WO2022007905A1

WO2022007905A1 - Novel vaccine adjuvant and use thereof in novel coronavirus vaccines and other vaccines

Info

Publication number: WO2022007905A1
Application number: PCT/CN2021/105297
Authority: WO
Inventors: 李艳梅; 吴军军; 陈永湘
Original assignee: 清华大学
Priority date: 2020-07-10
Filing date: 2021-07-08
Publication date: 2022-01-13
Also published as: CN111956797A; CN111956797B

Abstract

The present invention relates to the field of biomedicine, and in particular to a novel vaccine adjuvant and the use thereof in novel coronavirus disease vaccines and other vaccines. A chemically modified cyclic dinucleotide, i.e. an SF compound, can be used as a vaccine adjuvant to be combined with the novel coronavirus disease vaccines.

Description

新型疫苗佐剂及其在新冠肺炎疫苗和其他疫苗中的应用Novel vaccine adjuvants and their applications in COVID-19 vaccines and other vaccines

优先权信息priority information

本申请请求2020年07月10日向中国国家知识产权局提交的、专利申请号为202010663965.4的专利申请的优先权和权益，并且通过参照将其全文并入此处。This application claims the priority and rights of patent application No. 202010663965.4 filed with the State Intellectual Property Office of China on July 10, 2020, and is hereby incorporated by reference in its entirety.

技术领域technical field

本公开涉及生物医药领域，具体涉及新型疫苗佐剂及其在新型冠状病毒肺炎疫苗和其他疫苗中的应用。The present disclosure relates to the field of biomedicine, and in particular to a novel vaccine adjuvant and its application in the novel coronavirus pneumonia vaccine and other vaccines.

背景技术Background technique

新冠疫情仍在全球肆虐，确诊人数逐日攀升，目前已突破千万级别。除了积极研发临床治疗药物与方案，新冠疫苗的开发也成为当前应对新冠疫情重中之重的方向。新冠疫苗的开发策略具体可以分为DNA疫苗、重组蛋白疫苗、腺病毒载体疫苗、灭活疫苗和减活疫苗等。目前，全球已有多款新冠疫苗进入临床实验，其中国内有五款疫苗，成为疫苗研究的主力。这五款疫苗中，四款是灭活疫苗，一款是腺病毒疫苗，表明灭活疫苗是当前研究的主流，也是成功可能性比较高的策略。The new crown epidemic is still raging around the world, and the number of confirmed cases is increasing day by day, and it has now exceeded 10 million. In addition to actively developing clinical therapeutic drugs and programs, the development of a new crown vaccine has also become the most important direction in the current response to the new crown epidemic. The development strategies of new crown vaccines can be divided into DNA vaccines, recombinant protein vaccines, adenovirus vector vaccines, inactivated vaccines and attenuated vaccines. At present, a number of new crown vaccines have entered clinical trials around the world, of which five domestic vaccines have become the main force of vaccine research. Among the five vaccines, four are inactivated vaccines and one is an adenovirus vaccine, indicating that inactivated vaccines are the mainstream of current research and a strategy with a relatively high probability of success.

灭活疫苗的成分由灭活毒株和铝佐剂组成。铝佐剂对于提高灭活疫苗的免疫反应强度非常重要，也是目前少有的被FDA批准可以用于人体疫苗的佐剂。但是铝佐剂自身存在较大缺陷，一是需要高剂量才能引起足够强度的免疫反应，二是铝佐剂的免疫反应类型偏向于体液免疫，对于促进抗原特异性细胞免疫基本没有效果。因此亟需开发新的新冠疫苗佐剂来同时增强疫苗的抗体和细胞免疫反应，可以为机体提供固有和适应性免疫双重保护。The composition of the inactivated vaccine consists of the inactivated strain and an aluminum adjuvant. Aluminum adjuvant is very important to improve the immune response strength of inactivated vaccine, and it is also one of the few adjuvants approved by FDA for human vaccines. However, aluminum adjuvant itself has major defects. First, high doses are required to induce an immune response of sufficient strength. Second, the type of immune response of aluminum adjuvant is biased towards humoral immunity, which is basically ineffective in promoting antigen-specific cellular immunity. Therefore, it is urgent to develop new adjuvants for new coronavirus vaccines to enhance the antibody and cellular immune responses of the vaccine at the same time, which can provide the body with dual protection of innate and adaptive immunity.

发明内容SUMMARY OF THE INVENTION

为解决现有技术中铝佐剂在增强疫苗体液和细胞免疫方面的不足，本公开的发明人首次将STING激动剂环二核苷酸应用于新冠疫苗体系，并证明其显著优于铝佐剂的免疫强化效果。In order to solve the deficiencies of aluminum adjuvant in enhancing vaccine humoral and cellular immunity in the prior art, the inventors of the present disclosure applied the STING agonist cyclic dinucleotide to the new crown vaccine system for the first time, and proved that it is significantly better than aluminum adjuvant immune boosting effect.

对环二核苷酸进行化学修饰提高其稳定性和脂溶性是当前改善其药物特性的重点研发方向之一。其中，磷硫酰化和羟基氟代是最具代表性的化学修饰类型。本公开的发明人通过研究发现，对环二核苷酸进行磷硫酰化可有效提高其抵抗磷酸酯酶的水解；同时进行氟代修饰，氟原子强烈的吸电子特性则可增强环二核苷酸的脂溶性及稳定性。Chemical modification of cyclic dinucleotides to improve their stability and lipid solubility is currently one of the key research and development directions to improve their drug properties. Among them, phosphorylation and hydroxyfluorination are the most representative types of chemical modifications. The inventors of the present disclosure have found through research that phosphorothiolation of cyclic dinucleotides can effectively improve their resistance to hydrolysis by phosphatase; at the same time, fluorine modification, the strong electron-withdrawing properties of fluorine atoms can enhance the cyclic dinuclear Lipid solubility and stability of glucosides.

为了获得经过化学修饰的环二核苷酸，发明人利用液相一瓶法设计和合成了一类新型化学修饰的环二核苷酸(cyclic dinucleotides,CDNs)，其结构式如SF所示。其中碱基可为如A、G等各种天然和非天然类型碱基。所提到的液相一瓶法是指多步反应均在一个圆底烧瓶中进行。In order to obtain chemically modified cyclic dinucleotides, the inventors designed and synthesized a new class of chemically modified cyclic dinucleotides (CDNs) using a liquid-phase one-bottle method, the structural formula of which is shown in SF. The bases can be various natural and non-natural bases such as A, G, etc. The liquid-phase one-flask method mentioned means that the multi-step reactions are all carried out in a round-bottom flask.

后续免疫实验表明SF类化合物SF1(结构式SF中B ₁和B ₂基团均为G)作为新冠疫苗佐剂可提高抗原特异性抗体滴度和T细胞的产生。SF类化合物可作为疫苗佐剂广泛应用于各类肺炎、肿瘤、流感等重组蛋白疫苗、灭活疫苗、减活疫苗、核酸疫苗的佐剂等方面。 Subsequent immunization experiments showed that the SF compound SF1 (both B ₁ and B ₂ groups in the structural formula SF are G) as a new crown vaccine adjuvant can improve antigen-specific antibody titers and T cell production. SF compounds can be widely used as vaccine adjuvants in various recombinant protein vaccines such as pneumonia, tumor, and influenza, inactivated vaccines, attenuated vaccines, and nucleic acid vaccine adjuvants.

具体而言，本公开提供了如下技术方案：Specifically, the present disclosure provides the following technical solutions:

本公开第一方面提供一种化合物在制备疫苗佐剂中的用途。在本公开的一些实施方案中，所述化合物为式SF所示化合物或式SF所示化合物的立体异构体、互变异构体、位置异构体、氮氧化合物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药：A first aspect of the present disclosure provides use of a compound in the preparation of a vaccine adjuvant. In some embodiments of the present disclosure, the compound is a compound represented by formula SF or a stereoisomer, tautomer, positional isomer, nitroxide, hydrate, solvate of the compound represented by formula SF , metabolites, pharmaceutically acceptable salts or prodrugs:

其中，B ₁和B ₂各自独立地选自天然碱基A、T、C、G、U及非天然碱基中的任一种； Wherein, B ₁ and B ₂ are each independently selected from any one of natural bases A, T, C, G, U and unnatural bases;

Y ₁和Y ₂各自独立地选自-OH和-SH中的任一种； Y ₁ and Y ₂ are each independently selected from any one of -OH and -SH;

X ₁和X ₂各自独立地选自-H、-OH和-F中的任一种， X ₁ and X ₂ are each independently selected from any one of -H, -OH and -F,

所述疫苗佐剂为肺炎疫苗或流感疫苗的佐剂。The vaccine adjuvant is an adjuvant for a pneumonia vaccine or an influenza vaccine.

本公开所提供的化合物对环二核苷酸进行磷硫酰化修饰和羟基氟代修饰。通过对环二核苷酸进行磷硫酰化可以有效提高化合物抵抗磷酸酯酶的水解，而且由于氟代修饰，氟原子强烈的吸电子特性可以增强环二核苷酸的脂溶性以及稳定性。The compounds provided by the present disclosure are phosphorothiolated and hydroxyfluorinated on cyclic dinucleotides. Phosphoroylation of cyclic dinucleotides can effectively improve the compound's resistance to hydrolysis by phosphatase, and due to the fluorine modification, the strong electron-withdrawing properties of fluorine atoms can enhance the lipid solubility and stability of cyclic dinucleotides.

本公开提供的SF类化合物通过直接结合免疫细胞中的STING蛋白，从而激活下游干扰素基因通路，产生INF-β及其他促炎细胞因子，促进疫苗抗原的免疫提呈、抗体分泌及T细胞增殖。The SF compounds provided by the present disclosure directly bind to the STING protein in immune cells, thereby activating the downstream interferon gene pathway, producing INF-β and other pro-inflammatory cytokines, and promoting the immune presentation of vaccine antigens, antibody secretion and T cell proliferation. .

根据本公开实施例的化合物在制备疫苗佐剂中的用途，还可以具有以下附加技术特征的至少之一：The use of the compounds according to the embodiments of the present disclosure in the preparation of vaccine adjuvants may also have at least one of the following additional technical features:

在本公开的一些实施方案中，所述式SF所示化合物的位置异构体的结构式为：In some embodiments of the present disclosure, the structural formula of the positional isomer of the compound represented by the formula SF is:

X ₁和X ₂各自独立地选自-H、-OH和-F中的任一种。 X ₁ and X ₂ are each independently selected from any one of -H, -OH and -F.

根据本公开优选的实施例，B ₁和B ₂均为G。 According to a preferred embodiment of the present disclosure, both B ₁ and B ₂ are G.

根据本公开优选的实施例，当B ₁和B ₂均为G时，SF类化合物的化学结构式为： According to a preferred embodiment of the present disclosure, when both B ₁ and B ₂ are G, the chemical structural formula of the SF compound is:

位置异构体是由于取代基或官能团在碳链上或碳环上的位置不同而产生的异构现象，本公开中的用于制备疫苗佐剂的化合物，不仅包括如结构式SF的化合物，还包括结构式SF的化合物的位置异构体。结构式SF的化合物与其位置异构体的化学性质及免疫活性类似。Positional isomers are isomerisms caused by the different positions of substituents or functional groups on the carbon chain or carbocyclic ring. The compounds used for the preparation of vaccine adjuvants in the present disclosure include not only compounds of structural formula SF, but also compounds of structural formula SF. Positional isomers of compounds of formula SF are included. Compounds of structural formula SF are similar in chemical properties and immunological activities to their positional isomers.

在本公开的一些实施方案中，所述非天然碱基选自人造碱基或经修饰的天然碱基，优选地，所述经修饰的天然碱基为I(次黄嘌呤)或mC(5-甲基胞嘧啶)。In some embodiments of the present disclosure, the unnatural base is selected from artificial bases or modified natural bases, preferably, the modified natural bases are I (hypoxanthine) or mC (5 -methylcytosine).

在本公开的一些实施方案中，所述疫苗佐剂为核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、多肽疫苗、灭活疫苗或减活疫苗的佐剂。In some embodiments of the present disclosure, the vaccine adjuvant is an adjuvant for a nucleic acid vaccine, a recombinant protein vaccine, an adenovirus vector vaccine, a polypeptide vaccine, an inactivated vaccine, or a live attenuated vaccine.

在本公开的一些实施方案中，所述核酸疫苗为DNA疫苗和/或RNA疫苗。In some embodiments of the present disclosure, the nucleic acid vaccine is a DNA vaccine and/or an RNA vaccine.

本公开第二方面提供化合物在预防肺炎和/或流感中的用途。在本公开的一些实施方案中，所述化合物为式SF所示化合物或式SF所示化合物的立体异构体、互变异构体、位置异构体、氮氧化合物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药：A second aspect of the present disclosure provides the use of a compound for preventing pneumonia and/or influenza. In some embodiments of the present disclosure, the compound is a compound represented by formula SF or a stereoisomer, tautomer, positional isomer, nitroxide, hydrate, solvate of the compound represented by formula SF , metabolites, pharmaceutically acceptable salts or prodrugs:

在本公开的一些实施方案中，所述非天然碱基选自人造碱基或经修饰的天然碱基，优选地，所述经修饰的天然碱基为I或mC。In some embodiments of the present disclosure, the non-natural base is selected from artificial bases or modified natural bases, preferably, the modified natural base is I or mC.

本公开第三方面提供一种疫苗佐剂。在本公开的一些实施方案中，所述疫苗佐剂含有本公开第一方面所述的化合物，所述疫苗佐剂为肺炎疫苗或流感疫苗的佐剂。A third aspect of the present disclosure provides a vaccine adjuvant. In some embodiments of the present disclosure, the vaccine adjuvant comprises the compound described in the first aspect of the present disclosure, and the vaccine adjuvant is an adjuvant for a pneumonia vaccine or an influenza vaccine.

本公开第四方面提供第三方面所述的疫苗佐剂在预防肺炎和/或流感中的用途。A fourth aspect of the present disclosure provides the use of the vaccine adjuvant of the third aspect for preventing pneumonia and/or influenza.

本公开第五方面提供第三方面所述的疫苗佐剂在制备疫苗组合物中的用途。在本公开的一些实施方案中，所述疫苗组合物包括所述疫苗佐剂及疫苗活性成分。A fifth aspect of the present disclosure provides the use of the vaccine adjuvant of the third aspect in the preparation of a vaccine composition. In some embodiments of the present disclosure, the vaccine composition includes the vaccine adjuvant and a vaccine active ingredient.

在本公开的一些实施方案中，所述活性成分选自核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、多肽疫苗、灭活疫苗或减活疫苗中的至少之一。In some embodiments of the present disclosure, the active ingredient is selected from at least one of nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, polypeptide vaccines, inactivated vaccines or attenuated vaccines.

在本公开的一些实施方案中，所述疫苗活性成分为新型冠状病毒疫苗，所述新型冠状病毒疫苗以SARS-CoV-2的灭活病毒、减活病毒、刺突糖蛋白、包膜糖蛋白、膜糖蛋白、核衣壳蛋白、RBD蛋白、多肽表位以及表达这些蛋白和多肽的核酸序列中的至少之一作为疫苗抗原。In some embodiments of the present disclosure, the active ingredient of the vaccine is a novel coronavirus vaccine, and the novel coronavirus vaccine contains inactivated virus, attenuated virus, spike glycoprotein, envelope glycoprotein of SARS-CoV-2 , at least one of membrane glycoproteins, nucleocapsid proteins, RBD proteins, polypeptide epitopes, and nucleic acid sequences expressing these proteins and polypeptides as vaccine antigens.

在本公开的一些实施方案中，所述疫苗组合物中疫苗抗原与佐剂的用量比为1:10-10:1，优选为1:5-5:1，进一步优选为1:4。In some embodiments of the present disclosure, the dosage ratio of vaccine antigen to adjuvant in the vaccine composition is 1:10-10:1, preferably 1:5-5:1, more preferably 1:4.

在本公开的一些实施方案中，所述疫苗组合物为疫苗制剂，所述疫苗制剂以口服、静脉注射、动脉注射、粘膜给药、鼻腔给药、肌肉注射、皮下注射、器官注射或胸腹腔内注射施用。In some embodiments of the present disclosure, the vaccine composition is a vaccine formulation that is administered orally, intravenously, intraarterally, mucosally, nasally, intramuscularly, subcutaneously, to an organ, or intrathoracically Administration by intramuscular injection.

本公开第六方面提供一种疫苗组合物。在本公开的一些实施方案中，所述疫苗组合物包含本公开第三方面所述的疫苗佐剂及疫苗活性成分。A sixth aspect of the present disclosure provides a vaccine composition. In some embodiments of the present disclosure, the vaccine composition comprises the vaccine adjuvant of the third aspect of the present disclosure and a vaccine active ingredient.

根据本公开实施例的疫苗组合物，还可以具有以下附加技术特征的至少之一：The vaccine composition according to the embodiments of the present disclosure may further have at least one of the following additional technical features:

在本公开的一些实施方案中，所述疫苗活性成分选自核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、多肽疫苗、灭活疫苗或减活疫苗中的至少之一。In some embodiments of the present disclosure, the vaccine active ingredient is selected from at least one of nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, polypeptide vaccines, inactivated vaccines or attenuated vaccines.

在本公开的一些实施方案中，所述疫苗活性成分为新型冠状病毒疫苗，所述新型冠状病毒疫苗以SARS-CoV-2的灭活病毒、减活病毒、刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白)、核衣壳蛋白(N蛋白)、RBD蛋白、多肽表位以及表达这些蛋白和多肽的核酸序列中的至少之一作为疫苗抗原。In some embodiments of the present disclosure, the active ingredient of the vaccine is a novel coronavirus vaccine, and the novel coronavirus vaccine is a SARS-CoV-2 inactivated virus, attenuated virus, spike glycoprotein (S protein), At least one of envelope glycoprotein (E protein), membrane glycoprotein (M protein), nucleocapsid protein (N protein), RBD protein, polypeptide epitopes and nucleic acid sequences expressing these proteins and polypeptides is used as a vaccine antigen.

本公开第七方面提供疫苗组合物在预防肺炎和/或流感中的用途。在本公开的一些实施方案中，所述疫苗组合物为第六方面所述的疫苗组合物。A seventh aspect of the present disclosure provides the use of a vaccine composition for preventing pneumonia and/or influenza. In some embodiments of the present disclosure, the vaccine composition is the vaccine composition of the sixth aspect.

本公开第八方面提供一种预防肺炎和/或流感的方法。在本公开的一些实施方案中，所述方法包括向有预防肺炎和/或流感需求的受试者施用第六方面所述的疫苗组合物。An eighth aspect of the present disclosure provides a method of preventing pneumonia and/or influenza. In some embodiments of the present disclosure, the method comprises administering the vaccine composition of the sixth aspect to a subject in need of prophylaxis against pneumonia and/or influenza.

本公开中的佐剂可以作为肺炎疫苗或流感疫苗的佐剂。肺炎疫苗包括非典(SARS)疫苗、中东呼吸综合征(MERS)疫苗、新冠肺炎(SARS-CoV-2)疫苗等。所述流感疫苗包括甲型流感疫苗、乙型流感疫苗、丙型流感疫苗等。The adjuvants in the present disclosure can be used as adjuvants for pneumonia vaccines or influenza vaccines. Pneumonia vaccines include SARS (SARS) vaccine, Middle East respiratory syndrome (MERS) vaccine, new coronary pneumonia (SARS-CoV-2) vaccine, etc. The influenza vaccine includes influenza A vaccine, influenza B vaccine, influenza C vaccine and the like.

本公开还提供前述化合物的制备方法，包括：The present disclosure also provides methods for preparing the aforementioned compounds, including:

(1)使式S1所示化合物和吡啶-三氟乙酸盐、叔丁胺、二氯乙酸发生脱保护反应，以便获得式S2所示化合物；(1) deprotection reaction occurs between the compound shown in formula S1 and pyridine-trifluoroacetate, tert-butylamine, and dichloroacetic acid, so as to obtain the compound shown in formula S2;

(2)使式S2所示化合物和式S3所示化合物发生磷酰化反应，以便获得式S4所示化合物；(2) phosphorylation of the compound shown in formula S2 and the compound shown in formula S3 to obtain a compound shown in formula S4;

(3)使式S4所示化合物和DDTT、二氯乙酸发生氧化和脱保护反应，以便获得式S5所示化合物；(3) make compound shown in formula S4 and DDTT, dichloroacetic acid take place oxidation and deprotection reaction, in order to obtain compound shown in formula S5;

(4)使式S5所示化合物与环化试剂、氧化剂发生亲核取代和氧化反应，以便获得S6所示化合物；(4) nucleophilic substitution and oxidation reaction of the compound shown in formula S5 with a cyclization reagent and an oxidant, so as to obtain a compound shown in S6;

(5)使式S6所示化合物与叔丁胺发生亲核取代反应，以便获得S7所示化合物；(5) nucleophilic substitution reaction of the compound shown in formula S6 and tert-butylamine is made to obtain the compound shown in S7;

(6)使式S7所示化合物和甲胺和氢氟酸三乙胺盐发生脱保护反应，以便获得SF所示化合物；(6) deprotection reaction occurs between the compound shown in formula S7 and methylamine and triethylamine hydrofluoric acid salt, so as to obtain the compound shown in SF;

其中，式S1所示化合物、式S2所示化合物、式S3所示化合物、式S4所示化合物、式S5所示化合物、式S6所示化合物分别如下所示：Wherein, the compound represented by the formula S1, the compound represented by the formula S2, the compound represented by the formula S3, the compound represented by the formula S4, the compound represented by the formula S5, and the compound represented by the formula S6 are respectively as follows:

其中各化合物中L1，L2各自独立地选自

等碱基保护基； wherein L1 and L2 in each compound are independently selected from

isobasic protecting groups;

Z选自硅烷保护的羟基、无保护羟基、F原子以及H原子；Z is selected from the group consisting of silane-protected hydroxyl groups, unprotected hydroxyl groups, F atoms and H atoms;

在本公开的一些实施方案中，所述硅烷保护的羟基选自三甲基硅醚羟基、叔丁基二甲基硅醚羟基、叔丁基二苯基硅醚羟基。In some embodiments of the present disclosure, the silane-protected hydroxy group is selected from the group consisting of trimethylsilyl ether hydroxy, tert-butyldimethylsilyl ether hydroxy, tert-butyldiphenylsilyl ether hydroxy.

在本公开的一些实施方案中，所述环化试剂为5,5-二甲基-2-氯-1,3,2-二氧磷杂己内酰磷酸酯；In some embodiments of the present disclosure, the cyclizing reagent is 5,5-dimethyl-2-chloro-1,3,2-dioxaphosphacaprolactone phosphate;

所述氧化剂为选自碘或3H-1,2-苯并二硫醇-3-酮1,1-二氧化物中的至少一种。The oxidizing agent is at least one selected from iodine or 3H-1,2-benzodithiol-3-one 1,1-dioxide.

在本公开的一些实施方案中，步骤(1)中所述脱保护反应在室温条件下进行。In some embodiments of the present disclosure, the deprotection reaction in step (1) is performed at room temperature.

在本公开的一些实施方案中，步骤(2)中所述磷酰化反应在无水条件下进行；In some embodiments of the present disclosure, the phosphorylation reaction in step (2) is carried out under anhydrous conditions;

在本公开的一些实施方案中，步骤(3)中所述氧化和脱保护反应在室温条件下进行；In some embodiments of the present disclosure, the oxidation and deprotection reactions described in step (3) are carried out at room temperature;

在本公开的一些实施方案中，步骤(4)中所述亲核取代和氧化反应在室温条件下进行；In some embodiments of the present disclosure, the nucleophilic substitution and oxidation reactions described in step (4) are performed at room temperature;

在本公开的一些实施方案中，步骤(5)中所述亲核取代反应在室温条件下进行；In some embodiments of the present disclosure, the nucleophilic substitution reaction in step (5) is performed at room temperature;

在本公开的一些实施方案中，步骤(6)中所述脱保护反应在油浴50℃条件下进行。In some embodiments of the present disclosure, the deprotection reaction in step (6) is performed in an oil bath at 50°C.

新冠疫苗的开发策略具体可以分为DNA疫苗、重组蛋白疫苗、腺病毒载体疫苗、灭活疫苗和减活疫苗等。核酸疫苗，重组蛋白疫苗在往常研究中具有安全性，但其免疫原性不足，需要进行佐剂筛选以产生高效的体液和细胞免疫。腺病毒载体疫苗在冠状病毒疫苗开发中具有较大优势，然而大范围的人群接种较少，如何实现高效力的同时避免预存免疫也是目前新冠病毒开发的重大挑战。减活疫苗虽然具有高的免疫原性，但也同时有毒力恢复的风险，目前还没有证据表明减毒COVID-19的毒性不会返强。The development strategies of new crown vaccines can be divided into DNA vaccines, recombinant protein vaccines, adenovirus vector vaccines, inactivated vaccines and attenuated vaccines. Nucleic acid vaccines and recombinant protein vaccines are safe in previous studies, but their immunogenicity is insufficient, and adjuvant screening is required to generate efficient humoral and cellular immunity. Adenovirus vector vaccines have great advantages in the development of coronavirus vaccines, but a large number of people are less vaccinated. How to achieve high efficacy and avoid pre-existing immunity is also a major challenge for the development of new coronaviruses. Although live attenuated vaccines are highly immunogenic, they also have the risk of virulence recovery, and there is currently no evidence that the virulence of attenuated COVID-19 will not return to strong.

灭活疫苗是最为经典的疫苗形式。灭活疫苗的成分由灭活毒株和铝佐剂组成。然而铝佐剂需要高剂量才能引起足够强度的免疫反应，且其免疫反应类型偏向于体液免疫，对于促进抗原特异性细胞免疫基本没有效果。在以往SARS疫苗研究中，人们发现低效力的中和抗体及非中和抗体诱导往往会导致抗体依赖感染增强效应，从而扩大了病毒的感染。按照目前的临床研究来看，除了中和抗体以外，抗原特异性CD8+T细胞对于清除被感染的细胞和新冠病毒的作用也至关重要。CD8+T细胞可以通过清除被感染的肺细胞来显著降低由于细胞因子过量释放引起的肺部炎症损伤，为机体预防新冠感染提供细胞免疫防护。Inactivated vaccines are the most classic form of vaccines. The composition of the inactivated vaccine consists of the inactivated strain and an aluminum adjuvant. However, high doses of aluminum adjuvants are required to induce a sufficiently strong immune response, and the type of immune response is biased towards humoral immunity, which is basically ineffective in promoting antigen-specific cellular immunity. In previous SARS vaccine studies, it was found that the induction of neutralizing antibodies and non-neutralizing antibodies with low potency often resulted in antibody-dependent infection-enhancing effects, thereby expanding the infection of the virus. According to current clinical studies, in addition to neutralizing antibodies, antigen-specific CD8+ T cells are also crucial for clearing infected cells and the new coronavirus. CD8+ T cells can significantly reduce lung inflammation damage caused by excessive cytokine release by removing infected lung cells, and provide cellular immune protection for the body to prevent new coronary infection.

本公开的发明人首次将STING激动剂环二核苷酸应用于新冠疫苗体系，并证明其显著优于铝佐剂的免疫强化效果。为了获得经过化学修饰的环二核苷酸，发明人利用液相一瓶法设计和合成了一类新型化学修饰的环二核苷酸(cyclic dinucleotides,CDNs)。后续免疫实验表明SF类化合物SF1作为新冠重组S蛋白疫苗佐剂可提高抗原特异性抗体滴度和T细胞的产生。本公开提供的SF类化合物通过直接结合免疫细胞中的STING蛋白，从而激活下游干扰素基因通路，产生INF-β及其他促炎细胞因子，促进疫苗抗原的免疫提呈、抗体分泌及T细胞增殖。SF类化合物可作为疫苗佐剂广泛应用于各类肺炎、肿瘤、流感等重组蛋白疫苗、灭活疫苗的佐剂等方面。The inventors of the present disclosure applied the STING agonist cyclic dinucleotide to the new crown vaccine system for the first time, and proved that it is significantly superior to the immune enhancement effect of aluminum adjuvant. In order to obtain chemically modified cyclic dinucleotides, the inventors designed and synthesized a new class of chemically modified cyclic dinucleotides (CDNs) using a liquid-phase one-bottle method. Subsequent immunization experiments showed that the SF compound SF1, as an adjuvant for the new crown recombinant S protein vaccine, can improve the antigen-specific antibody titer and the production of T cells. The SF compounds provided by the present disclosure directly bind to the STING protein in immune cells, thereby activating the downstream interferon gene pathway, producing INF-β and other pro-inflammatory cytokines, and promoting the immune presentation of vaccine antigens, antibody secretion and T cell proliferation. . SF compounds can be widely used as vaccine adjuvants in various types of pneumonia, tumor, influenza and other recombinant protein vaccines, adjuvants for inactivated vaccines, etc.

本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。Additional aspects and advantages of the present disclosure will be set forth, in part, from the following description, and in part will become apparent from the following description, or may be learned by practice of the present disclosure.

附图说明Description of drawings

本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present disclosure will become apparent and readily understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein:

图1显示了SF类化合物SF1的合成过程；Figure 1 shows the synthesis process of the SF compound SF1;

图2显示了分别向Babl/c小鼠施用S蛋白抗原、S蛋白抗原和SF1佐剂、S蛋白抗原和铝佐剂以及空白对照直到取样的时间进程，其中，D0、D14、D28、D35分别代表第0天、第14天、第28天、第35天；Figure 2 shows the time course of administration of S protein antigen, S protein antigen and SF1 adjuvant, S protein antigen and aluminum adjuvant, and blank control to Babl/c mice, respectively, until sampling, wherein D0, D14, D28, D35, respectively Represents Day 0, Day 14, Day 28, Day 35;

图3A显示了空白对照组(Blank)、S蛋白抗原组(S)、S蛋白抗原+SF1佐剂组(S+SF1)、S蛋白抗原+铝佐剂组(S+Alum)脾脏的形态；Figure 3A shows the spleen morphology of blank control group (Blank), S protein antigen group (S), S protein antigen + SF1 adjuvant group (S + SF1), and S protein antigen + aluminum adjuvant group (S + Alum);

图3B显示了空白对照组(Blank)、S蛋白抗原组(S)、S蛋白抗原+SF1佐剂组(S+SF1)、S蛋白抗原+铝佐剂组(S+Alum)脾脏的重量统计结果；Figure 3B shows the weight statistics of the spleen in the blank control group (Blank), the S protein antigen group (S), the S protein antigen + SF1 adjuvant group (S + SF1), and the S protein antigen + aluminum adjuvant group (S + Alum). result;

图4A显示了空白对照组(Blank)、S蛋白抗原组(S)、S蛋白抗原+SF1佐剂组(S+SF1)、S蛋白抗原+铝佐剂组(S+Alum)取脾脏细胞进行酶联免疫吸附斑点测定的结果；Figure 4A shows the blank control group (Blank), S protein antigen group (S), S protein antigen + SF1 adjuvant group (S + SF1), S protein antigen + aluminum adjuvant group (S + Alum) to take spleen cells for Results of ELISA spot assay;

图4B显示了空白对照组(Blank)、S蛋白抗原组(S)、S蛋白抗原+SF1佐剂组(S+SF1)、S蛋白抗原+铝佐剂组(S+Alum)脾脏细胞IFN-γ斑点计数结果；Figure 4B shows the blank control group (Blank), S protein antigen group (S), S protein antigen + SF1 adjuvant group (S + SF1), S protein antigen + aluminum adjuvant group (S + Alum) spleen cells IFN- gamma spot count results;

图5显示了S蛋白抗原组(S)、S蛋白抗原+SF1佐剂组(S+SF1)、S蛋白抗原+铝佐剂组(S+Alum)SARS-CoV-2膜外域S蛋白抗原的IgG抗体滴度。Figure 5 shows the S protein antigen group (S), S protein antigen + SF1 adjuvant group (S + SF1), S protein antigen + aluminum adjuvant group (S + Alum) SARS-CoV-2 membrane ectodomain S protein antigen IgG antibody titers.

发明详细描述Detailed description of the invention

现在详细描述本公开的某些实施方案，其实例由随附的结构式和化学式说明。本公开意图涵盖所有的替代、修改和等同技术方案，它们均包括在如权利要求定义的本公开范围内。本领域技术人员应认识到，许多与本文所述类似或等同的方法和材料能够用于实践本公开。本公开绝不限于本文所述的方法和材料。在所结合的文献、专利和类似材料的一篇或多篇与本申请不同或相矛盾的情况下(包括但不限于所定义的术语、术语应用、所描述的技术，等等)，以本申请为准。Certain embodiments of the present disclosure will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulae. This disclosure is intended to cover all alternatives, modifications and equivalents, which are included within the scope of this disclosure as defined by the claims. One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application (including, but not limited to, terms defined, uses of terms, techniques described, etc.), this Application shall prevail.

应进一步认识到，本公开的某些特征，为清楚可见，在多个独立的实施方案中进行了描述，但也可以在单个实施例中以组合形式提供。反之，本公开的各种特征，为简洁起见，在单个实施方案中进行了描述，但也可以单独或以任意适合的子组合提供。It should further be appreciated that certain features of the present disclosure, which are, for clarity, described in the context of multiple separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

除非另外说明，本公开所使用的所有科技术语具有与本公开所属领域技术人员的通常理解相同的含义。本公开涉及的所有专利和公开出版物通过引用方式整体并入本公开。Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications referred to in this disclosure are incorporated by reference in their entirety.

定义或一般术语Definition or General Terms

术语“包含”、“包括”为开放式表达，即包括本公开所指明的内容，但并不排除其他方面的内容。The terms "comprising" and "including" are open-ended expressions, that is, the contents specified in the present disclosure are included, but other aspects are not excluded.

“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。立体异构体包括对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体(顺/反)异构体、阻转异构体，等等。"Stereoisomers" refer to compounds that have the same chemical structure, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans), atropisomers, etc. .

“对映异构体”是指一个化合物的两个不能重叠但互成镜像关系的异构体。"Enantiomer" refers to two nonsuperimposable, but mirror-image isomers of a compound.

“非对映异构体”是指有两个或多个手性中心并且其分子不互为镜像的立体异构体。非对映异构体具有不同的物理性质，如熔点、沸点、光谱性质和反应性。非对映异构体混合物可通过高分辨分析操作如电泳和色谱，例如HPLC来分离。"Diastereomer" refers to a stereoisomer having two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactivity. Diastereomeric mixtures can be separated by high resolution analytical procedures such as electrophoresis and chromatography, eg HPLC.

“手性”是具有与其镜像不能重叠性质的分子；而“非手性”是指与其镜像可以重叠的分子。"Chiral" is a molecule that has the property of being non-superimposable with its mirror image; while "achiral" refers to a molecule that is superimposable with its mirror image.

本公开所使用的立体化学定义和规则一般遵循S.P.Parker，Ed.，McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company，New York；and Eliel，E.and Wilen，S.，“Stereochemistry of Organic Compounds”，John Wiley&Sons，Inc.，New York，1994。Stereochemistry definitions and rules used in this disclosure generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

许多有机化合物以光学活性形式存在，即它们具有使平面偏振光的平面发生旋转的能力。在描述光学活性化合物时，使用前缀D和L或R和S来表示分子关于其一个或多个手性中心的绝对构型。前缀d和l或(+)和(-)是用于指定化合物所致平面偏振光旋转的符号，其中(-)或l表示化合物是左旋的。前缀为(+)或d的化合物是右旋的。一种具体的立体异构体是对映异构体，这种异构体的混合物称作对映异构体混合物。对映异构体的50:50混合物称为外消旋混合物或外消旋体，当在化学反应或过程中没有立体选择性或立体特异性时，可出现这种情况。Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about one or more of its chiral centers. The prefixes d and 1 or (+) and (-) are symbols used to designate the rotation of plane polarized light by the compound, where (-) or 1 indicates that the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. A specific stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

本公开公开化合物的任何不对称原子(例如，碳等)都可以以外消旋或对映体富集的形式存在，例如(R)-、(S)-或(R,S)-构型形式存在。在某些实施方案中，各不对称原子在(R)-或(S)-构型方面具有至少50％对映体过量，至少60％对映体过量，至少70％对映体过量，至少80％对映体过量，至少90％对映体过量，至少95％对映体过量，或至少99％对映体过量。Any asymmetric atom (eg, carbon, etc.) of the compounds of the present disclosure may exist in racemic or enantiomerically enriched forms, such as (R)-, (S)- or (R,S)-configurations exist. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 70% enantiomeric excess in the (R)- or (S)-configuration 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.

依据起始物料和方法的选择，本公开化合物可以以可能的异构体中的一个或它们的混合物，例如外消旋体和非对应异构体混合物(这取决于不对称碳原子的数量)的形式存在。光学活性的(R)-或(S)-异构体可使用手性合成子或手性试剂制备，或使用常规技术拆分。如果化合物含有一个双键，取代基可能为E或Z构型；如果化合物中含有二取代的环烷基，环烷基的取代基可能有顺式或反式构型。Depending on the choice of starting materials and methods, the compounds of the present disclosure may be present as one of the possible isomers or as mixtures thereof, such as racemates and mixtures of diastereomers (depending on the number of asymmetric carbon atoms) form exists. Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have the cis or trans configuration.

所得的任何立体异构体的混合物可以依据组分物理化学性质上的差异被分离成纯的或基本纯的几何异构体，对映异构体，非对映异构体，例如，通过色谱法和/或分步结晶法。The resulting mixtures of any stereoisomers may be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers on the basis of differences in the physicochemical properties of the components, for example, by chromatography and/or fractional crystallization.

可以用已知的方法将任何所得终产物或中间体的外消旋体通过本领域技术人员熟悉的方法拆分成光学对映体，如，通过对获得的其非对映异构的盐进行分离。外消旋的产物也可以通过手性色谱来分离，如，使用手性吸附剂的高效液相色谱(HPLC)。特别地，对映异构体可以通过不对称合成制备，例如，可参考Jacques,et al.,Enantiomers,Racemates and Resolutions(Wiley Interscience,New York,1981)；Principles of Asymmetric Synthesis(2 ^nd Ed. Robert E.Gawley,Jeffrey Aubé,Elsevier,Oxford,UK,2012)；Eliel,E.L.Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962)；Wilen,S.H.Tables of Resolving Agents and Optical Resolutions p.268(E.L.Eliel,Ed.,Univ.of Notre Dame Press,Notre Dame,IN 1972)；Chiral Separation Techniques:A Practical Approach(Subramanian,G.Ed.,Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim,Germany,2007)。 Any resulting racemate of the final product or intermediate can be resolved into the optical enantiomers by known methods by methods familiar to those skilled in the art, eg, by performing diastereomeric salts thereof on the resulting salts. separation. Racemic products can also be separated by chiral chromatography, eg, high performance liquid chromatography (HPLC) using chiral adsorbents. In particular, enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 ^nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SH Tables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

术语“互变异构体”或“互变异构形式”是指具有不同能量的可通过低能垒(low energy barrier)互相转化的结构异构体。若互变异构是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(protontautomer)(也称为质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组来进行的互相转化。酮-烯醇互变异构的具体实例是戊烷-2,4-二酮和4-羟基戊-3-烯-2-酮互变异构体的互变。互变异构的另一个实例是酚-酮互变异构。酚-酮互变异构的一个具体实例是吡啶-4-醇和吡啶-4(1H)-酮互变异构体的互变。除非另外指出，本公开化合物的所有互变异构体形式都在本公开的范围之内。The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that are interconvertible through a low energy barrier. A chemical equilibrium of tautomers can be achieved if tautomerism is possible (eg, in solution). For example, protontautomers (also known as prototropic tautomers) include interconversions by migration of protons, such as keto-enol isomerization and imine-ene Amine isomerization. Valence tautomers include interconversions by recombination of some of the bonding electrons. A specific example of keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-ketone tautomerism. A specific example of phenol-ketone tautomerism is the interconversion of pyridin-4-ol and pyridin-4(lH)-one tautomers. Unless otherwise indicated, all tautomeric forms of the disclosed compounds are within the scope of this disclosure.

“位置异构体”是由于取代基或官能团在碳链上或碳环上的位置不同而产生的异构现象，本公开中的用于制备疫苗佐剂的化合物，不仅包括如结构式SF的化合物，还包括结构式SF的化合物的位置异构体。"Positional isomers" are the isomerism caused by the different positions of substituents or functional groups on the carbon chain or carbocyclic ring. The compounds used for the preparation of vaccine adjuvants in the present disclosure not only include compounds of structural formula SF , and also includes positional isomers of compounds of formula SF.

在本说明书的各部分，本公开公开化合物的取代基按照基团种类或范围公开。特别指出，本公开包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C _1～6烷基”特别指各自独立的甲基、乙基、C ₃烷基、C ₄烷基、C ₅烷基和C ₆烷基。 In various parts of this specification, substituents of compounds disclosed in the present disclosure are disclosed in terms of group type or scope. Specifically, this disclosure includes each individual subcombination of each member of these group species and ranges. For example, the term "C _{1 ~ 6} alkyl group" refers particularly each independently methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C ₆ alkyl.

在本公开的各部分，描述了连接取代基。当该结构清楚地需要连接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”，则应该理解，该“烷基”分别代表连接的亚烷基基团或亚芳基基团。In various sections of this disclosure, linking substituents are described. When the structure clearly requires a linking group, the Markush variables listed for that group should be understood to be the linking group. For example, if the structure requires a linking group and an "alkyl" group is listed for the definition of a Markush group for that variable, it should be understood that the "alkyl" represents the attached alkylene group or arylene group, respectively group.

另外，需要说明的是，除非以其他方式明确指出，在本文中通篇采用的描述方式“各…和…独立地为”、“…和…各自独立地为”和“…和…分别独立地为”可以互换，应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。In addition, it should be noted that, unless expressly stated otherwise, the descriptions used throughout this document "each ... and ... are independently", "... and ... are each independently" and "... and ... are each independently "" can be interchanged and should be understood in a broad sense. It can either mean that in different groups, the specific options expressed by the same symbol do not affect each other, or it can mean that in the same group, the same symbol is the same. The specific options expressed between them do not affect each other.

术语“药学上可接受的”是指当给人施用时生理上可耐受的并且一般不产生过敏或相似不适当的反应，例如肠胃不适、眩晕等的分子实体和组合物。优选地，本文所用的术语“药学上可接受的”是指联邦监管机构或国家政府批准的或美国药典或其他一般认可的药典上列举的在动物中、更特别在人体中使用的。The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergic or similar inappropriate reactions, such as gastrointestinal upset, dizziness, and the like. Preferably, the term "pharmaceutically acceptable" as used herein means approved by a federal regulatory agency or a national government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly in humans.

本公开中立体化学的定义和惯例的使用通常参考以下文献：S.P.Parker,Ed.,McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company,New York；and Eliel,E.and Wilen,S.,"Stereochemistry of Organic Compounds",John Wiley&Sons,Inc.,New York,1994.本公开的化合物可以包含不对称中心或手性中心，因此存在不同的立体异构体。本公开的化合物所有的立体异构形式，包括但绝不限于，非对映体，对映异构体，阻转异构体，和它们的混合物，如外消旋混合物，组成了本公开的一部分。很多有机化合物都以光学活性形式存在，即它们有能力旋转平面偏振光的平面。在描述光学活性化合物时，前缀D、L或R、S用来表示分子手性中心的绝对构型。前缀d、l或(+)、(-)用来命名化合物平面偏振光旋转的符号，(-)或l是指化合物是左旋的，前缀(+)或d是指化合物是右旋的。这些立体异构体的化学结构是相同的，但是它们的立体结构不一样。特定的立体异构体可以是对映体，异构体的混合物通常称为对映异构体混合物。50:50的对映体混合物被称为外消旋混合物或外消旋体，这可能导致化学反应过程中没有立体选择性或立体定向性。术语“外消旋混合物”和“外消旋体”是指等摩尔的两个对映异构体的混合物，缺乏光学活性。Definitions and conventions for stereochemistry in this disclosure are generally referenced by SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S. ., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present disclosure may contain asymmetric centers or chiral centers and therefore exist as different stereoisomers. All stereoisomeric forms of the compounds of the present disclosure, including, but not limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, constitute the part. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D, L or R, S are used to denote the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to designate the sign of the plane-polarized light rotation of the compound, (-) or l means the compound is levorotatory, and the prefix (+) or d means the compound is dextrorotatory. The chemical structures of these stereoisomers are the same, but their steric structures are not the same. A specific stereoisomer may be an enantiomer, and a mixture of isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can result in no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, devoid of optical activity.

“异构体”为具有相同分子式的不同化合物。“立体异构体”为仅仅原子的空间排列方式不同的异构体。如本文使用的术语“异构体”包括任何和所有的几何异构体和立体异构体。例如，“异构体”包括顺式和反式异构体、E-和Z-异构体、R-和S-对映异构体、非对映异构体、(d)异构体、(l)-异构体、其外消旋混合物、及落入本说明书范围的其它其混合物。"Isomers" are different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the arrangement of the atoms in space. The term "isomer" as used herein includes any and all geometric and stereoisomers. For example, "isomers" include cis and trans isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (d) isomers , (l)-isomers, racemic mixtures thereof, and other mixtures thereof falling within the scope of this specification.

本公开的“水合物”是指本公开所提供的化合物或其盐，其还包括化学量或非化学当量通过非共价分子间力结合的水，也可说是溶剂分子是水所形成的缔合物。The "hydrate" of the present disclosure refers to the compound or its salt provided by the present disclosure, which also includes water bound by non-covalent intermolecular force in a stoichiometric or non-stoichiometric amount, or it can be said that the solvent molecule is formed by water associates.

本公开的“溶剂化物”是指一个或多个溶剂分子与本公开的化合物所形成的缔合物。形成溶剂化物的溶剂包括，但并不限于，水，异丙醇，乙醇，甲醇，二甲亚砜，乙酸乙酯，乙酸，氨基乙醇。A "solvate" of the present disclosure refers to an association of one or more solvent molecules with a compound of the present disclosure. Solvate-forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.

本公开的“氮氧化物”是指当化合物含几个胺官能团时，可将1个或大于1个的氮原子氧化形成N-氧化物。N-氧化物的特殊实例是叔胺的N-氧化物或含氮杂环氮原子的N-氧化物。可用氧化剂例如过氧化氢或过酸(例如过氧羧酸)处理相应的胺形成N-氧化物(参见Advanced Organic Chemistry,Wiley Interscience,第4版,Jerry March,pages)。尤其是，N-氧化物可用L.W.Deady的方法制备(Syn.Comm.1977,7,509-514)，其中例如在惰性溶剂例如二氯甲烷中，使胺化合物与间-氯过氧苯甲酸(MCPBA)反应。"Nitrogen oxide" in the present disclosure means that when the compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form an N-oxide. Particular examples of N-oxides are N-oxides of tertiary amines or N-oxides containing nitrogen heterocyclic nitrogen atoms. The corresponding amines can be treated with oxidizing agents such as hydrogen peroxide or peracids (eg, peroxycarboxylic acids) to form N-oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared by the method of LWDeady (Syn. Comm. 1977, 7, 509-514) in which, for example, an amine compound is combined with m-chloroperoxybenzoic acid (MCPBA) in an inert solvent such as dichloromethane. reaction.

化合物可存在多种不同几何异构体和互变异构体，所述式(I)-式(III)化合物包括所有此类形式。为避免疑惑，当化合物以几种几何异构体或互变异构体之一存在并且只具体描述或显示一种时，显然所有其它形式包括在式(I)-式(III)中。Compounds may exist in a number of different geometric isomers and tautomers, and the compounds of formula (I)-(III) include all such forms. For the avoidance of doubt, when a compound exists as one of several geometric isomers or tautomers and only one is specifically described or shown, it is clear that all other forms are included in formula (I)-(III).

本公开所使用的术语“前药”，代表一个化合物在体内转化为本公开所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。As used in this disclosure, the term "prodrug" refers to the in vivo conversion of a compound to the compound shown in this disclosure. Such conversion is effected by hydrolysis of the prodrug in blood or enzymatic conversion to the parent structure in blood or tissue.

除非其他方面表明，本公开的化合物的所有互变异构形式都包含在本公开的范围之内。Unless otherwise indicated, all tautomeric forms of the compounds of the present disclosure are included within the scope of the present disclosure.

“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本公开所描述的那样采用试验的方法进行表征。这样的产物可以是通过给药化合物经过氧化，还原，水解，酰氨化，脱酰氨作用，酯化，脱脂作用，酶裂解等等方法得到。相应地，本公开包括化合物的代谢产物，包括将本公开的化合物与哺乳动物充分接触一段时间所产生的代谢产物。"Metabolite" refers to a product obtained by metabolism of a specific compound or salt thereof in vivo. Metabolites of a compound can be identified by techniques well known in the art, and their activity can be characterized using experimental methods as described in this disclosure. Such products may be obtained by subjecting the administered compound to oxidation, reduction, hydrolysis, amidation, deamidation, esterification, delipidation, enzymatic cleavage, and the like. Accordingly, the present disclosure includes metabolites of compounds, including metabolites produced by contacting a compound of the present disclosure with a mammal for a sufficient period of time.

本公开化合物的各种药学上可接受的盐形式都是有用的。术语“药学上可接受的盐”是指那些盐形式对于制药化学家而言是显而易见的，即它们基本上无毒并能提供所需的药代动力学性质、适口性、吸收、分布、代谢或***。其他因素，在性质上更加实用，对于选择也很重要，这些是：原材料的成本、结晶的容易、产率、稳定性、吸湿性和结果原料药的流动性。简单地讲，药物组合物可以通过有效成分与药学上可接受的载体制备得到。Various pharmaceutically acceptable salt forms of the compounds of the present disclosure are useful. The term "pharmaceutically acceptable salts" refers to those salt forms that are obvious to the pharmaceutical chemist, ie they are substantially non-toxic and provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion. Other factors, more practical in nature, are also important for selection, these are: cost of raw materials, ease of crystallization, yield, stability, hygroscopicity and resulting drug substance flowability. Briefly, pharmaceutical compositions can be prepared by using active ingredients and pharmaceutically acceptable carriers.

本公开所使用的“药学上可接受的盐”是指本公开的化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，如文献：S.M.Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences,66:1-19,1977.所记载的。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，硝酸盐等，和有机酸盐如乙酸盐，丙酸盐，乙醇酸盐，草酸盐，马来酸盐，丙二酸盐，琥珀酸盐，富马酸盐，酒石酸盐，枸橼酸盐，苯甲酸盐，扁桃酸盐，甲磺酸盐，乙磺酸盐，甲苯磺酸盐，磺基水杨酸盐等，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。As used in this disclosure, "pharmaceutically acceptable salts" refer to both organic and inorganic salts of the compounds of this disclosure. Pharmaceutically acceptable salts are well known in the art, as described in the literature: S.M.Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19, 1977. Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, inorganic acid salts formed by reaction with amino groups including hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, Nitrates, etc., and organic acid salts such as acetate, propionate, glycolate, oxalate, maleate, malonate, succinate, fumarate, tartrate, citric acid Salts, benzoates, mandelates, methanesulfonates, ethanesulfonates, toluenesulfonates, sulfosalicylates, etc., or obtained by other methods such as ion exchange methods described in books and literature these salts.

其他药学上可接受的盐包括己二酸盐、苹果酸盐、2-羟基丙酸、藻酸盐、抗坏血酸盐、天冬氨酸盐、苯磺酸盐、苯甲酸盐、重硫酸盐、硼酸盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、环戊基丙酸盐、二葡萄糖酸盐、十二烷基硫酸盐、乙磺酸盐、甲酸盐、反丁烯二酸盐、葡庚糖酸盐、甘油磷酸盐、葡萄糖酸盐、半硫酸盐、庚酸盐、己酸盐、氢碘酸盐、2-羟基-乙磺酸盐、乳糖醛酸盐、乳酸盐、月桂酸盐、月桂基硫酸盐、苹果酸盐、丙二酸盐、甲磺酸盐、2-萘磺酸盐、烟酸盐、硝酸盐、油酸盐、棕榈酸盐、扑酸盐、果胶酸盐、过硫酸盐、3-苯基丙酸盐、苦味酸盐、特戊酸盐、丙酸盐、硬脂酸盐、硫氰酸盐、对甲苯磺酸盐、十一酸盐、戊酸盐、等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N ⁺(C _1-4烷基) ₄的盐。 Other pharmaceutically acceptable salts include adipate, malate, 2-hydroxypropionic acid, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, Borate, Butyrate, Camphorate, Camphorsulfonate, Cyclopentylpropionate, Digluconate, Lauryl Sulfate, Ethanesulfonate, Formate, Fumaric Acid Salt, Glucoheptonate, Glycerophosphate, Gluconate, Hemisulfate, Heptanoate, Caproate, Hydroiodide, 2-Hydroxy-ethanesulfonate, Lacturonate, Lactate , laurate, lauryl sulfate, malate, malonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, Pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate , valerate, etc. Salts obtained with appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts.

本公开也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产物可以通过季铵化作用得到。碱金属或碱土金属盐包括钠盐、锂盐、钾盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等等。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C _1-8磺酸化物和芳香磺酸化物。胺盐，例如但不限于N,N’-二苄基乙二胺，氯普鲁卡因，胆碱，氨，二乙醇胺和其它羟烷基胺，乙二胺，N-甲基还原葡糖胺，普鲁卡因，N-苄基苯乙胺，1-对-氯苄基-2-吡咯烷-1’-基甲基-苯并咪唑，二乙胺和其它烷基胺，哌嗪和三(羟甲基)氨基甲烷；碱土金属盐，例如但不限于钡，钙和镁；过渡金属盐，例如但不限于锌。 The present disclosure also contemplates the quaternary ammonium salts formed by any compound containing an N-containing group. Water- or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Pharmaceutically acceptable salts further comprise suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion, such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C _{1 -8} Sulfonates and aromatic sulfonates. Amine salts such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylreduced glucose Amines, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkaline earth metal salts such as but not limited to barium, calcium and magnesium; transition metal salts such as but not limited to zinc.

在本说明书中，如果在化学名称和化学结构间存在任何差异，结构是占优的。In this specification, if there is any difference between chemical name and chemical structure, the structure will prevail.

本公开所使用的任何保护基团、氨基酸和其它化合物的缩写，除非另有说明，都以它们通常使用的、公认的缩写为准，或参照IUPAC-IUB Commission on Biochemical Nomenclature(参见Biochem.1972，11：942-944)。Any abbreviations for protecting groups, amino acids and other compounds used in this disclosure, unless otherwise stated, are based on their commonly used, recognized abbreviations, or by reference to the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 1972, 11:942-944).

下面将结合实施例对本公开的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照本领域常用的技术手段进行。The solutions of the present disclosure will be explained below with reference to the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the technical means commonly used in the field is used.

以下给出的实施例采用鸟苷酸进行验证。实验过程中合成CDN(SF)1(缩写为SF1)。SF1为同侧单硫单氟修饰环二核苷酸。后续免疫实验表明SF类化合物SF1作为新冠重组S蛋白疫苗佐剂可提高抗原特异性抗体滴度和T细胞的产生。SF类化合物可作为疫苗佐剂广泛应用于各类肺炎、肿瘤、流感等重组蛋白疫苗、灭活疫苗的佐剂等方面。The examples given below were validated using guanylic acid. CDN(SF)1 (abbreviated as SF1) was synthesized during the experiment. SF1 is a monosulfur-monofluorine-modified cyclic dinucleotide on the same side. Subsequent immunization experiments showed that the SF compound SF1, as an adjuvant for the new crown recombinant S protein vaccine, can improve the antigen-specific antibody titer and the production of T cells. SF compounds can be widely used as vaccine adjuvants in various types of pneumonia, tumor, influenza and other recombinant protein vaccines, adjuvants for inactivated vaccines, etc.

实施例1 SF1的合成Example 1 Synthesis of SF1

本实施例以鸟苷酸作为示例，合成了SF1。SF1为同侧单硫单氟修饰的环二核苷酸，其制备方法如下，具体步骤可以参考附图1反应式：In this example, guanylate was used as an example to synthesize SF1. SF1 is a cyclic dinucleotide modified with monosulfur and monofluorine on the same side.

化合物D2Compound D2

称取0.5mmol鸟苷酸亚磷酰胺单体(S1)和0.116g(0.6mmol)吡啶三氟乙酸盐(pyridine-TFA)放入50mL圆底烧瓶中，加入磁子和2.5mL乙腈搅拌溶解，再加入18μL水搅拌反应1min。之后加入2.5mL t-BuNH ₂搅拌反应10min。反应结束后负压旋除溶剂至蓬松状，再加入5mL乙腈旋至蓬松状，重复操作两次除去t-BuNH ₂。加入6mL二氯甲烷(DCM)溶解固体，之后加入90μL水和6mL 6％二氯乙酸(DCA)的DCM溶液(4.4mmol)搅拌反应10min，中途取样进行ESI-MS检测判断DMTr保护基脱除情况。加入0.7mL吡啶中和DCA，负压浓缩至2mL，再加入4mL无水乙腈溶解样品，负压旋除溶剂，重复操作三次，最后一次剩余1mL时停止旋蒸，使用橡胶塞塞住待用。 Weigh 0.5mmol of guanylate phosphoramidite monomer (S1) and 0.116g (0.6mmol) of pyridine trifluoroacetate (pyridine-TFA) into a 50mL round-bottom flask, add magnetron and 2.5mL of acetonitrile and stir to dissolve , and then 18 μL of water was added to stir the reaction for 1 min. Then 2.5 mL of t-BuNH _{2 was} added and the reaction was stirred for 10 min. After the completion of the reaction, the solvent was removed under negative pressure until it was fluffy, and 5 mL of acetonitrile was added and it was rotated until it was fluffy, and the operation was repeated twice to remove t-BuNH ₂ . Add 6 mL of dichloromethane (DCM) to dissolve the solid, then add 90 μL of water and 6 mL of 6% dichloroacetic acid (DCA) in DCM (4.4 mmol) to stir the reaction for 10 min, take samples halfway through ESI-MS detection to determine the removal of the DMTr protecting group . Add 0.7 mL of pyridine to neutralize DCA, concentrate to 2 mL under negative pressure, then add 4 mL of anhydrous acetonitrile to dissolve the sample, and spin to remove the solvent under negative pressure.

化合物D4Compound D4

另外称取0.557g(0.65mmol)氟代鸟苷酸亚磷酰胺单体(D3)放入10mL圆底烧瓶中，加入4mL无水乙腈溶解固体，负压旋除乙腈至蓬松状，重复操作四次，在最后一次剩余2mL时停止旋蒸，使用橡胶塞封闭。使用注射器快速吸取上述干燥好的单体加入到含有D2的圆底烧瓶中，搅拌反应2min。In addition, weigh 0.557g (0.65mmol) of the fluoroguanylate phosphoramidite monomer (D3) into a 10mL round-bottom flask, add 4mL of anhydrous acetonitrile to dissolve the solid, remove the acetonitrile under negative pressure until it is fluffy, repeat operation four The rotary evaporation was stopped when the last 2 mL was left, and it was closed with a rubber stopper. Use a syringe to quickly draw the above-mentioned dried monomers into a round-bottomed flask containing D2, and stir for 2 min.

化合物D5Compound D5

向含有D4的圆底烧瓶中加入0.113g DDTT(((二甲基氨基-亚甲基)氨基)-3H-1,2,4-二噻唑-3-硫酮，0.55mmol，1.1当量)硫化三价磷，搅拌反应30min，负压浓缩至油状。加入8mL DCM溶解油状物，在加入0.09mL水和8mL 6％DCA的DCM溶液(1.5mmol)搅拌反应10min，中途取样进行ESI-MS检测判断DMTr保护基脱除情况。反应完全后加入5mL吡啶，负压浓缩至2mL，再加入15mL吡啶浓缩至10mL，使用橡胶塞塞住。To the round bottom flask containing D4 was added 0.113 g DDTT (((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione, 0.55 mmol, 1.1 equiv) sulfurized Trivalent phosphorus, stirred and reacted for 30min, and concentrated to oil under negative pressure. Add 8 mL of DCM to dissolve the oily substance, add 0.09 mL of water and 8 mL of a DCM solution of 6% DCA (1.5 mmol), and stir the reaction for 10 min, take samples halfway through and carry out ESI-MS detection to determine the removal of the DMTr protecting group. After the reaction was completed, 5 mL of pyridine was added, concentrated to 2 mL under negative pressure, and then 15 mL of pyridine was added and concentrated to 10 mL, which was plugged with a rubber stopper.

化合物D6Compound D6

称取0.34g 5,5-二甲基-2-氯-1,3,2-二氧磷杂己内酰磷酸酯(DMOCP，1.75mmol)加入到含有D5的圆底烧瓶中，搅拌反应10min。加入0.32mL水和165mg碘单质，搅拌反应5min，中途取样进行ESI-MS检测判断氧化完成情况。反应完成后，将混合物倒入含有0.1g亚硫酸氢钠的70mL水溶液中，搅拌反应5min消耗掉多余氧化剂，再缓慢加入2g碳酸氢钠中和反应体系，继续搅拌5min；将反应体系倒入含有80mL***/乙酸乙酯(体积1/1)混合溶剂的分液漏斗中，加入80mL水进行萃取分离操作，收集有机相之后负压旋除溶剂得到油状液体即为化合物D6。Weigh 0.34g of 5,5-dimethyl-2-chloro-1,3,2-dioxaphosphacaprolactone phosphate (DMOCP, 1.75mmol) into a round-bottomed flask containing D5, and stir the reaction for 10min . 0.32 mL of water and 165 mg of iodine were added, and the reaction was stirred for 5 min. A sample was taken in the middle for ESI-MS detection to determine the completion of the oxidation. After the reaction was completed, the mixture was poured into a 70 mL aqueous solution containing 0.1 g of sodium bisulfite, and the excess oxidant was consumed by stirring the reaction for 5 min. Then 2 g of sodium bicarbonate was slowly added to neutralize the reaction system, and stirring was continued for 5 min; the reaction system was poured into the reaction system containing 80mL of ether/ethyl acetate (volume 1/1) mixed solvent was placed in a separating funnel, and 80mL of water was added to carry out extraction and separation operation. After collecting the organic phase, the solvent was removed under negative pressure to obtain an oily liquid, which was compound D6.

化合物D7Compound D7

将D6转移至25mL圆底烧瓶中，加入2mL乙酸乙酯油泵负压旋除，重复操作三次以去除吡啶。加入2.5mL乙腈溶解油状物，再加入2.5mL t-BuNH ₂搅拌反应10min。之后加入2.5mL乙腈负压旋干，重复操作三次去除t-BuNH ₂。再加入2.5mL甲醇溶解样品，同样负压旋至泡沫状。 Transfer D6 to a 25mL round-bottomed flask, add 2mL of ethyl acetate oil pump and spin off under negative pressure, and repeat the operation three times to remove pyridine. 2.5 mL of acetonitrile was added to dissolve the oil, and then 2.5 mL of t-BuNH ₂ was added to stir the reaction for 10 min. Then, 2.5 mL of acetonitrile was added to dry under negative pressure, and the operation was repeated three times to remove t-BuNH ₂ . Then add 2.5mL methanol to dissolve the sample, and spin to foam under the same negative pressure.

化合物SF1Compound SF1

向含有D6的圆底烧瓶中加入10mL 33％甲胺无水乙醇溶液(质量比)，塞上橡胶塞搅拌反应1.5h，中途取样进行ESI-MS检测判断异丁酰基(iBu)保护基脱除情况。反应完成后，浓缩至油状，加入400μL吡啶和200μL三乙胺继续旋蒸至油状，重复操作三次将产物由t-BuNH ₂盐形式转变为三乙胺盐形式。接着加入400μL吡啶溶解油状物，橡胶塞塞住瓶口，将圆底烧瓶至于50℃油浴锅中搅拌。使用注射器分别吸取1.4mL三乙胺和0.83mL三乙胺氢氟酸盐并将二者同步缓慢注射到圆底烧瓶中，持续1min。注射完成后搅拌反应1h，中途取样进行ESI-MS检测判断TBS保护基脱除情况。反应完成后，趁热将反应溶液缓慢滴到两个含有30mL色谱纯丙酮并且正在搅拌的50mL离心管中，可以观察到白色固体慢慢析出。沉淀完成之后，取出磁子，7000rpm离心10min。小心去掉上清，加入新的丙酮洗涤离心，重复两次，负压抽干溶剂即以40％的产率得到最终产物SF1。 ¹H NMR(400M,D ₂O)δ8.26–7.71(m,2H),6.38–5.87(m,2H),5.86–5.45(m,2H),4.58–4.32(m,4H),4.07(d,J＝11.1Hz,2H). ³¹P NMR(400M,D ₂O)δ55.35,54.96,-1.05. ¹⁹F NMR(400M,D ₂O)δ-122.42,-130.51.ESI-HRMS(负电模式):C ₂₀H ₂₂FN ₁₀O ₁₂P ₂S ^-，[M-H] ^-理论分子量707.0604；实测分子量707.0606。核磁共振结果表明通过实施例1中的方法合成的化合物确实为化合物SF1。 Add 10 mL of 33% methylamine anhydrous ethanol solution (mass ratio) to the round-bottomed flask containing D6, plug it with a rubber stopper, and stir the reaction for 1.5 h, take samples halfway through ESI-MS detection to determine the removal of the isobutyryl (iBu) protecting group condition. After the reaction was completed, it was concentrated to an oily state, 400 μL of pyridine and 200 μL of triethylamine were added to continue rotary evaporation to an oily state, and the operation was repeated three times to convert the product from the t-BuNH ₂ salt form to the triethylamine salt form. Then, 400 μL of pyridine was added to dissolve the oily substance, the bottle mouth was plugged with a rubber stopper, and the round-bottomed flask was stirred in an oil bath at 50°C. Use a syringe to draw 1.4 mL of triethylamine and 0.83 mL of triethylamine hydrofluoride, respectively, and inject the two into the round-bottomed flask synchronously and slowly for 1 min. After the injection was completed, the reaction was stirred for 1 h, and samples were taken in the middle for ESI-MS detection to determine the removal of the TBS protecting group. After the reaction was completed, the reaction solution was slowly dropped into two 50 mL centrifuge tubes containing 30 mL of chromatographically pure acetone while still being stirred, and a white solid could be observed to slowly separate out. After the precipitation was completed, the magnet was taken out and centrifuged at 7000 rpm for 10 min. Carefully remove the supernatant, add new acetone to wash and centrifuge, repeat twice, and vacuum dry the solvent to obtain the final product SF1 with a yield of 40%. ¹ H NMR(400M, D ₂ O)δ8.26-7.71(m,2H),6.38-5.87(m,2H),5.86-5.45(m,2H),4.58-4.32(m,4H),4.07( d, J=11.1Hz, 2H). ³¹ P NMR(400M, D ₂ O) δ55.35, 54.96, -1.05. ¹⁹ F NMR(400M, D ₂ O) δ-122.42, -130.51.ESI-HRMS( Negative mode): C ₂₀ H ₂₂ FN ₁₀ O ₁₂ P ₂ S ^- , [MH] ^- Theoretical molecular weight 707.0604; measured molecular weight 707.0606. The results of nuclear magnetic resonance indicated that the compound synthesized by the method in Example 1 was indeed compound SF1.

实施例2 SF1作为新冠疫苗佐剂的免疫反应效果评价Example 2 Evaluation of the immune response effect of SF1 as a new crown vaccine adjuvant

本实施例以实施例1制备的SF1作为新冠疫苗佐剂对于增强抗原免疫反应效果进行了评价。In this example, the SF1 prepared in Example 1 was used as a new crown vaccine adjuvant to evaluate the effect of enhancing the antigen immune response.

1、免疫小鼠1. Immune mice

实验采用SARS-CoV-2膜外域S蛋白作为疫苗抗原(该疫苗抗原购自北京义翘神州生物技术有限公司Sino Biological Inc)，进行Babl/c小鼠免疫评价。小鼠为6-8周龄，共20只，分为四组，每组5只。四组分别为空白对照组、S蛋白抗原组、S蛋白抗原+SF1佐剂组、S蛋白抗原+铝佐剂组，其中，空白对照组指未施用抗原和佐剂，S蛋白抗原组指仅施用了SARS-CoV-2膜外域S蛋白抗原，S蛋白抗原+SF1佐剂组指施用了S蛋白抗原和SF1佐剂，S蛋白抗原+铝佐剂组指施用了S蛋白抗原和铝佐剂。S蛋白抗原用量250μg/kg，SF1用量1mg/kg，铝佐剂5mg/kg，间隔两周免疫三次。免疫完成后隔一周对小鼠进行处理，进行免疫相关表征。对小鼠进行处理的时间轴见图2。In the experiment, the SARS-CoV-2 membrane ectodomain S protein was used as the vaccine antigen (the vaccine antigen was purchased from Sino Biological Inc, Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) to conduct immune evaluation of Babl/c mice. The mice were 6-8 weeks old, with a total of 20 mice, divided into four groups with 5 mice in each group. The four groups are blank control group, S protein antigen group, S protein antigen + SF1 adjuvant group, and S protein antigen + aluminum adjuvant group. The blank control group refers to no administration of antigen and adjuvant, and the S protein antigen group refers to only administration of antigen and adjuvant. SARS-CoV-2 ectodomain S protein antigen, S protein antigen + SF1 adjuvant group refers to the administration of S protein antigen and SF1 adjuvant, S protein antigen + aluminum adjuvant group refers to the administration of S protein antigen and aluminum adjuvant. The dosage of S protein antigen was 250 μg/kg, the dosage of SF1 was 1 mg/kg, and the dosage of aluminum adjuvant was 5 mg/kg. Mice were treated at one-week intervals after immunization for immune-related characterization. The timeline for the treatment of mice is shown in Figure 2.

2、各组小鼠脾脏的形态观察和重量统计2. Morphological observation and weight statistics of spleen of mice in each group

分别从各组中随机选取四只小鼠，处死后取其脾脏，图3A显示了空白对照组、S蛋白抗原组、S蛋白抗原+SF1佐剂组、S蛋白抗原+铝佐剂组脾脏的形态，图3B显示了空白对照组、S蛋白抗原组、S蛋白抗原+SF1佐剂组、S蛋白抗原+铝佐剂组脾脏的重量统计结果。图中空白对照代表未施用疫苗抗原和佐剂，“S”代表仅向小鼠施用了SARS-CoV-2膜外域S蛋白抗原，“S+SF1”代表向小鼠施用了SARS-CoV-2膜外域S蛋白抗原和SF1佐剂，“S+Alum”代表向小鼠施用了SARS-CoV-2膜外域S蛋白抗原和铝佐剂。图3A结果表明SF1免疫后无明显毒副作用，脾脏形态正常。图3B结果表明S+SF1组小鼠免疫后脾脏重量高于其他组。Four mice were randomly selected from each group, and their spleens were taken after sacrifice. Figure 3B shows the weight statistics of the spleen in the blank control group, the S protein antigen group, the S protein antigen + SF1 adjuvant group, and the S protein antigen + aluminum adjuvant group. The blank control in the figure represents that no vaccine antigen and adjuvant were administered, "S" represents that only the SARS-CoV-2 membrane ectodomain S protein antigen was administered to mice, and "S+SF1" represents that SARS-CoV-2 membrane was administered to mice The ectodomain S protein antigen and SF1 adjuvant, "S+Alum" represents administration of the SARS-CoV-2 membrane ectodomain S protein antigen and aluminum adjuvant to mice. Figure 3A shows that SF1 has no obvious side effects after immunization, and the spleen has a normal shape. The results of Figure 3B showed that the spleen weight of the mice in the S+SF1 group was higher than that in the other groups after immunization.

3、进行酶联免疫吸附斑点测定实验3. Perform enzyme-linked immunosorbent assay assay

取各组小鼠脾脏细胞进行酶联免疫吸附斑点测定(ELISPOT)，表征疫苗的细胞免疫情况，实验方法如下：The spleen cells of each group of mice were taken for enzyme-linked immunosorbent spot assay (ELISPOT) to characterize the cellular immunity of the vaccine. The experimental method is as follows:

将小鼠脾脏研磨过滤得到单细胞悬液，按照每孔100万细胞加入到IFN-γ抗体(达科为生物技术有限公司)预包被96孔板中，使用50μg/mL S蛋白抗原刺激36h，最后进行酶联显色形成斑点，读板统计所得斑点数。The mouse spleen was ground and filtered to obtain a single-cell suspension, and 1 million cells per well were added to a 96-well plate pre-coated with IFN-γ antibody (Dakewe Biotechnology Co., Ltd.), and stimulated with 50 μg/mL S protein antigen for 36 h , and finally carry out enzyme-linked color development to form spots, and the number of spots obtained is counted by reading the plate.

结果如附图4A和4B所示，图4A中空白对照组、S蛋白抗原组、S蛋白抗原+SF1佐剂组、S蛋白抗原+铝佐剂组酶联免疫吸附斑点测定的斑点数的结果表明，SF1化合物可以显著增强SARS-CoV-2膜外域S蛋白抗原的抗原特异性T细胞免疫反应，效果显著优于铝佐剂。附图4B中结果表明，SF1免疫刺激产生的S蛋白特异性T细胞斑点数显著多于铝佐剂组以及单独S蛋白组，证明SF1具有优秀的T细胞免疫活化效果。The results are shown in Figures 4A and 4B. In Figure 4A, the results of the number of spots measured by ELISA in the blank control group, the S protein antigen group, the S protein antigen + SF1 adjuvant group, and the S protein antigen + aluminum adjuvant group It was shown that the SF1 compound can significantly enhance the antigen-specific T cell immune response of the SARS-CoV-2 membrane ectodomain S protein antigen, and the effect is significantly better than that of the aluminum adjuvant. The results in Figure 4B show that the number of S protein-specific T cell spots produced by SF1 immunostimulation is significantly more than the aluminum adjuvant group and the S protein alone group, which proves that SF1 has an excellent T cell immune activation effect.

因此，通过上述的小鼠脾脏的形态观察以及酶联免疫吸附斑点测定实验，均能够证实SF1化合物作为SARS-CoV-2膜外域S蛋白抗原的佐剂，具有优秀的T细胞免疫活化效果，可以显著增强S蛋白抗原的抗原特异性T细胞免疫反应，效果显著优于铝佐剂。Therefore, through the above-mentioned morphological observation of mouse spleen and ELISA spot assay experiments, it can be confirmed that SF1 compound, as an adjuvant for SARS-CoV-2 ectodomain S protein antigen, has excellent T cell immune activation effect and can be used as an adjuvant. Significantly enhance the antigen-specific T cell immune response of S protein antigen, the effect is significantly better than that of aluminum adjuvant.

4、酶联免疫吸附测定4. ELISA

采用酶联免疫吸附测定，测定免疫后血清中抗原特异性抗体滴度情况，实验方法如下：Enzyme-linked immunosorbent assay was used to determine the antigen-specific antibody titer in the serum after immunization. The experimental method is as follows:

先对96孔板进行S蛋白抗原包被，用量0.1μg/孔，静置过夜。使用明胶封闭3h后，分别加入梯度稀释的各组免疫血清孵育1.5h，洗涤，再加入HRP标记兔抗鼠IgG二抗孵育1.5h，洗涤。最后加入TMB底物显色，在OD450波长处读取吸光度值，计算各组滴度。The 96-well plate was first coated with S protein antigen at an dosage of 0.1 μg/well, and allowed to stand overnight. After blocking with gelatin for 3 hours, the serially diluted immune sera of each group were added to incubate for 1.5 hours, washed, and then HRP-labeled rabbit anti-mouse IgG secondary antibody was added to incubate for 1.5 hours and washed. Finally, TMB substrate was added to develop color, and the absorbance value was read at the wavelength of OD450, and the titer of each group was calculated.

附图5结果表明SF1可以显著提高SARS-CoV-2膜外域S蛋白抗原的特异性抗体滴度，平均滴度在20万左右，显著优于铝佐剂组的滴度。Figure 5 shows that SF1 can significantly increase the specific antibody titer of SARS-CoV-2 ectodomain S protein antigen, with an average titer of about 200,000, which is significantly better than that of the aluminum adjuvant group.

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., means a specific feature described in connection with the embodiment or example, A structure, material, or feature is included in at least one embodiment or example of the present disclosure. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present disclosure have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present disclosure. Embodiments are subject to variations, modifications, substitutions and variations.

Claims

化合物在制备疫苗佐剂中的用途，其中，所述化合物为式SF所示化合物或式SF所示化合物的立体异构体、互变异构体、位置异构体、氮氧化合物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药：Use of a compound in the preparation of a vaccine adjuvant, wherein the compound is a compound represented by formula SF or a stereoisomer, tautomer, positional isomer, nitroxide, hydrate of a compound represented by formula SF , solvates, metabolites, pharmaceutically acceptable salts or prodrugs:

其中，B ₁和B ₂各自独立地选自天然碱基A、T、C、G、U及非天然碱基中的任一种； Wherein, B ₁ and B ₂ are each independently selected from any one of natural bases A, T, C, G, U and unnatural bases;

Y ₁和Y ₂各自独立地选自-OH和-SH中的任一种； Y ₁ and Y ₂ are each independently selected from any one of -OH and -SH;

X ₁和X ₂各自独立地选自-H、-OH和-F中的任一种， X ₁ and X ₂ are each independently selected from any one of -H, -OH and -F,

所述疫苗佐剂为肺炎疫苗或流感疫苗的佐剂。The vaccine adjuvant is an adjuvant for a pneumonia vaccine or an influenza vaccine.
根据权利要求1所述的用途，其中，所述式SF所示化合物的位置异构体的结构式为：The use according to claim 1, wherein the structural formula of the positional isomer of the compound represented by the formula SF is:

其中，B ₁和B ₂各自独立地选自天然碱基A、T、C、G、U及非天然碱基中的任一种； Wherein, B ₁ and B ₂ are each independently selected from any one of natural bases A, T, C, G, U and unnatural bases;

Y ₁和Y ₂各自独立地选自-OH和-SH中的任一种； Y ₁ and Y ₂ are each independently selected from any one of -OH and -SH;

X ₁和X ₂各自独立地选自-H、-OH和-F中的任一种。 X ₁ and X ₂ are each independently selected from any one of -H, -OH and -F.
根据权利要求1或2所述的用途，其中，所述非天然碱基选自人造碱基或经修饰的天然碱基，优选地，所述经修饰的天然碱基为I或mC。The use according to claim 1 or 2, wherein the unnatural base is selected from artificial bases or modified natural bases, preferably, the modified natural base is I or mC.
根据权利要求1或2所述的用途，其中，所述疫苗佐剂为核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、多肽疫苗、灭活疫苗或减活疫苗的佐剂。The use according to claim 1 or 2, wherein the vaccine adjuvant is an adjuvant for nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, polypeptide vaccines, inactivated vaccines or attenuated vaccines.
化合物在预防肺炎和/或流感中的用途，其中，所述化合物为式SF所示化合物或式SF所示化合物的立体异构体、互变异构体、位置异构体、氮氧化合物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药：Use of a compound in the prevention of pneumonia and/or influenza, wherein the compound is a compound represented by formula SF or a stereoisomer, tautomer, positional isomer, nitroxide, or compound represented by formula SF. Hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs:

其中，B ₁和B ₂各自独立地选自天然碱基A、T、C、G、U及非天然碱基中的任一种； Wherein, B ₁ and B ₂ are each independently selected from any one of natural bases A, T, C, G, U and unnatural bases;

Y ₁和Y ₂各自独立地选自-OH和-SH中的任一种； Y ₁ and Y ₂ are each independently selected from any one of -OH and -SH;

X ₁和X ₂各自独立地选自-H、-OH和-F中的任一种。 X ₁ and X ₂ are each independently selected from any one of -H, -OH and -F.
根据权利要求5所述的用途，其中，所述式SF所示化合物的位置异构体的结构式为：Use according to claim 5, wherein, the structural formula of the positional isomer of the compound represented by the formula SF is:

其中，B ₁和B ₂各自独立地选自天然碱基A、T、C、G、U及非天然碱基中的任一种； Wherein, B ₁ and B ₂ are each independently selected from any one of natural bases A, T, C, G, U and unnatural bases;

Y ₁和Y ₂各自独立地选自-OH和-SH中的任一种； Y ₁ and Y ₂ are each independently selected from any one of -OH and -SH;

X ₁和X ₂各自独立地选自-H、-OH和-F中的任一种。 X ₁ and X ₂ are each independently selected from any one of -H, -OH and -F.
根据权利要求5或6所述的用途，其中，所述非天然碱基选自人造碱基或经修饰的天然碱基，优选地，所述经修饰的天然碱基为I或mC。The use according to claim 5 or 6, wherein the unnatural base is selected from artificial bases or modified natural bases, preferably, the modified natural base is I or mC.
一种疫苗佐剂，其中，所述疫苗佐剂含有权利要求1-4中任一项所述的化合物，所述疫苗佐剂为肺炎疫苗或流感疫苗的佐剂。A vaccine adjuvant, wherein the vaccine adjuvant contains the compound of any one of claims 1-4, and the vaccine adjuvant is an adjuvant for a pneumonia vaccine or an influenza vaccine.
权利要求8所述的疫苗佐剂在预防肺炎和/或流感中的用途。Use of the vaccine adjuvant of claim 8 in preventing pneumonia and/or influenza.
权利要求8所述的疫苗佐剂在制备疫苗组合物中的用途，其中，所述疫苗组合物包括所述疫苗佐剂及疫苗活性成分。The use of the vaccine adjuvant of claim 8 in the preparation of a vaccine composition, wherein the vaccine composition comprises the vaccine adjuvant and a vaccine active ingredient.
根据权利要求10所述的用途，其中，所述活性成分选自核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、多肽疫苗、灭活疫苗或减活疫苗中的至少之一。The use according to claim 10, wherein the active ingredient is selected from at least one of nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, polypeptide vaccines, inactivated vaccines or attenuated vaccines.
根据权利要求10所述的用途，其中，所述疫苗活性成分为新型冠状病毒疫苗，所述新型冠状病毒疫苗以SARS-CoV-2的灭活病毒、减活病毒、刺突糖蛋白、包膜糖蛋白、膜糖蛋白、核衣壳蛋白、RBD蛋白、多肽表位以及表达这些蛋白和多肽的核酸序列中的至少之一作为疫苗抗原。The use according to claim 10, wherein the active ingredient of the vaccine is a novel coronavirus vaccine, and the novel coronavirus vaccine is composed of inactivated virus, attenuated virus, spike glycoprotein, envelope of SARS-CoV-2 At least one of glycoproteins, membrane glycoproteins, nucleocapsid proteins, RBD proteins, polypeptide epitopes, and nucleic acid sequences expressing these proteins and polypeptides are used as vaccine antigens.
根据权利要求12所述的用途，其中，所述疫苗组合物中疫苗抗原与佐剂的用量比为1:10-10:1，优选为1:5-5:1，进一步优选为1:4。The use according to claim 12, wherein the dosage ratio of vaccine antigen to adjuvant in the vaccine composition is 1:10-10:1, preferably 1:5-5:1, more preferably 1:4 .
根据权利要求10所述的用途，其中，所述疫苗组合物为疫苗制剂，所述疫苗制剂以口服、静脉注射、动脉注射、粘膜给药、鼻腔给药、肌肉注射、皮下注射、器官注射或胸腹腔内注射施用。The use according to claim 10, wherein the vaccine composition is a vaccine formulation, and the vaccine formulation is administered orally, intravenously, intraarterially, mucosally, nasally, intramuscularly, subcutaneously, intraorganically, or by injection. Administration by intrathoracic intraperitoneal injection.
一种疫苗组合物，其中，所述疫苗组合物包含权利要求8所述的疫苗佐剂及疫苗活性成分。A vaccine composition, wherein the vaccine composition comprises the vaccine adjuvant of claim 8 and a vaccine active ingredient.
根据权利要求15所述的疫苗组合物，其中，所述疫苗活性成分选自核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、多肽疫苗、灭活疫苗或减活疫苗中的至少之一。The vaccine composition according to claim 15, wherein the vaccine active ingredient is selected from at least one of nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, polypeptide vaccines, inactivated vaccines or attenuated vaccines.
根据权利要求15所述的疫苗组合物，其中，所述疫苗活性成分为新型冠状病毒疫苗，所述新型冠状病毒疫苗以SARS-CoV-2的灭活病毒、减活病毒、刺突糖蛋白、包膜糖蛋白、膜糖蛋白、核衣壳蛋白、RBD蛋白、多肽表位以及表达这些蛋白和多肽的核酸序列中的至少之一作为疫苗抗原。The vaccine composition according to claim 15, wherein the active ingredient of the vaccine is a novel coronavirus vaccine, and the novel coronavirus vaccine is composed of inactivated virus, attenuated virus, spike glycoprotein, At least one of envelope glycoproteins, membrane glycoproteins, nucleocapsid proteins, RBD proteins, polypeptide epitopes, and nucleic acid sequences expressing these proteins and polypeptides are used as vaccine antigens.
根据权利要求17所述的疫苗组合物，其中，所述疫苗组合物中疫苗抗原与佐剂的用量比为1:10-10:1，优选为1:5-5:1，进一步优选为1:4。The vaccine composition according to claim 17, wherein the dosage ratio of vaccine antigen to adjuvant in the vaccine composition is 1:10-10:1, preferably 1:5-5:1, more preferably 1 :4.
疫苗组合物在预防肺炎和/或流感中的用途，其中，所述疫苗组合物为权利要求15-18中任一项所述的疫苗组合物。Use of a vaccine composition for preventing pneumonia and/or influenza, wherein the vaccine composition is the vaccine composition of any one of claims 15-18.
一种预防肺炎和/或流感的方法，其中，所述方法包括向有预防肺炎和/或流感需求的受试者施用权利要求15-18中任一项所述的疫苗组合物。A method of preventing pneumonia and/or influenza, wherein the method comprises administering the vaccine composition of any one of claims 15-18 to a subject in need of preventing pneumonia and/or influenza.