Triterpenoid saponin vaccine adjuvant having high efficiency, low toxicity and good stability, preparation method therefor, and use thereof

Abstract

The present invention aims to provide a novel triterpenoid saponin vaccine adjuvant. By means of molecular design, synthesis, activity evaluation, and other means, the adjuvant significantly improves immunostimulatory activity thereof while reducing the potential toxicity thereof and significantly enhancing the stability of a product. Specifically, the adjuvant of the present invention can induce a Th1 / Th2 mixed immune response, effectively improve the ability of an antigen presenting cell (such as a dendritic cell) to uptake, treat, and present an antigen (including but not limited to a microbial pathogen, a cancer cell, etc.), thereby enhancing the cellular immune efficacy and exhibiting great potential for preventing and treating a disease caused by a related pathogen.

Description

High-efficiency low-toxicity stable triterpenoid saponin vaccine adjuvant and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a high-efficiency low-toxicity stable triterpenoid saponin vaccine adjuvant and a preparation method and application thereof. BACKGROUND

[0002] The widespread use of vaccines has ended smallpox in humans; the gradual implementation of expanded immunization programs has eliminated polio caused by wild strains in China, and has controlled diseases such as hepatitis B, measles, diphtheria, pertussis, tetanus, tuberculosis, and Japanese encephalitis. The emergence of vaccines has directly and indirectly saved countless lives, greatly reducing disease incidence and mortality. At the same time, vaccines are not only the cornerstone of disease prevention and control, but also a public health strategy that demonstrates excellent cost-effectiveness. With minimal economic investment, it has effectively curbed large-scale disease outbreaks and built a solid defense for social health and well-being.

[0003] Adjuvants play a crucial role in vaccines, as they can enhance the immunogenicity of vaccines, i.e., the ability of vaccines to stimulate the body's immune response. Therefore, the selection and optimization of adjuvants play a decisive role in improving the protective efficiency of vaccines.

[0004] Whether a vaccine can be successfully developed and applied in clinical practice largely depends on whether a suitable adjuvant can be found. A suitable adjuvant not only improves the safety and effectiveness of the vaccine, but also reduces the incidence of adverse reactions, thereby increasing public acceptance of the vaccine.

[0005] Currently, only six new adjuvants, MF59, AS04, AS03, AS01, CpG1018, and Matrix-M, have been approved for marketing by the FDA, with extremely high thresholds (Nature Reviews Drug Discovery, 2021, 20, 54-475).

[0006] New adjuvants are divided into different components: emulsion adjuvants such as MF59 and AS03; Toll-like receptor agonists (CpG and MPLA); adsorbed on aluminum salt colloids such as AS04; combinations of immune enhancers (QS-21 and MPL in the AS01 series); and the like.

[0007] The most prominent new adjuvant in the AS01 series contains two independent immune-stimulating molecules: MPL and QS-21. QS-21 is a triterpenoid saponin purified from the bark extract of the Chilean soap tree. Studies have found that it has the ability to enhance antibody responses and promote specific T cell responses.

[0008] QS-21 has hemolytic toxicity, MPL and QS-21 are formulated into liposomes with cholesterol and phospholipids in the presence of cholesterol, which eliminates the hemolytic toxicity of part of QS-21 (Human vaccines & Immunotherapeutics, 2017, 13, 19-33). The molecular mechanism of QS-21 adjuvanticity is unknown, preliminary studies have shown that after intramuscular injection, QS-21 will target macrophages in the lymph node, attract them to flow to the injection site, and activate caspase-1 at the site, regulate and balance cellular immunity and humoral immunity (Nat. Chem. 2014, 6, 635-43.).

[0009] QS-21 has certain instability due to the presence of ester groups, easy hydrolysis and 1,2-migration (Carbohydrate Research 1996, 280, 1-14), which brings difficulties to storage, transportation and quality control.

[0010] QS-21 is naturally contained and rare, which needs to be extracted from a soapbark tree with an age of more than 15 years, and its content is 0.03 / 100,000. QS-21 contains complex triterpenoids, left-wing trisaccharides, right-wing tetrasaccharides and complex side chains modified by arabinose, which brings challenges to macro synthesis, and the price is extremely expensive, and the current market price is about 500,000 yuan per gram.

[0011] Triterpenoid saponin adjuvants such as QS-21 have attracted much attention due to their strong immune stimulating ability, but their high toxicity and relatively unstable properties limit their wide application (Acc. Chem. Res. 2016, 49, 1741-1756). Therefore, it is particularly important to develop a new adjuvant that maintains high immune stimulating activity while having low toxicity and high stability. SUMMARY

[0012] The purpose of the present application is to provide a new type of triterpenoid saponin vaccine adjuvant, which realizes the significant reduction of toxicity and the improvement of stability while maintaining high immune stimulating activity through careful design of molecular structure adjustment. Specifically, the adjuvant of the present application can induce Th1 / Th2 mixed immune response, effectively improve the uptake, processing and presentation ability of antigen-presenting cells (such as dendritic cells) to antigens (including but not limited to microbial pathogens, cancer cells, etc.), and further enhance the cellular immune efficacy, which shows great potential for preventing and treating diseases caused by related pathogens.

[0013] In the first aspect of the present application, a triterpenoid saponin vaccine adjuvant is provided, which has the structure shown in general formula I:

[0014] wherein

[0015] R1 may be hydrogen, alkyl, aryl, glycosyl;

[0016] R 2 may be hydrogen, glycosyl;

[0017] R 3 may be hydrogen, hydroxyl;

[0018] U is methylene, nitrogen hydrogen, oxygen atom;

[0019] V is methylene, carbonyl, nitrogen hydrogen, oxygen atom;

[0020] W is methylene, carbonyl, nitrogen hydrogen, oxygen atom;

[0021] X is methylene, oxygen atom;

[0022] I is an integer from 1 to 6;

[0023] m is 0 and 1;

[0024] n is 0, 3 and 4;

[0025] and when m is 0, n is 3 or 4; when m is 1, n is 0;

[0026] and UV and VW are not both adjacent ketone carbonyl, oxygen atom or nitrogen oxygen σ bond.

[0027] In another preferred example, the triterpenoid saponin adjuvant is selected from the group consisting of:

[0028] R 1 is:

[0029] optionally one of:

[0030] R 2 is:

[0031] optionally one of:

[0032] R 3 is hydrogen, deuterium, hydroxyl;

[0033] R 4 is alkyl, cycloalkyl, phenyl;

[0034] o is an integer greater than or equal to 1;

[0035] p is an integer greater than or equal to 1;

[0036] R 5 is hydrogen, hydroxyl, alkoxy, glycoside;

[0037] R 6 is hydrogen, hydroxyl, alkoxy, glycoside;

[0038] R 7 is hydrogen, hydroxyl, alkoxy, glycoside;

[0039] R 8 is hydrogen, hydroxyl, alkoxy, glycoside;

[0040] R 9 is hydrogen, hydroxyl, alkoxy, glycoside;

[0041] R 10 is hydrogen, hydroxyl, alkoxy, glycoside;

[0042] R 11 is hydrogen, methyl, hydroxymethylene, alkyl, carboxyl;

[0043] R 12 is hydrogen, methyl, hydroxymethylene, alkyl, carboxyl;

[0044] and R 5 is different from R 6 , R 7 is different from R 8 , R 9 is different from R 10 is both hydroxyl, alkoxy or glycoside,

[0045] and R 11 is different from R 12 is methyl, hydroxymethylene, alkyl, carboxyl.

[0046] In another preferred embodiment, the compound of general formula I, the triterpene skeleton comprises an aldehyde group and a hydroxyl group, but is not limited to these functional groups.

[0047] In another preferred embodiment, U is methylene, V is methylene, W is methylene, X is methylene.

[0048] In another preferred embodiment, m is 1, n is 0.

[0049] In another preferred embodiment, I is 1, 2, 3, 4, 5 or 6; preferably, I is 2, 3, 4.

[0050] In another preferred embodiment, U is methylene, V is methylene, W is methylene, X is methylene, I is an integer from 1 to 6, m is 1, n is 0.

[0051] In another preferred embodiment, U is methylene, V is methylene, W is methylene, X is methylene, I is 2, 3, 4, 5 or 6, m is 1, n is 0.

[0052] In another preferred embodiment, U is methylene, V is methylene, W is methylene, X is methylene, I is 2, 3, 4, or 5, m is 1, n is 0. In another preferred embodiment, R 2 is hydrogen.

[0053] In another preferred embodiment, R 3 is hydroxy.

[0054] In another preferred embodiment, R 1 , R 2 , R 3 , U, V, W, X, I, m, n, o and p are each independently the corresponding group or corresponding value (for I, m, n, o and p) of each of the specific compounds of the embodiments (such as Adl-Ad49) or of each of the specific compounds of Table A.

[0055] In a preferred embodiment of the application, the compound of formula I is selected from Table A:

[0056] Table A

[0057] In a preferred embodiment of the application, the compound of formula I is selected from:

[0058] In another preferred embodiment, the structure of said 8 is the structure of 9 is

[0059] wherein compound 8 needs to be an alpha type sugar donor to react with carboxylic acid compound 9 to obtain the beta glycosidic bond in compound 10.

[0060] In a second aspect of the application, there is provided an adjuvant composition comprising a compound or a pharmaceutically acceptable salt thereof as described above, in combination with at least one pharmaceutically acceptable carrier or diluent.

[0061] The triterpenoid saponin adjuvants of the application can be used to prevent or treat diseases caused by the relevant pathogen.

[0062] In a third aspect of the application, there is provided an adjuvant combination comprising: (1) one or more triterpenoid saponin adjuvants of the application, and (2) 3-O-deacylated-4'-monophosphoryl lipid A (MPL).

[0063] In another preferred embodiment, the triterpenoid saponin adjuvant is compound Ad5-Ad9.

[0064] In another preferred embodiment, the triterpenoid saponin adjuvant is compound Ad6 and / or Ad8.

[0065] In a fourth aspect of the present application, there is provided an immunological composition comprising: (i) an adjuvant combination as described herein; and (ii) an antigen.

[0066] In another preferred embodiment, the antigen comprises a protein antigen.

[0067] In another preferred embodiment, the antigen comprises an antigen derived from a pathogen or a tumor cell.

[0068] In another preferred embodiment, the pathogen comprises a virus, including but not limited to VSV virus, CSFV virus.

[0069] In another preferred embodiment, the antigen comprises an ovalbumin (OVA) antigen, a keyhole limpet hemocyanin (KLH) antigen, a VSV-gE antigen, a CSFV-E2 antigen.

[0070] In a fifth aspect of the present application, there is provided the use of the triterpenoid saponin adjuvant, the immunological composition for the preparation of an antibody, a vaccine, an immunotherapeutic drug and / or an immunostimulant.

[0071] In a sixth aspect of the present application, there is provided a method for treating a disease, the method comprising: administering to a subject in need thereof an adjuvant composition of the present application or an immunological composition of the present application.

[0072] It should be understood that, within the scope of the present application, each of the technical features described above and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 is an antibody change graph, Ad1-Ad12, Ad16, Ad20, Ad37 and Ad38 and OVA antigen are used in combination, which obviously promotes the body to produce IgGl antibody relative to other compounds.

[0074] Figure 2 is an adjuvant combination antibody change graph, Ad6 and Ad8 have no toxic side effects relative to QS21; Ad6 and QS21 induce IgG antibody titer significantly higher than QS21 in the second week after immunization under the condition of equal immunization dose, which shows that Ad6 preferentially induces immune response of the body; Ad8 relative to QS21, after three immunizations, the IgG and IgM antibody titers induced by the body are comparable to QS21 under the condition of reducing the immunization dose by 10 times, which shows that the use of Ad8 can reduce the immunization dose and save economic cost.

[0075] Figure 3 is an adjuvant combination antibody change graph, Ad5-8 are used in combination with KLH antigen, which can all promote the body to produce IgG and IgM antibodies comparable to QS21.

[0076] Figure 4 is a plot of the change in body weight of mice.

[0077] Figure 5 is a plot of the change in long-term antibody of Ad5, Ad46, Ad47, Ad48, Ad49, Ad8 adjuvant combination.

[0078] Figure 6 is a plot of the change in long-term antibody of Ad5-9 adjuvant combination.

[0079] Figure 7 is the effect of Ad6 (VA05) + MPL, Ad8 (VA06) + MPL and OVA antigen.

[0080] Figure 8 is the effect of Ad6 (VA05) + MPL, Ad8 (VA06) + MPL and VZV-gE antigen. DETAILED DESCRIPTION:

[0081] Through extensive and in-depth research, through a large number of screening, the present inventors accidentally obtained a triterpenoid saponin vaccine adjuvant with a structure shown in general formula I, which has a significantly improved immune stimulating activity by adjusting the length of the carbon chain in the molecular structure, and has a significantly reduced toxicity. The adjuvant of the present application can induce a mixed Th1 / Th2 immune response, effectively improve the uptake, processing and presentation capacity of antigen-presenting cells to antigens, and thus enhance the cellular immune efficacy, and has a broad application prospect in the field of immunological preparations.

[0082] Terminology

[0083] As used herein, the terms "containing", "comprising", or "including" mean that various components can be applied together in a mixture or composition of the present application. Therefore, the terms "consisting essentially of and "consisting of are included in the term "containing".

[0084] As used herein, the term "alkyl" refers to a straight chain (i.e., unbranched) or branched saturated hydrocarbon group containing only carbon atoms, or a combination of straight chain and branched groups. When an alkyl group is preceded by a number indicating the number of carbon atoms (e.g., C1-C6 alkyl), it is intended that the alkyl group contain from 1 to 6 carbon atoms, for example, C1-C4 alkyl means an alkyl group containing from 1 to 4 carbon atoms, representative examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, or the like.

[0085] The term "aryl" refers to all-carbon monocyclic or fused polycyclic (that is, rings which share adjacent pairs of carbon atoms) rings having a completely conjugated pi-electron system, and is an aromatic, ring hydrocarbon group. When an aryl group is preceded by a number designating the number of carbon atoms, e.g., C6-C12 aryl, it is intended that the aryl group have from 6 to 12 ring carbon atoms, such as phenyl and naphthyl. The aryl ring can be fused to other ring groups (including saturated or unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment of the radical must be at a carbon atom of the ring having the completely conjugated pi-electron system.

[0086] As used herein, the term "glycosyl" refers to a group or residue formed by the attachment of one or more monosaccharides or derivatives thereof through a glycosidic linkage. This term encompasses the reactive monovalent group obtained by removing the hydrogen atom on the terminal group of saccharide molecules ranging from monosaccharides to oligosaccharides, polysaccharides, and the like.

[0087] As used herein, the term "carboxyl" refers to a -COOH group or a -alkyl-COOH group, alkyl being as defined herein above, e.g., "C2-C4 carboxyl" refers to a group of the structure -C1-C3 alkyl-COOH, representative examples of carboxyl groups include, but are not limited to: -COOH, -CH2COOH, -C2H4COOH, or the like.

[0088] As used herein, the term "carbonyl" refers to a divalent functional group consisting of a carbon atom and an oxygen atom connected by a double bond (i.e., C=O).

[0089] As used herein, the term "methylene" has the general structure -CH2-.

[0090] As used herein, the term "nitrogen hydrogen" refers to a -NH- group.

[0091] Triterpenoid saponin adjuvants of the invention

[0092] The triterpenoid saponin adjuvants of the invention are compounds of Formula I, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a prodrug thereof.

[0093] As used herein, "adjuvants of the invention", "compounds of the invention", "compounds of Formula I of the invention", or "compounds of Formula I" are used interchangeably to refer to compounds of Formula I, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a prodrug thereof. It is understood that the term also includes mixtures of the above components.

[0094] In particular, the compounds of Formula I are as described above in the first aspect of the invention.

[0095] The term "pharmaceutically acceptable salt" refers to those salts of the compounds of the present application which are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts are those formed from the compounds of the present application with acids, including but not limited to hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, and the like inorganic acids, formic, acetic, propionic, oxalic, malonic, succinic, fumaric, maleic, lactic, malic, tartaric, citric, picric, methanesulfonic, benzenesulfonic, benzenesulfonic, and the like organic acids; and aspartic, glutamic, and the like acidic amino acids. One preferred class of salts are metal salts formed from the compounds of the present application with bases, including but not limited to sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, and the like inorganic bases, aqueous ammonia, triethylamine, diethylamine, and the like organic bases.

[0096] The compounds of the present application as described herein can be converted to their pharmaceutically acceptable salts by conventional means, for example, by adding a solution of the appropriate acid to a solution of the above compound, and removing the solvent after the salt formation is complete.

[0097] Preferred compounds of the present application are those compounds shown in Table A, preferably compounds Ad5, Ad6, Ad7, Ad8, or Ad9, for example Ad6 or Ad8.

[0098] Preparation Methods

[0099] The preparation of the compounds of the present application of the structure of Formula I is described in more detail below, but these specific methods are not to be construed as limiting the present application in any way. The compounds of the present application can also be readily prepared by combining various synthetic methods described in this specification or known in the art, as such combinations would be readily apparent to one skilled in the art to which the present application pertains.

[0100] Generally, in the preparation schemes, each reaction is typically carried out in an inert solvent at a temperature in the range of -60 °C to the reflux temperature (e.g., -60 °C to 0 °C, preferably -50 °C to 10 °C), or at a temperature in the range of room temperature to the reflux temperature (e.g., 0 °C to 80 °C, preferably 0 °C to 50 °C). The reaction time is typically in the range of 0.1 hour to 60 hours, preferably 0.5 to 48 hours.

[0101] The following general preparation schemes can be used to synthesize the compounds of the present application of the structure of Formula I.

[0102] Typically, the method comprises the steps of:

[0103] (a) reacting compound 1 and compound 2 in an inert solvent to form compound 3;

[0104] (b) removing the Ac group from compound 3 by hydrolysis to form compound 4;

[0105] (c) reacting compound 4 and compound 5 in an inert solvent to form compound 6;

[0106] (d) removing the protecting group (e.g. TIPS) from compound 6 in an inert solvent to form compound 7;

[0107] (e) reacting compound 7 with trichloroacetonitrile in an inert solvent to form compound 8;

[0108] (f) reacting compound 8 and compound 9 in an inert solvent to form compound 10;

[0109] (g) reducing compound 10 (reducing the azido group to -NH2) in an inert solvent to form compound 11;

[0110] (h) reacting compound 11 and compound 12 in an inert solvent to form compound 13;

[0111] (i) removing the protecting group (e.g. Bn, acid sensitive protecting group TES) from compound 13 in an inert solvent to form a compound of Formula I.

[0112] In the present application, representative inert solvents include, but are not limited to, methanol, tetrahydrofuran, toluene, or a combination thereof.

[0113] In the present application, representative compound 9 includes, but is not limited to, compounds shown in the following Formula 9-a, 9-b and 9-c:

[0114] In the present application, representative compound 12 includes, but is not limited to, the following:

[0115] Adjuvant combination

[0116] In the present application, an adjuvant combination is also provided, comprising: (1) one or more triterpene saponin adjuvants of the present application, and (2) 3-O-deacyl-4'-monophosphoryl lipid A (MPL).

[0117] In the present application, the ratio of each component in the adjuvant combination is not limited. For example, in the adjuvant combination, the molar ratio of the triterpene saponin adjuvant to MPL is 1:20 to 20:1.

[0118] Vaccine composition of the present application

[0119] The present application also provides a vaccine composition prepared by the method of the present application. The vaccine composition of the present application includes prophylactic vaccine composition and therapeutic vaccine composition. The vaccine composition of the present application can be a monovalent or a multivalent vaccine composition.

[0120] These vaccines contain high purity, high titer antigens, and are usually combined with "pharmaceutically acceptable carriers" which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive viral particles. These carriers are well known to those skilled in the art.

[0121] In the present application, the vaccine composition or pharmaceutical composition contains the compound of formula I of the first aspect of the present application as the only adjuvant or as one of the adjuvants in a multi-adjuvant (e.g., containing the adjuvant combination of the third aspect of the present application).

[0122] The vaccine composition of the present application (including viruses, pharmaceutically acceptable carriers and / or adjuvants) usually contains diluents such as water, saline, glycerol, ethanol and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like can be present in the vaccine composition.

[0123] More specifically, the vaccine, including the immunogenic composition, contains an immunologically effective amount of the virus, and other desired components as described above. An "immunologically effective amount" means the amount that is effective to treat or prevent when administered to an individual in a single dose or in a series of doses. The amount will depend on the physiological condition of the animal (e.g., human), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, and other relevant factors.

[0124] In the present application, the vaccine composition or immunogenic composition can be prepared in injectable forms, such as liquid solutions or emulsions; or in solid forms suitable for reconstitution or suspension in a liquid excipient prior to injection. The preparation can also be emulsified or encapsulated in liposomes, enhancing the adjuvant effect in the above-mentioned pharmaceutically acceptable carriers.

[0125] The conventional method is to administer the immunogenic composition by injection from a parenteral (subcutaneous or intramuscular) route. Other formulations suitable for other modes of administration include oral and transdermal applications, etc. The therapeutic dose can be a single dose regimen or a multiple dose regimen. The vaccine composition of the present application can be administered in combination with other immunomodulators.

[0126] The main advantages of the present application include:

[0127] 1. The triterpenoid saponin vaccine adjuvant having the structure shown in general formula I in the present application has significantly improved immune stimulating activity.

[0128] 2. The triterpene saponin vaccine adjuvant of the present application has the structure shown in Formula I, and has significantly reduced toxicity.

[0129] 3. The triterpene saponin vaccine adjuvant of the present application can induce a Th1 / Th2 mixed immune response, effectively improve the uptake, processing and presentation capacity of antigen-presenting cells to antigens, and thus enhance the cellular immune efficacy.

[0130] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0131] The experimental methods described in the following examples, if not specifically stated, are conventional methods; the reagents and materials, if not specifically stated, can be obtained commercially.

[0132] The experimental methods described in the following examples, the solvents and chemicals used are analytical or chemical pure; anhydrous solvents are treated according to standard methods; column chromatography and silica gel plates are of conventional types. The organic phase obtained by extraction is dried with anhydrous sodium sulfate, if not specified. LC-MS uses Agilent 1260 high performance liquid-ion trap mass spectrometry (ESI source), diode array detector (DAD), detection wavelength 210 nm and 254 nm. The system model, if not specifically stated, is a conventional method; the reagents and materials, if not specifically stated, can be obtained commercially.

[0133] Example 1: Synthesis and characterization of compounds of Formula I

[0134] Technical route summary: Sugar acceptor 1 and sugar donor 2 are activated by acid to obtain disaccharide 3; disaccharide acceptor 4 is obtained by removing the protection, and reacted with sugar donor 5 to obtain oligosaccharide fragment 6; alcohol 7 is obtained by removing the anomeric TIPS protecting group, and then preparing sugar donor 8; 8 is reacted with acid 9 by glycosylation to obtain 10 compounds; then the azide is reduced to amino to obtain compound 11; compound 11 is reacted with acid 12 by amidation to obtain compound 13; the benzyl group and acid-sensitive protecting group of compound 13 are removed to obtain the final compound of Formula I.

[0135] For the preparation of Ad1, the structure of sugar donor 1 is Sugar donor 1 (1.14 g, 2.1 mmol) and sugar donor 2 (1.25 g, 3.2 mmol) were mixed, dissolved in toluene and concentrated in vacuo, followed by the addition of dry dichloromethane and molecular sieves and stirred at room temperature for 1 h. The reaction was cooled to -45 °C, followed by the dropwise addition of freshly distilled boron trifluoride etherate (25 μL, 0.42 mmol) via syringe. After stirring for 3 h, the reaction was followed by TLC, quenched with triethylamine, filtered, concentrated and purified by column chromatography to give compound 3 (1.20 g, 73%). TLC: Rf= 0.4 (5:1 hexanes / EtOAc).1H NMR (400 MHz, CDCI3) δ 7.42-7.28 (m, 10H), 5.71 (s, 1H), 4.82 (dd, J = 10.4, 7.4 Hz, 1H), 4.74 (d, J = 11.7 Hz, 1H), 4.55 (d, J = 4.8 Hz, 2H), 4.53 (d, J = 3.0 Hz, 2H), 4.10-4.04 (m, 2H), 4.04-4.01 (m, 2H), 3.88 (dd, J = 9.4, 7.3 Hz, 1H), 3.70-3.62 (m, 2H), 3.62-3.54 (m, 2H), 2.06 (s, 3H), 1.56 (s, 3H), 1.34 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H). MS (ESI) m / z: (M + Na)+ 792.4, found 792.4.

[0136] Compound 3 (0.838 g, 1.09 mmol) was dissolved in methanol (50 mL) and water (5 mL), followed by the addition of potassium carbonate (0.3 g, 2.18 mmol). After stirring at room temperature for 1.5 h, dichloromethane was added, dried, filtered, concentrated and purified by column chromatography to give compound 4 (674 mg, 84%). TLC: Rf= 0.6 (3:1 hexanes / EtOAc).1H NMR (400 MHz, CDCI3) δ 7.43-7.28 (m, 10H), 5.65 (s, 1H), 4.73 (d, J = 11.6 Hz, 1H), 4.58-4.48 (m, 4H), 4.08 (d, J = 5.7 Hz, 1H), 4.02 (d, J = 3.5 Hz, 1H), 3.98-3.85 (m, 3H), 3.62 (ddq, J = 28.9, 11.4, 6.3, 5.4 Hz, 4H), 3.35 (ddd, J = 9.6, 7.4, 4.2 Hz, 1H), 1.51 (s, 3H), 1.34 (s, 3H), 1.25 (d, J = 6.3 Hz, 3H), 1.17-0.83 (m, 21H). MS (ESI) m / z: (M + Na)+ 750.4, found 750.2.

[0137] The structure of sugar donor 5 is Sugar donor 5 (845 mg, 1.5 mmol) and sugar acceptor 4 (727 mg, 1.0 mmol) were mixed, dissolved in toluene and concentrated under vacuum three times, then dry dichloromethane and molecular sieves were added and stirred at room temperature for 1 h. The reaction was cooled to -45 °C, then freshly distilled boron trifluoride etherate (21 μL, 0.2 mmol) was added dropwise via syringe. After stirring for 3 h, the reaction was complete by TLC, then the reaction was quenched with triethylamine, filtered, concentrated and purified by column chromatography (PE / EAc, 30:1 to 8:1) to give compound 6 (632 mg, 59%). TLC: Rf= 0.2 (8:1 hexanes / EtOAc).1H NMR (400 MHz, CDCI3) δ 7.38 - 7.22 (m, 25H), 5.68 (s, 1H), 4.92 (d, J = 2.8 Hz, 1H), 4.90 (s, 1H), 4.82 (d, J = 11.0 Hz, 1H), 4.79 (d, J = 11.1 Hz, 1H), 4.72 (d, J = 6.7 Hz, 1H), 4.69 (d, J = 6.8 Hz, 1H), 4.63 (d, J = 3.4 Hz, 1H), 4.60 (d, J = 4.1 Hz, 1H), 4.56 - 4.48 (m, 4H), 4.17 (dd, J = 7.6, 5.5 Hz, 1H), 4.05 (d, J = 5.6 Hz, 1H), 4.01 (d, J = 3.5 Hz, 1H), 3.97 - 3.85 (m, 3H), 3.68 - 3.51 (m, 7H), 3.30 (td, J = 7.4, 2.5 Hz, 1H), 3.23 - 3.16 (m, 1H), 1.51 (s, 3H), 1.35 (s, 3H), 1.23 (d, J = 6.1 Hz, 3H), 1.07 - 0.92 (m, 21H). MS (ESI) m / z: (M + Na)+ 1152.6, found 1152.9.

[0138] Compound 6 (575 mg, 0.51 mmol) was dissolved in tetrahydrofuran (50 mL), then cooled to 0 °C, then TBAF (1 M in THF, 0.76 mL, 0.76 mmol) and acetic acid (35 μL, 0.61 mmol) were added. After the reaction was complete, it was quenched with saturated sodium bicarbonate, extracted with ethyl acetate, dried, filtered, concentrated and purified by column chromatography to give the reduced trisaccharide 7 (400 mg, 82%).

[0139] The reducing anomer was dissolved in 7 (400 mg, 0.42 mmol) in dichloromethane (32 mL), cooled to 0 °C, and then tri chloroacetonitrile (0.34 mL, 0.34 mmol) and DBU (0.1 mL, 0.67 mmol) were added. It was stirred at room temperature overnight, concentrated, and column chromatographed (PE / EA 5:1) to give compound 8 (199 mg, 85%).

[0140] Triterpene 9 (653 mg, 0.32 mmol) and sugar donor 8 (230 mg, 0.21 mmol) were dissolved in toluene and concentrated in vacuo, then dry dichloromethane and molecular sieves were added and stirred at room temperature for 1 h. The reaction was cooled to -78 °C, then freshly distilled boron trifluoride etherate (15 μL, 0.23 mmol) was added dropwise via syringe. After 3 h, the reaction was followed by TLC, then quenched by the addition of triethylamine, filtered, concentrated, and column chromatographed to give compound 10 (322 mg, 73%).

[0141] Compound (1.0 equiv.) was dissolved in triethylamine, freshly prepared PhSeH (20-30 equiv.) was added, then stirred at 38 °C for about 8 h, directly concentrated, and column chromatographed on silica gel (5:1 to 3:2 PE / EA) to give compound 11 (about 80% yield).

[0142] The structure of compound 12 is Compound 12 (11.5 equiv.) was dissolved in tetrahydrofuran, cooled to 0 °C, then triethylamine (90 equiv.) and ethyl chloroformate (10.0 equiv.) were added. After 2-3 h, compound 11 (1.0 equiv.) was added at 0 °C, stirred for 2 h, then quenched by the addition of water. It was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed on silica gel (3:1 PE / EA) to give compound 13 (85-90% yield).

[0143] Compound 12 (11.5 equiv.) was dissolved in tetrahydrofuran, cooled to 0 °C, then triethylamine (90 equiv.) and ethyl chloroformate (10.0 equiv.) were added. After 2-3 h, compound 11 (1.0 equiv.) was added at 0 °C, stirred for 2 h, then quenched by the addition of water. It was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed on silica gel (3:1 PE / EA) to give compound 13 (85-90% yield).

[0144] Compound 13 (1.0. equiv.) was dissolved in tetrahydrofuran / ethanol mixture, 10% Pd / C was added, and hydrogenation was carried out at 50 psi for 24 hours. The reaction solution was diluted with methanol, and the Pd / C was removed by filtration. The crude product was obtained by concentration. Then 75% trifluoroacetic acid in water was added, and the mixture was stirred at 0°C for about 2 hours. The trifluoroacetic acid solution was removed at low temperature, and the final compound (yield 42-55%) was obtained by column chromatography. The compound was Adl.

[0145] For the preparation of Ad2-Ad49, the R 1 , R 2 , R 3 , X, U, V, W groups were replaced by the corresponding groups, respectively, and the same procedure as for the preparation of Adl was used to prepare Ad2-Ad49, respectively.

[0146] The characteristic NMR data and mass spectrometry data of the standard structure of Adl-Ad49 in the examples are as follows:

[0147] Example 2

[0148] Immunization and detection process of ovalbumin (OVA) antigen and saponin adjuvant: 6-8 weeks old female C57BL / 6J mice were used in this study, with four mice in each group. OVA antigen (100 μg) and different saponin adjuvants (50 μg) were used in combination in 5% Tween-80 solution, and OVA antigen without saponin adjuvant was used as a negative control. Through this combination of antigen and adjuvant, the mice were immunized by intramuscular injection three times on days 0, 7, and 14, respectively, and then the blood of the mice was collected on days 14, 28, and 42. The serum was collected after overnight incubation at 4°C, and then stored at -40°C. Ovalbumin Specific IgG, Ovalbumin Specific IgGl, Ovalbumin Specific IgG2b, and Ovalbumin Specific IgG2a antibody titers were detected by enzyme-linked immunosorbent assay (ELISA) related kits.

[0149] The antibody levels of OVA-specific IgG and its subclasses in serum were detected by ELISA. The steps of ELISA kit were as follows: before adding standard, sample and control (blank) wells, the plate was washed twice. Then 100 μl of standard or sample was added to each well, and after sealing the plate, incubated at 37°C for 90 minutes. The plate was washed 3 times, each time for 1 minute. 100 μl of HRP-labeled antibody working solution was added to each well, and after sealing the plate, incubated at 37°C for 30 minutes. The plate was washed 5 times, each time for 1 minute. 90 μl of TMB substrate solution was added, and after sealing the plate, incubated at 37°C for 10-20 minutes. 50 μl of stop solution was added. Read immediately at 450 nm wavelength and calculate the results.

[0150] The results are shown in Figure 1, Ad1-Ad12, Ad16, Ad20, Ad37 and Ad38 were combined with OVA antigen, among which Ad5-9, Ad20, Ad37, Ad38 significantly promoted the production of IgG1 antibody in the body relative to other compounds.

[0151] The immunization and detection process of keyhole limpet hemocyanin (KLH) antigen with saponin adjuvant: 6-8 week old female C57BL / 6J mice were used in this study, with four mice in each group. KLH antigen (20 μg) and different saponin adjuvants (50 μg) were used in combination with 5% Tween-80 solution, and KLH antigen without saponin adjuvant was used as a negative control. Through this method of antigen and adjuvant combination, the mice were immunized by intramuscular injection three times on days 0, 7 and 14, respectively, and then the mouse blood was collected on days 14, 28 and 42, and the serum was collected after overnight incubation at 4°C and then stored at -40°C. Keyhole Limpet Hemocyanin IgM and Keyhole Limpet Hemocyanin IgG antibody titers were detected by enzyme-linked immunosorbent assay (ELISA) related kit.

[0152] The antibody levels of KLH-specific IgG and IgM in serum were detected by ELISA. The steps of ELISA kit were as follows: before adding standard, sample and control (blank) wells, the plate was washed twice. Then 100 μl of standard or sample was added to each well, and after sealing the plate, incubated at 37°C for 90 minutes. The plate was washed 3 times, each time for 1 minute. 100 μl of HRP-labeled antibody working solution was added to each well, and after sealing the plate, incubated at 37°C for 30 minutes. The plate was washed 5 times, each time for 1 minute. 90 μl of TMB substrate solution was added, and after sealing the plate, incubated at 37°C for 10-20 minutes. 50 μl of stop solution was added. Read immediately at 450 nm wavelength and calculate the results.

[0153] Results are shown in Figures 2 and 3. Figure 2 shows that Ad6 and Ad8 have no toxic side effects relative to QS21. Ad6 and QS21 induce significantly higher IgG antibody titers than QS21 at the second week post-immunization at the same immunization dose, indicating that Ad6 preferentially induces immune responses. Ad8 induces IgG and IgM antibody titers comparable to QS21 after three immunizations at a 10-fold reduced immunization dose relative to QS21, indicating that the use of Ad8 can reduce the immunization dose and save economic costs. Figure 3 shows that Ad5-8, when combined with KLH antigen, can promote the production of IgG and IgM antibodies comparable to QS21.

[0154] During the experiment, the body weight of the mice was monitored, and the results are shown in Figure 4, indicating that Ad45, Ad48, Ad50, Ad52, and Ad8 have high safety.

[0155] For mice immunized with OVA antigen, the titers of each antibody subtype were detected at 21 days, 28 days, 42 days, and 3 months. The results of Ad5, Ad46, Ad47, Ad48, Ad49, and Ad8 are shown in Figure 5, and the results of Ad5-9 are shown in Figure 6.

[0156] Example 3

[0157] In this example, the effects of the adjuvants Ad6 (hereinafter referred to as VA05) and Ad8 (hereinafter referred to as VA06) combined with MPL were verified. A three-week evaluation method was used to compare the effects of different adjuvants combined with MPL. Female C57BL / 6 mice (6-8 weeks old, 4 per group) were subcutaneously immunized with OVA antigen (5 μg) alone or combined with AS01b [liposome: QS-21 (5 μg) + MPL (5 μg) + DOPC (100 μg) + cholesterol (25 μg)], QS-21 + MPL [QS-21 (5 μg) + MPL (5 μg)], VA05 + MPL [VA05 (5 μg) + MPL (5 μg)], or VA06 + MPL [VA06 (5 μg) + MPL (5 μg)] on days 0, 7, and 14. Serum samples were collected from each mouse on day 42 and analyzed for anti-OVA activity.

[0158] The results are shown in Figure 7. VA05 + MPL induced serum IgG and its subclass anti-OVA antibody responses comparable to those induced by AS01. VA06 + MPL was superior to AS01 and VA05 + MPL in inducing responses, confirming its strong ability to activate humoral immunity (Figure 7A). Figures 7B and 7C show the analysis of CD4 +Representative flow cytometry dot plots of cytokine (TNF-a, IFN-g, and IL-2) expression in T lymphocytes. These plots visually demonstrate that VA06 + MPL increased CD4 + T cell frequencies. FIGS. 7D and 7E show that analysis of CD8 + Representative flow cytometry dot plots of cytokine (TNF-a, IFN-g, and IL-2) expression in T lymphocytes. These plots visually demonstrate that VA06 + MPL increased CD8 + T cell frequencies, demonstrating that VA06 + MPL induced a balanced Thl / Th2 response and robust CD8+ cytotoxic T lymphocyte (CTL) production. Despite the absence of liposomes, VA06 was able to maintain the synergistic immunostimulatory effect of MPL, with potency comparable to AS01b, while offering a more flexible adjuvant design.

[0159] To further demonstrate its efficacy, VZV-gE antigen was used in combination with VA05 + MPL and VA06 + MPL, in comparison to Shingrix, with a focus on humoral and cellular immunity. The results, shown in FIG. 8, demonstrate that all adjuvant groups (AS01b, QS-21 + MPL, VA05 + MPL, VA06 + MPL) induced higher VZV-gE-specific IgG, IgGl, IgG2a, and IgG2b titers than the PBS control group. Notably, VA06 + MPL exhibited slightly higher antibody levels than Shingrix, confirming its ability to drive strong humoral immunity against the key VZV-gE antigen.

[0160] Regarding cellular immunity (FIGS. 8C-8E), flow cytometry and quantitative data showed that adjuvant treatment enhanced VZV-gE-specific CD4 + and CD8 + T cell responses. Specifically, AS01b, VA05 + MPL, and VA06 + MPL increased CD4 + / CD8 + T cell percentages that produced TNF-a, IFN-g, and IL-2 (key cytokines for antiviral immunity) compared to PBS. In particular, VA06 + MPL was comparable to AS01b in stimulating VZV-gE-specific cytokine-producing T cells, highlighting its potential as a Shingrix replacement adjuvant: it retains the ability to activate cellular immunity that is crucial for controlling varicella-zoster virus reactivation, while offering the previously mentioned advantages over AS01b, including better stability and a simpler production process.

[0161] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the application from the teaching- presented herein.

Claims

1. A triterpenoid saponin adjuvant, such as a compound of Formula I, and pharmaceutically acceptable salts, isotopes, and isomers thereof, having Formula I: ###0001### Formula I wherein R 1 may be hydrogen, alkyl, aryl, glycosyl; R 2 is hydrogen, a sugar group; R 3 is hydrogen, hydroxyl; U is methylene, nitrogen hydrogen, oxygen atom; V is methylene, carbonyl, nitrogen hydrogen, oxygen atom; W is methylene, carbonyl, nitrogen hydrogen, oxygen atom; X is methylene, oxygen atom; l is an integer from 1 to 6; m is 0 and 1; n is 0, 3 and 4; and when m is 0, n is 3 or 4; and when m is 1, n is 0; and UV and VW are not both adjacent keto carbonyl, oxygen atom or nitrogen oxygen sigma bond.

2. The triterpene saponin adjuvant of claim 1, wherein R 1 is: optionally one of the following: R 2 is: optionally one of the following: R 3 is hydrogen, deuterium, hydroxyl; R 4 is alkyl, cycloalkyl, phenyl; o is an integer greater than or equal to 1; p is an integer greater than or equal to 1; R 5 is hydrogen, hydroxyl, alkoxy, glycoside; R 6 is hydrogen, hydroxyl, alkoxy, glycoside; R 7 is hydrogen, hydroxyl, alkoxy, glycoside; R 8 is hydrogen, hydroxyl, alkoxy, glycoside; R 9 is hydrogen, hydroxyl, alkoxy, glycoside; R 10 is hydrogen, hydroxyl, alkoxy, glycoside; R 11 is hydrogen, methyl, hydroxymethyl, alkyl, carboxyl; R 12 is hydrogen, methyl, hydroxymethyl, alkyl, carboxyl; and R 5 with R 6 , R 7 with R 8 , R 9 with R 10 are both hydroxyl, alkoxy or glycoside, and R 11 and R 12 are different from each other and are methyl, hydroxymethyl, alkyl, carboxyl.

3. The triterpene saponin adjuvant of claim 1, wherein the triterpene skeleton comprises an aldehyde group and a hydroxyl group.

4. The triterpenoid adjuvant of claim 1, wherein, U is methylene, V is methylene, W is methylene, and X is methylene.

5. The triterpenoid adjuvant of claim 1, wherein, m is 1, and n is 0.

6. The triterpenoid adjuvant of claim 1, wherein, l is 2, 3, 4, 5 or 6.

7. The triterpenoid adjuvant of claim 1, wherein, U is methylene, V is methylene, W is methylene, and X is methylene, l is 2, 3, 4, 5 or 6, m is 1, and n is 0.

8. The triterpenoid saponin adjuvant of claim 1, wherein comprising the following compounds:

9. The triterpenoid adjuvant of claim 1, wherein, The triterpenoid saponin adjuvants are selected from the group consisting of:

10. An adjuvant composition comprising the triterpene saponin adjuvant of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, in combination with at least one pharmaceutically acceptable carrier or diluent.

11. The triterpene saponin adjuvant of any one of claims 1-9, and pharmaceutically acceptable salts thereof, or the composition of claim 10, for use as a vaccine adjuvant for the prevention or treatment of a disease associated therewith.

12. An adjuvant combination, characterized in that, The adjuvant combination comprises: (1) one or more triterpene saponin adjuvants as described in any one of claims 1-9, and (2) 3-O-deacyl-4'-monophosphoryl lipid A (MPL).

13. The adjuvant combination of claim 12, wherein, The triterpenoid saponin adjuvants are selected from the group consisting of:

14. An immunological composition comprising, in admixture, a polypeptide of claim 1 and a pharmaceutically acceptable carrier. The immunological composition comprises: (i) the adjuvant combination of claim 12; and (ii) an antigen.

15. Use of the triterpenoid saponin adjuvant of any one of claims 1-9, the adjuvant composition of claim 12, or the immunological composition of claim 14, wherein, for the preparation of antibodies, vaccines, immunotherapeutic drugs and / or immunostimulants. for the preparation of antibodies, vaccines, immunotherapeutic drugs and / or immunostimulants.

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