day 14, respectively, at day 1 and day 2. Each mouse was subcutaneously injected with 100. Mu.L final volume of vaccine (PBS containing 10. Mu.g of OVA or 1 mM pentabiphenyl [3] containing 10. Mu.g of OVA]100 μl each of the aromatic ammonium salt vaccine). On day 21, serum was taken for antibody detection and spleen cells were taken for cytokine detection. ELISA method for detecting OVA specific antibody reaction. 96-well ELISA plates were coated with 10. Mu.g/mL OVA antigen and stored overnight at 4 ℃. After 3 washes with PBST (PBS buffer containing 0.05% Tween-20), 1 h was blocked with blocking buffer (1% BSA in PBST solution) at room temperature. Mouse serum was collected, diluted in a blocking buffer gradient and incubated at room temperature for 2 h. After 5 washes of PBST, goat anti-mouse IgG horseradish peroxidase was added and incubated 1 h. After 5 washes of PBST, 100. Mu.L of 3,3', 5' -tetramethylbenzidine peroxidase substrate (TMB) was added to each well, and the mixture was allowed to stand for 20 min to evaluate the binding of the antibody. Finally add 50. Mu.L of 2M H 2 SO 4 The substrate reaction was terminated and its OD value was measured at 450 nm using an ELISA instrument. The above procedure was repeated with goat anti-mouse IgG1, igG2a and IgG2b horseradish peroxidase to determine OVA antibody isotype. Antibody titer was calculated as the reciprocal of serum dilution and OD value.
Example 4
Treatment experiment of pentabiphenyl [3] arene ammonium salt and tumor model of mice
A mouse lymphoma model was established using C57BL/6 mice to evaluate pentabiphenyl [3]]Tumor treatment effect of aromatic ammonium salt as vaccine adjuvant. Mouse lymphoma cells (EG 7-OVA) were inoculated subcutaneously (5X 10) in the buttocks of C57BL/6 mice 5 And/or just). When the tumor volume reaches 50 mm 3 On the left and right, tumor-bearing mice were randomly divided into 2 groups (n=5) blank control group and pentabiphenyl [3]]Aromatic hydrocarbon ammonium salt group.On days 6 and 13, 100 μl of each of the two vaccines was injected in the inguinal region of each mouse, during which tumor volumes were measured and recorded every 2 days. After 20 days, the mouse tumor experiments were terminated. Pentobiphenyl [3]]The aromatic hydrocarbon ammonium salt vaccine shows good treatment effect, and the tumor inhibition rate is up to about 82%.
The foregoing description is only for the convenience of those skilled in the art to understand the technical solution of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A water-soluble pentabiphenyl [3] arene shown in a formula III is characterized by having the following structural characteristics:
。
2. the process for preparing a water-soluble pentabiphenyl aromatic hydrocarbon of formula iii according to claim 1, comprising the steps of:
(1) The pentabiphenyl full-hydroxy monomer, 1, 3-dibromopropane and cesium carbonate are added into acetonitrile according to the proportion of 1:40:12, N 2 Stirring and refluxing 12 h under the condition of protecting 90 ℃, naturally cooling to room temperature after the reaction is finished, carrying out suction filtration, extracting the obtained solid with dichloromethane and water for 3 times, mixing an organic phase with silica gel, and separating by a column chromatography to obtain a compound shown in a formula I;
(2) Adding a compound shown in a formula I and paraformaldehyde into dichloromethane according to the proportion of 1:1, stirring until the raw materials are completely dissolved, adding a Lewis acid catalyst, reacting for 25 min at room temperature, quenching the reaction by using a saturated sodium bicarbonate solution, washing an organic phase by using the saturated sodium bicarbonate solution and the saturated sodium chloride solution in sequence, stirring the organic phase by using silica gel, and separating the organic phase by using a column chromatography method to obtain the compound shown in the formula II;
wherein n=1-2;
(3) Adding a compound of formula II to acetonitrile at N 2 Heating and refluxing under protection, then adding 30% trimethylamine (20 eq) ethanol solution into the mixture, and reacting overnight; naturally cooling to room temperature after the completion, spin-drying the reaction solution, and washing the solid with acetonitrile and dichloromethane respectively to obtain a water-soluble pentabiphenyl arene macrocycle shown in a formula III;
。
3. the process for preparing a compound of formula II according to claim 1, wherein the Lewis acid catalyst is boron trifluoride diethyl etherate, p-toluene sulphonic acid or trifluoroacetic acid.
4. Use of a water-soluble pentabiphenyl [3] arene of formula iii according to claim 1 as a vaccine adjuvant for the treatment of lymphomas: wherein said as vaccine adjuvant means: the water-soluble pentabiphenyl [3] arene shown in the formula III can form strong electrostatic interaction with the OVA antigen with net negative charge, and after the vaccine consisting of the water-soluble pentabiphenyl [3] arene and the OVA antigen is injected into a mouse body, the lymphatic tumor of the mouse is effectively treated.
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| CN108610243A (en) * | 2018-05-04 | 2018-10-02 | 上海大学 | Water-soluble cationic biphenyl aromatic hydrocarbons, its inclusion compound and preparation method thereof |
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| CN108610243A (en) * | 2018-05-04 | 2018-10-02 | 上海大学 | Water-soluble cationic biphenyl aromatic hydrocarbons, its inclusion compound and preparation method thereof |
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