CN116903479B - 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
- Publication number
- CN116903479B CN116903479B CN202310891469.8A CN202310891469A CN116903479B CN 116903479 B CN116903479 B CN 116903479B CN 202310891469 A CN202310891469 A CN 202310891469A CN 116903479 B CN116903479 B CN 116903479B
- Authority
- CN
- China
- Prior art keywords
- pentabiphenyl
- arene
- ammonium salt
- formula
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000004945 aromatic hydrocarbons Chemical class 0.000 title claims abstract description 20
- 239000012646 vaccine adjuvant Substances 0.000 title abstract description 21
- 229940124931 vaccine adjuvant Drugs 0.000 title abstract description 21
- 238000001308 synthesis method Methods 0.000 title abstract description 5
- -1 arene ammonium salt Chemical class 0.000 claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 37
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 239000000178 monomer Substances 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 10
- 239000012074 organic phase Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 5
- 229920002866 paraformaldehyde Polymers 0.000 claims description 5
- VEFLKXRACNJHOV-UHFFFAOYSA-N 1,3-dibromopropane Chemical compound BrCCCBr VEFLKXRACNJHOV-UHFFFAOYSA-N 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 4
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- RVPAGRVOSVAXSH-UHFFFAOYSA-N n,n-dimethylmethanamine;ethanol Chemical compound CCO.CN(C)C RVPAGRVOSVAXSH-UHFFFAOYSA-N 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 claims description 3
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000010791 quenching Methods 0.000 claims description 2
- 230000000171 quenching effect Effects 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 33
- 101000609767 Dromaius novaehollandiae Ovalbumin Proteins 0.000 abstract description 17
- 239000000427 antigen Substances 0.000 abstract description 15
- 102000036639 antigens Human genes 0.000 abstract description 15
- 108091007433 antigens Proteins 0.000 abstract description 15
- 229960005486 vaccine Drugs 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 6
- 230000001926 lymphatic effect Effects 0.000 abstract description 4
- 230000009881 electrostatic interaction Effects 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 239000002671 adjuvant Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 150000002678 macrocyclic compounds Chemical class 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000005934 immune activation Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 239000011968 lewis acid catalyst Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003242 quaternary ammonium salts Chemical group 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000012265 solid product Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 239000002841 Lewis acid Substances 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001217 buttock Anatomy 0.000 description 1
- VTJUKNSKBAOEHE-UHFFFAOYSA-N calixarene Chemical compound COC(=O)COC1=C(CC=2C(=C(CC=3C(=C(C4)C=C(C=3)C(C)(C)C)OCC(=O)OC)C=C(C=2)C(C)(C)C)OCC(=O)OC)C=C(C(C)(C)C)C=C1CC1=C(OCC(=O)OC)C4=CC(C(C)(C)C)=C1 VTJUKNSKBAOEHE-UHFFFAOYSA-N 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- MSBXTPRURXJCPF-DQWIULQBSA-N cucurbit[6]uril Chemical compound N1([C@@H]2[C@@H]3N(C1=O)CN1[C@@H]4[C@@H]5N(C1=O)CN1[C@@H]6[C@@H]7N(C1=O)CN1[C@@H]8[C@@H]9N(C1=O)CN([C@H]1N(C%10=O)CN9C(=O)N8CN7C(=O)N6CN5C(=O)N4CN3C(=O)N2C2)C3=O)CN4C(=O)N5[C@@H]6[C@H]4N2C(=O)N6CN%10[C@H]1N3C5 MSBXTPRURXJCPF-DQWIULQBSA-N 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000007517 lewis acids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000001768 microscale thermophoresis Methods 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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) Adding a pentabiphenyl full-hydroxyl monomer, 1, 3-dibromopropane and cesium carbonate into acetonitrile according to the proportion of 1:40:12, stirring and refluxing for 12: 12h under the condition of N 2 at the temperature of 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 at room temperature for 25 min, 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 2O)n represents paraformaldehyde.
(3) The compound shown in the formula II is added into acetonitrile, heated and refluxed under the protection of N 2, and then 30% trimethylamine (20 eq) ethanol solution is added into the mixture to react 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:
To a 250 mL round bottom flask were added 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 100mL acetonitrile as solvent was added thereto, stirred until the starting material was completely dissolved, heated to 90 ℃, refluxed under stirring under protection of N 2 for 12 h, 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 (3X 50 mL), merging filtrate, distilling under reduced pressure to remove solvent, 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/v, yield 84%）.1H NMR (400 MHz, CDCl3)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:
to a 150 mL round bottom flask was added bromopentabiphenyl monomer (0.20 g,1 mmol), paraformaldehyde (0.01 g, 2 mmol), then 50mL 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, 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 organic phase 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), thus obtaining the white solid product, namely the bromopentabiphenyl [3] arene (0.40 g, yield 83%).
1H NMR (400 MHz, CDCl3)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:
Into a 250 mL two-mouth bottle, bromopentabiphenyl [3] arene (0.20, g, 0.07, mmol) was added, 120mL of acetonitrile was added as a solvent for dissolution, and heated to 90℃under the protection of N 2, then 30% trimethylamine ethanol solution 4mL was added thereto, and the mixture was stirred and refluxed for 5 days. After the reaction is finished, naturally cooling to room temperature, spin-drying the reaction solution, dispersing the solid in acetonitrile, ultrasonically washing (2X 50 mL), then washing (50 mL) with dichloromethane, and centrifuging to obtain a solid product, namely the pentabiphenyl [3] arene ammonium salt (0.21 g, 84%), wherein the melting point is 335 ℃.
1H NMR (400 MHz, DMSO-d6)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
The binding constant of pentabiphenyl [3] arene ammonium salt and OVA protein is determined by micro-scale thermophoresis, and the OVA protein is marked by fluorescent dye NT-647 by using a single molecule NT protein marking kit. PBS buffer containing 0.05% Tween-20 (pH 7.4) was used as detection buffer. The concentration of OVA protein with fluorescent label is kept unchanged, and the concentration of pentabiphenyl [3] arene ammonium salt solution is diluted to 0.03 mu M from 1 mM times to obtain 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 for analysis using the nt.115 monomer system. The K D value was calculated using NanoTemper software package. The binding constant obtained is 9.58 mu M, which shows that the pentabiphenyl [3] arene 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 of vaccine (100. Mu.L each of PBS containing 10. Mu.g of OVA or 1mM pentabiphenyl [3] arene ammonium salt vaccine containing 10. Mu.g of OVA). 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), 1h was blocked with blocking buffer (1% BSA in PBST solution) at room temperature. Mouse serum was collected, diluted with blocking buffer, 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 antibody binding. Finally, 50. Mu.L of 2M H 2SO4 was added to terminate the substrate reaction, and the OD at 450 nm was measured by ELISA. 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 lymphatic tumor model is established by using a C57BL/6 mouse to evaluate the tumor treatment effect of the pentabiphenyl [3] arene ammonium salt as a vaccine adjuvant. Mouse lymphoma cells (EG 7-OVA) were inoculated subcutaneously (5X 10 5/mouse) in the buttocks of C57BL/6 mice. When the tumor volume reached about 50mm 3, tumor-bearing mice were randomly divided into 2 groups (n=5) a blank control group and a pentabiphenyl [3] arene 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. The pentabiphenyl [3] arene ammonium salt vaccine shows excellent treating effect and tumor inhibiting rate as high as 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 (2)
1. The pentabiphenyl [3] arene ammonium salt shown in the formula III is characterized by having the following structural characteristics:
。
2. The process for preparing an ammonium salt of a pentabiphenyl [3] arene represented by the formula iii according to claim 1, comprising the steps of:
(1) Adding pentabiphenyl full-hydroxy monomer, 1, 3-dibromopropane and cesium carbonate into acetonitrile according to the proportion of 1:40:12, stirring and refluxing for 12: 12h under the condition of N 2 at the protection of 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 as a formula I
；
(2) Adding a compound shown in a formula I and paraformaldehyde into dichloromethane according to a ratio of 1:1, stirring until the raw materials are completely dissolved, adding trifluoromethanesulfonic acid, reacting at room temperature for 25 min, quenching the reaction by using saturated sodium bicarbonate solution, washing an organic phase by using saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence, stirring the organic phase by using silica gel, and separating by using a column chromatography to obtain the compound shown in a formula II
；
(3) Adding a compound shown in a formula II into acetonitrile, heating and refluxing under the protection of N 2, and then adding a 30% trimethylamine ethanol solution of 20: 20 eq into the mixture to react for 7 days; naturally cooling to room temperature after finishing, spin-drying the reaction solution, and washing the solid with acetonitrile and dichloromethane respectively to obtain pentabiphenyl [3] arene ammonium salt shown in formula III;
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310891469.8A CN116903479B (en) | 2023-07-20 | 2023-07-20 | Water-soluble pentabiphenyl aromatic hydrocarbon, synthesis method thereof and application thereof in vaccine adjuvant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310891469.8A CN116903479B (en) | 2023-07-20 | 2023-07-20 | Water-soluble pentabiphenyl aromatic hydrocarbon, synthesis method thereof and application thereof in vaccine adjuvant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116903479A CN116903479A (en) | 2023-10-20 |
| CN116903479B true CN116903479B (en) | 2024-05-07 |
Family
ID=88352697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310891469.8A Active CN116903479B (en) | 2023-07-20 | 2023-07-20 | Water-soluble pentabiphenyl aromatic hydrocarbon, synthesis method thereof and application thereof in vaccine adjuvant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116903479B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110642684A (en) * | 2019-10-15 | 2020-01-03 | 天津师范大学 | Macrocyclic and cage-shaped molecules based on biphenyl arene and derivative compounds thereof, and synthetic method and application thereof |
| CN112479835A (en) * | 2020-11-20 | 2021-03-12 | 上海应用技术大学 | Pentabiphenyl macrocyclic compound and preparation and application thereof |
| CN113979876A (en) * | 2021-11-19 | 2022-01-28 | 天津师范大学 | Water-soluble quaterphenyl aromatic hydrocarbon macrocyclic compound and preparation method and application thereof |
-
2023
- 2023-07-20 CN CN202310891469.8A patent/CN116903479B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110642684A (en) * | 2019-10-15 | 2020-01-03 | 天津师范大学 | Macrocyclic and cage-shaped molecules based on biphenyl arene and derivative compounds thereof, and synthetic method and application thereof |
| CN112479835A (en) * | 2020-11-20 | 2021-03-12 | 上海应用技术大学 | Pentabiphenyl macrocyclic compound and preparation and application thereof |
| CN113979876A (en) * | 2021-11-19 | 2022-01-28 | 天津师范大学 | Water-soluble quaterphenyl aromatic hydrocarbon macrocyclic compound and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| A Modular Synthetic Strategy for Functional Macrocycles;Kaidi Xu等;《Angew. Chem. Int. Ed.》;20200427;第59卷(第18期);7214 –7218 * |
| 免疫佐剂在肿瘤免疫疗法中的应用进展;钱颖;《中国生物工程杂志》;20201231;第40卷(第3期);96-103 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116903479A (en) | 2023-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1165237A (en) | Particles of lipoid-soluble substances, compositions containing said particles and biologically active substances adsorbed thereon, and a process for the preparation thereof | |
| JP3421337B2 (en) | Synthetic conjugate vaccine for influenza | |
| CN113993839A (en) | Ionizable lipid molecule, preparation method thereof and application thereof in preparation of lipid nanoparticles | |
| CN106008667B (en) | Small peptide, its application and vaccine as vaccine adjuvant | |
| CN107522772B (en) | Short peptide, application of short peptide as vaccine adjuvant and vaccine using short peptide as vaccine adjuvant | |
| CA2361009A1 (en) | Her-2/neu fusion proteins | |
| CN105497891A (en) | Application of polypeptide hydrogel serving as protein vaccine adjuvant and protein vaccine | |
| Yang et al. | Preparation and antitumor effects of nanovaccines with MAGE-3 peptides in transplanted gastric cancer in mice | |
| CN111760021B (en) | Conjugate containing alpha-galactosylceramide analogue and saccharide antigen, and preparation method and application thereof | |
| CN108047071B (en) | Carcinone artificial hapten, artificial antigen, preparation method and application thereof | |
| CN116903479B (en) | Water-soluble pentabiphenyl aromatic hydrocarbon, synthesis method thereof and application thereof in vaccine adjuvant | |
| CN113577255B (en) | Tumor nano vaccine, preparation method and application thereof | |
| CN116554046B (en) | Ionizable lipid compound and lipid nanoparticle thereof | |
| CN113663060B (en) | Whole-cell tumor nano vaccine, preparation method and application thereof | |
| CN109265679B (en) | Poly (glutamic acid-choline phosphoric acid) and application thereof as vaccine immunologic adjuvant | |
| CN116693632A (en) | African swine fever virus epitope peptide, monoclonal antibody and application thereof | |
| CN115590836A (en) | Lipid nanoparticle for improving mRNA vaccine induced immune response capability and application thereof | |
| US9499798B2 (en) | Method for inducing fusion of dendritic cell with tumour cell | |
| JP7405983B2 (en) | Use of oligosaccharide compounds containing heptose chains in the production of vaccines against Helicobacter pylori | |
| CN111303076A (en) | Preparation method of methylene blue hapten and methylene blue immunogen | |
| CN113307762A (en) | Preparation and application of broad-spectrum antibody for simultaneously detecting three illegal additives in weight-losing health-care food | |
| CN114259559B (en) | Synthetic tumor vaccine containing alpha-GalCer endogenous adjuvant | |
| CN115381938A (en) | Vaccine adjuvant system and application thereof in new corona and monkeypox virus vaccine | |
| CN101890162A (en) | New use of PEG-PCL-PEG (polyethylene glycol-polycaprolactone-polyethylene glycol) and antigen-assembled mixture | |
| US20220289667A1 (en) | Pregabalin artificial hapten, artificial antigen and preparation method therefor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |