CN111909223A

CN111909223A - Cyclic dinucleotide covalent modifier and preparation method and application thereof

Info

Publication number: CN111909223A
Application number: CN202010694692.XA
Authority: CN
Inventors: 李艳梅; 吴军军; 张伯斗; 胡洪果; 李文浩; 赵朗; 陈永湘
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-11-10
Also published as: WO2022012402A1

Abstract

The invention relates to a ringDinucleotide covalent modification products, and a preparation method and application thereof. In particular to a method for connecting a connecting arm on a compound shown as a formula SF, which is to connect the connecting arm on S in the phosphorothioate of the compound shown as the formula SF,

Description

Cyclic dinucleotide covalent modifier and preparation method and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a cyclic dinucleotide covalent modifier and a preparation method and application thereof.

Background

Tumor immunotherapy has now gradually evolved into an important and highly potential direction for cancer therapy. In immunotherapy, how to effectively enhance the immune response of the tumor microenvironment and relieve immune tolerance is a core problem about the treatment effect. As a new pattern recognition receptor recently discovered, the interferon gene stimulatory protein (STING) pathway has attracted research and development interest of many large drug enterprises. Nuohua, merck and Baishimeibao companies successively invest in the development of STING pathway agonists for clinical immunotherapy of tumors. STING proteins, which are localized on the endoplasmic reticulum and highly expressed in T cells and antigen presenting cells, can be activated by their natural agonist Cyclic Dinucleotides (CDNs), thereby facilitating the transcriptional translation of downstream host defense genes, including type I interferons and other proinflammatory cytokines. Based on this, cyclic dinucleotides can be ideal candidates as therapeutic agents or vaccine adjuvants. However, the negative charge and the esterase hydrolysis susceptibility of CDNs phosphodiesters greatly hinder their clinical application. Therefore, the design and development of a proper method for improving the stability and transmembrane efficiency of the cyclic dinucleotide have important research values.

Disclosure of Invention

The invention aims to solve at least one of the technical problems in the prior art and provides a cyclic dinucleotide covalent modification substance, a preparation method and application thereof. Specifically provided is a method for attaching a linker arm to a compound of formula SF.

The chemical modification of cyclic dinucleotides to improve their stability and lipid solubility is one of the major research and development directions for improving their pharmaceutical properties. Of these, phosphothioylation and hydroxyfluorination are the most representative types of chemical modification. The inventor finds in the research process that the resistance of cyclic dinucleotides to hydrolysis of phosphatase can be effectively improved by carrying out phosphothioylation modification on the cyclic dinucleotides; furthermore, by introducing fluorine atoms, the strong electron-withdrawing property of the fluorine atoms can enhance the lipid solubility and stability of the cyclic dinucleotide. The stability of cyclic dinucleotides can be increased by these modifications. Meanwhile, the inventor also finds that although the phosphorothioate hydroxyl group fluoro modification can improve the stability of the cyclic-di-nucleotide, the modification is still insufficient for improving the transmembrane efficiency of the cyclic-di-nucleotide, so that a suitable delivery vector needs to be designed and developed to further improve the stimulation efficiency of the cyclic-di-nucleotide on the STING pathway.

The conventional delivery method is to use carriers such as macromolecules, liposomes and the like for non-covalent coating and loading. Non-covalent loading is characterized by insufficient physical homogeneity and rapid release of the encapsulated cyclic dinucleotides. Too fast release rate can cause high-intensity activation of STING pathway, which is not favorable for application in fields of vaccine adjuvant, etc. Meanwhile, high activation may further cause the side effect of cytokine storm, which endangers life and health. The covalent loading can avoid the above situation to a certain extent, so that the development of the cyclic dinucleotide covalent loading method has important application value. However, there are currently few methods for covalent ligation of cyclic dinucleotides, mainly for biotin ligation, and few studies relating to the STING pathway activity of the linker molecules. Then how to select a proper connection site, design a high-efficiency connection method and not influence the activity of the cyclic-dinucleotide is a core problem to be solved for developing the covalent loading of the cyclic-dinucleotide.

Phosphothioylation of phosphate esters in oligonucleotides is a common method for increasing the stability of nucleotide nucleases. The existing literature shows that sulfydryl in the phosphorothioate can be efficiently reacted with an alpha-halogenated compound, so that the idea of using an alpha halogenated connecting arm to react with the phosphorothioated cyclic dinucleotide to realize chemical connection is formed. However, the presence of the hydroxyl group at the 2-position can cause intramolecular nucleophilic substitution reaction to lead to leaving of the linker arm. In order to solve the problem, a novel class of covalently modified Cyclic Dinucleotides (CDNs) is designed and synthesized by a liquid phase one-bottle method. The provided hydroxyl at the 2-position on the same side is substituted by fluorine atoms, so that nucleophilic substitution reaction in molecules on the phosphorothioate can be completely avoided, and the connecting arm stably exists, so that the antigen presenting cells can be obviously activated. Meanwhile, p-hydroxymethyl benzyl bromide is used as a model connecting arm for connection and activity verification. Experimental research shows that the stability and transmembrane efficiency of the cyclic dinucleotide can be improved by carrying out covalent modification and connection on the cyclic dinucleotide, and the activity of the cyclic dinucleotide is not influenced. The connection method can be used for connecting other active molecules or covalent load and the like, and has wide application prospect.

To this end, according to one aspect of the invention, there is provided a method of attaching a linker arm to a compound of formula SF, according to an embodiment of the invention, attaching the linker arm to S in the phosphorothioate of the compound of formula SF,

the inventor finds that the connecting arm is connected to the S in the phosphorothioate of the compound shown in the formula SF, so that the influence on the activity of the compound shown in the formula SF is small, and the connecting arm connected to the S in the phosphorothioate can be used for covalently connecting target units such as synthetic joint adjuvants, high-molecular covalent loading, fluorescent labels and the like.

According to a specific embodiment of the present invention, the method specifically includes: reacting the S group of the compound of formula SF with a halide by nucleophilic substitution to form the linker arm. The inventor utilizes nucleophilic substitution reaction of S in the phosphorothioate and the halide to ensure the accuracy and uniqueness of site connection of the connecting arm, so that the connecting arm stably exists and is used for covalent connection of target units.

According to one embodiment of the invention, the halide is an alpha halide. According to an embodiment of the present invention, the α halide may be a benzyl halide or a haloacetyl group, such as p-bromomethylbenzyl alcohol, p-bromomethylbenzoic acid, iodoacetamido, and the like. These linkers are all highly reactive with compounds of formula SF, and the groups on the opposite side of the linker can be conveniently used to covalently link target units.

According to a particular embodiment of the invention, B of the compound of formula SF above₁Or B₂Each independently is a base;

x is selected from-H, -OH, -F; y is selected from-OH and-SH. Specifically, the base is selected from a natural base or a non-natural base, and the natural base is selected from a base A, a base G, a base C, a base T and a base U.

According to a particular embodiment of the invention, the compound of formula SF above is prepared by the following steps:

(1) subjecting the compound of formula S1 to a deprotection reaction to obtain a compound of formula S2;

(2) subjecting a compound of formula S2 and a compound of formula S3 to a phosphorylation reaction to obtain a compound of formula S4;

(3) subjecting a compound of formula S4 to oxidation and deprotection reactions with DDTT and dichloroacetic acid to obtain a compound of formula S5;

(4) subjecting the compound represented by the formula S5 to nucleophilic substitution and oxidation reaction with a cyclizing reagent and an oxidizing agent to obtain a compound represented by the formula S6;

(5) subjecting the compound of formula S6 to nucleophilic substitution reaction with tert-butylamine to obtain a compound of formula S7;

(6) subjecting a compound represented by formula S7, methylamine and hydrofluoric acid triethylamine salt to deprotection reaction so as to obtain a compound represented by formula SF;

wherein, the compound shown as the formula S1, the compound shown as the formula S2, the compound shown as the formula S3, the compound shown as the formula S4, the compound shown as the formula S5 and the compound shown as the formula S6 are respectively shown as follows:

wherein L1 and L2 in each compound are respectively and independently base protecting groups, preferably L1 and L2 in each compound are respectively and independently base protecting groups

n is 2, and Z is selected from silicon hydroxyl and F.

According to a specific embodiment of the present invention, in the step (4), the cyclization agent is 5, 5-dimethyl-2-chloro-1, 3, 2-dioxaphosphorinanyl phosphate; the oxidant is at least one selected from iodine or 3H-1, 2-benzodithiol-3-one 1, 1-dioxide.

According to a specific embodiment of the present invention, the deprotection reaction of the compound represented by formula S1, pyridine trifluoroacetate, tert-butylamine, and dichloroacetic acid is performed in step (1), and the deprotection reaction is performed at room temperature.

According to a specific embodiment of the present invention, the phosphorylation reaction in step (2) is carried out under anhydrous conditions.

According to a specific embodiment of the present invention, the oxidation and deprotection reaction in step (3) is performed under room temperature conditions.

According to a specific embodiment of the present invention, the nucleophilic substitution and oxidation reaction in step (4) are performed under room temperature conditions.

According to a specific embodiment of the present invention, the nucleophilic substitution reaction in step (5) is performed under room temperature conditions.

According to the specific embodiment of the present invention, the deprotection reaction in the step (6) is performed under the condition of 50 ℃ in an oil bath.

Therefore, the invention designs and synthesizes a novel covalently modified Cyclic Dinucleotides (CDNs) by the method and by using a liquid phase one-bottle method. The provided hydroxyl at the 2-position on the same side is substituted by fluorine atoms, so that nucleophilic substitution reaction in molecules on the phosphorothioate can be completely avoided, and the connecting arm stably exists, so that the antigen presenting cells can be obviously activated. Although, phosphothioylation of phosphate esters in oligonucleotides is a common method to increase the stability of nucleotide nucleases. The prior literature indicates that the sulfhydryl group in the phosphorothioate can be efficiently reacted with the alpha-halogenated compound. However, in practice, when an α -halogenated linker arm is used to react with a phosphorothioated cyclic dinucleotide, intramolecular nucleophilic substitution occurs in the presence of the hydroxyl group at the 2-position, resulting in the removal of the linker arm. To this end, the inventors of the present invention have designed and synthesized a novel class of covalently modified Cyclic Dinucleotides (CDNs) by a liquid phase one-bottle method, i.e., the method for preparing the compound represented by SF as described in the previous examples. The SF compound prepared by the method is connected with the connecting arm through affinity substitution, so that the problem that the connecting arm leaves due to intramolecular nucleophilic substitution reaction in the presence of 2-position hydroxyl can be effectively solved.

According to another aspect of the invention, the invention provides a compound obtained by attaching a linker arm to a compound of formula SF using the methods described above. The compound can be used for subsequent covalent connection or covalent loading of other targeting molecules, and has little influence on the activity of the CDNs compounds.

According to yet another aspect of the present invention, the present invention also provides a pharmaceutical composition comprising: a compound; and pharmaceutically acceptable adjuvants, carriers, excipients, vehicles or combinations thereof, which are the compounds described in the previous examples, i.e., SF compound attached to the linker arm. The compound can be specifically represented by the following compound shown in the formula I:

according to a further aspect of the invention, the invention also proposes the use of a compound as defined above for the preparation of a medicament for the treatment of an immune disorder.

According to a particular embodiment of the invention, the medicament is for the treatment of tumors or for antiviral, antibacterial;

according to a specific example of the present invention, the drug is used for activating an interferon gene stimulating protein.

According to a further aspect of the invention, there is also provided a pharmaceutical combination comprising: a compound as described in the preceding examples and at least one drug for the treatment of immune disorders.

According to a specific embodiment of the invention, the at least one drug for treating an immune disease is selected from at least one of an immune checkpoint blocking antibody, an immunostimulant, a vaccine, a chimeric antigen receptor T cell, radiotherapy and a chemotherapeutic drug.

According to an embodiment of the invention, the at least one drug for treating an immune disease is selected from at least one of an immune checkpoint blocking antibody, an immunostimulant, a vaccine, a chimeric antigen receptor T cell, radiotherapy and a chemotherapeutic drug. Immune checkpoints are protective factors in the human immune system that can prevent inflammatory damage and the like caused by T cell over-activation. Tumor cells can use this mechanism to escape immune surveillance and killing of the human body, thereby promoting their own growth. Immune checkpoint blocking antibodies, which may also be commonly referred to as immune checkpoint inhibitory antibodies, exert an anti-tumor effect by inhibiting immune checkpoint activity, activating the immune response of T cells against tumors. The immune checkpoint blocking antibody provided may be, for example, an anti-PD-L1 antibody, an anti-CTLA-4 antibody. The immunostimulant provided may be, for example, CpG, imidazoquinoline and monophosphoryl a. The radiotherapy and chemotherapy drugs provided may be doxorubicin, paclitaxel, cisplatin, and the like.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a graph showing the activation effect of murine macrophages in different treatment groups according to an embodiment of the present invention.

FIG. 2 is a graph showing the results of flow assays of cells treated with different compounds according to an embodiment of the present invention.

FIG. 3 is a graph of the flow results of antibodies in mice treated with different compounds according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ or contradict this application (including but not limited to defined terminology, application of terminology, described techniques, and the like), this application controls.

It will be further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

Definitions or general terms

The terms "comprising", "including" and "comprises" are open-ended expressions that include what is specified in the present invention, but do not exclude other aspects.

"stereoisomers" refers to compounds having the same chemical structure but differing in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.

"chiral" is a molecule having the property of not overlapping its mirror image; and "achiral" refers to a molecule that can overlap with its mirror image.

"enantiomer" refers to two isomers of a compound that are not overlapping but are in mirror image relationship to each other.

"diastereomer" refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may be separated by high resolution analytical procedures such as electrophoresis and chromatography, e.g., HPLC.

The stereochemical definitions and rules used in the present invention generally follow the general definitions of 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.

Many organic compounds exist in an optically active form, i.e., 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 a molecule with respect to one or more of its chiral centers. The prefixes d and l or (+) and (-) are the symbols used to specify the rotation of plane polarized light by the compound, where (-) or l indicates that the compound is left-handed. Compounds prefixed with (+) or d are dextrorotatory. A particular stereoisomer is an enantiomer and a mixture of such isomers is referred to as an enantiomeric mixture. A50: 50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in the chemical reaction or process.

Any asymmetric atom (e.g., carbon, etc.) of a compound disclosed herein can exist in racemic or enantiomerically enriched forms, such as the (R) -, (S) -or (R, S) -configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R) -or (S) -configuration.

Depending on the choice of starting materials and methods, the compounds of the invention may exist as one of the possible isomers or as mixtures thereof, for example as racemates and mixtures of non-corresponding isomers (depending on the number of asymmetric carbon atoms). 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 substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the substituents of the cycloalkyl group may have cis or trans configuration.

Any resulting mixture of stereoisomers may be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, depending on differences in the physicochemical properties of the components, for example, by chromatography and/or fractional crystallization.

The racemates of any of the resulting end products or intermediates can be resolved into the optical enantiomers by known methods using methods familiar to those skilled in the art, e.g., by separation of the diastereomeric salts obtained. The racemic product can also be separated by chiral chromatography, e.g., High Performance Liquid Chromatography (HPLC) using a chiral adsorbent. In particular, Enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al, Enantiomers, racemes and solutions (Wiley Interscience, New York, 1981); PrinciplesofAsymmetric Synthesis (2)^nd Ed.Robert E.Gawley,JeffreyAubé,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)。

The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that can interconvert by a low energy barrier (low energy barrier). If tautomerism is possible (e.g., in solution), then the chemical equilibrium of the tautomer can be reached. For example, proton tautomers (also known as proton transfer tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valenctautomers) include interconversion by recombination of some of the bonding electrons. A specific example of keto-enol tautomerism is the tautomerism of the pentan-2, 4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-ketone tautomerism. One specific example of phenol-ketone tautomerism is the tautomerism of pyridin-4-ol and pyridin-4 (1H) -one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

In the various parts of this specification, substituents of the disclosed compounds are disclosed in terms of group type or range. It is specifically intended that the invention includes each and every independent subcombination of the various members of these groups and ranges. For example, the term "C_1～6Alkyl "in particular denotes each independently of the other methyl, ethyl, C₃Alkyl radical, C₄Alkyl radical, C₅Alkyl and C₆An alkyl group.

In each of the parts of the invention, linking substituents are described. Where the structure clearly requires a linking group, the markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the markush group definition for the variable recites "alkyl," it is to be understood that "alkyl" represents an attached alkylene group or arylene group, respectively.

In addition, unless otherwise expressly indicated, the descriptions "… and … are each independently," "… and … are each independently," and "… and … are each independently" used throughout this document are interchangeable and should be broadly construed to mean that particular items expressed between the same symbols in different groups do not affect each other, or that particular items expressed between the same symbols in the same groups do not affect each other.

The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastrointestinal upset, dizziness and the like, when administered to a human. Preferably, the term "pharmaceutically acceptable" as used herein refers to those approved by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or matrix with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly injectable solutions. Suitable Pharmaceutical carriers are described in e.w. martin, "Remington's Pharmaceutical Sciences".

The definition and convention of stereochemistry in the present invention is generally used with reference to the following documents: S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E.and Wilen, S., "stereoschemistry of Organic Compounds", John Wiley & Sons, Inc., New York,1994. All stereoisomeric forms of the compounds of the present invention, including, but in no way limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefix D, L or R, S is used to indicate the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to designate the sign of the rotation of plane polarized light of the compound, with (-) or l indicating that the compound is left-handed and the prefix (+) or d indicating that the compound is right-handed. The chemical structures of these stereoisomers are identical, but their stereo structures are different. A particular stereoisomer may be an enantiomer, and a mixture of isomers is commonly referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during the chemical reaction. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.

"isomers" are different compounds having the same molecular formula. "stereoisomers" are isomers that differ only in the spatial arrangement of the atoms. The term "isomer" as used herein includes any and all geometric isomers 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 the present specification.

The "hydrate" of the present invention refers to the compound or salt thereof provided by the present invention, which further comprises water bonded by non-covalent intermolecular forces in a chemical amount or in a non-chemical equivalent amount, and may be said to be an association of solvent molecules with water.

"solvate" of the present invention refers to an association of one or more solvent molecules with a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.

"nitroxide" in the context of the present invention means that when a compound contains several amine functional groups, 1 or more than 1 nitrogen atom can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. The corresponding amines can be treated with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid) to form the N-oxide (see Advanced Organic Chemistry, Wiley Interscience, 4 th edition, Jerry March, pages). In particular, the N-oxide may be prepared by the method of L.W.Deady (Syn.Comm.1977,7,509-514) in which an amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.

The compounds may exist in a number of different geometric isomers and tautomers and the compounds of formula (I) -formula (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) -formula (III).

The term "prodrug", as used herein, means a compound that is converted in vivo to the compound shown in the present invention. Such conversion is effected by hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissue.

Unless otherwise indicated, all tautomeric forms of the compounds of the invention are included within the scope of the invention.

"metabolite" refers to the product of a particular compound or salt thereof obtained by metabolism in vivo. Metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by assay methods as described herein. Such products may be obtained by administering the compound by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a sufficient period of time.

Various pharmaceutically acceptable salt forms of the compounds of the present invention are useful. The term "pharmaceutically acceptable salts" means those salt forms that are readily apparent to the pharmaceutical chemist as being substantially non-toxic and providing 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, as a result, flowability of the drug substance. Briefly, the pharmaceutical composition can be prepared by combining the active ingredient with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as are: berge et al, description of the descriptive pharmaceutical 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 such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, nitrate and the like, and organic acid salts such as acetate, propionate, glycolate, oxalate, maleate, malonate, succinate, fumarate, tartrate, citrate, benzoate, mandelate, methanesulfonate, ethanesulfonate, toluenesulfonate, sulfosalicylate and the like, or obtained by other methods described in the literature such as ion exchange.

Other pharmaceutically acceptable salts include adipates, malates, 2-hydroxypropionic acid, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, cyclopentylpropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodiates, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurylsulfates, malates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, palmitates, embonate, pectinates, persulfates, 3-phenylpropionates, picrates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, glucarates, half sulfates, heptanates, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, decaMono-acid salts, valeric acid salts, and the like. Salts obtained with appropriate bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄A salt.

The present invention also contemplates quaternary ammonium salts formed from compounds containing groups of N. Water-soluble or oil-soluble or dispersion products can be obtained by quaternization. The alkali metal or alkaline earth metal salt includes sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt, iron salt, zinc salt, copper salt, manganese salt, aluminum salt and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations resistant to formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C_1-8Sulfonates and aromatic sulfonates. Amine salts such as, but not limited to, N '-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methyl reduced glucamine, 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; a transition metal salt such as, but not limited to, zinc.

In this specification, a structure is dominant if there is any difference between the chemical name and the chemical structure.

Abbreviations for any protecting groups, amino acids and other compounds used in the present invention shall be based on their commonly used, accepted abbreviations unless otherwise indicated, or refer to IUPAC-IUB Commission on Biochemical Nomenclature (see biochem.1972, 11: 942-944).

The invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of a compound shown in formula (I) or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials, carriers, excipients, menstruum or a combination thereof. When the compound of the present invention is administered in the form of a medicament to a mammal such as a human, it may be administered in the form of the compound itself or may be administered in the form of a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of an active ingredient and a pharmaceutically acceptable carrier.

"combination" means a fixed combination or a kit of parts for combined administration in the form of a single dosage unit, wherein the compounds disclosed herein and the combination partners (drugs for the treatment of tumor diseases, AIDS, inflammatory reactions and immunodeficiency diseases) can be administered separately at the same time or can be administered separately at certain intervals, in particular such that the combination partners show a cooperative, e.g. synergistic, effect. The term "pharmaceutical composition" as used herein means a product resulting from mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, such as the disclosed compounds and combination partners, are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, such as the compounds disclosed in this invention and the combination partners, are both administered to the patient as separate entities simultaneously, jointly or sequentially with no specific time limitation.

The phrase "pharmaceutically acceptable carrier" is art-recognized and includes pharmaceutically acceptable materials, compositions or carriers suitable for administration of the compounds of the invention to a mammal. The carrier comprises a liquid or solid filler, diluent, excipient, solvent or encapsulating material which is involved in carrying the subject substance or transferring it from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline water; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible materials used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical, buccal, sublingual, rectal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form is generally that amount of the compound which produces a therapeutic effect. Generally, the amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10 to about 30%, in units of one percent.

The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug containing a compound described herein to an individual in need thereof.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are carried out according to techniques or conditions described in literature in the art or according to techniques commonly used in the art.

Example 1

1. Chemical Synthesis of SF1

The phosphoramidite liquid phase one-bottle method synthesis strategy is adopted, and the synthesis route is shown as follows. The first partially fully protected phosphoramidite monomer D1(0.5mmol) was subjected to pyridine-TFA (pyridine-trifluoroacetate, 0.6mmol), water (18. mu.L) and t-BuNH₂(tert-butylamine, 2.5mL) after treatment, the phosphoramidite was changed to phosphorous acid; further, DMTr (4,4' -dimethoxytrityl) protecting group was removed by DCA (dichloroacetic acid, 6mL of 6% DCA in dichloromethane) to expose the hydroxyl group at the 6-position, yielding Compound D2. The second part of phosphoramidite monomer D3(0.6mmol) reacts with the 6-hydroxyl of the first part of phosphorous acid monomer through active trivalent phosphorus (D4), and forms dinucleotide phosphorothioate after DDTT (3- ((methylenethioadene) -amino) -3H-1,2,4-dithiazole-5-thione, 0.55mmol) is oxidized for 30 min. The second DMTr protecting group (D5) was also removed by DCA (8mL of 6% DCA in methylene chloride) and the cyclized ligation of the hydroxyl group at the 6-position to phosphorous acid was achieved by the addition of the cyclization agent DMOCP (5, 5-dimethyl-2-chloro-1, 3, 2-dioxaphosphorinanyl phosphate, 1.75 mmol). Subsequent addition of iodine (165mg) effected oxidation of the phosphite to afford the phosphate and phosphorothioate (D6), respectively. Finally, the rest of other protecting groups are removed by tert-butylamine (2.5mL), methylamine (10mL) and hydrofluoric acid triethylamine salt (0.83mL), and the corresponding cyclic dinucleotide product SF1 can be obtained by crystallization in acetone. The yield was about 40%. 1H NMR (400M, D)₂O)8.26–7.71(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,D₂O)55.35,54.96,-1.05.¹⁹F NMR(400M,D₂O) -122.42, -130.51.ESI-HRMS (negative mode): C₂₀H₂₂FN₁₀O₁₂P₂S^-[M-H]^-Theoretical value 707.0604; found 707.0606.

2. Covalent attachment of SF1 to alpha-halo linker arm

Dissolving SF1 in DMF, adding 2 times of equivalent of p-hydroxymethyl benzyl bromide, reacting for 2 hours, separating liquid phase to obtain product, and separating gradient 2-40% of mobile phase B for 30min, wherein the mobile phase A is 100% acetonitrile, and the mobile phase B is triethylamine acetate buffer (pH 7). ESI-HRMS (negative mode): C₂₈H₃₀FN₁₀O₁₃P₂S^-[M-H]^-Theoretical 827.1168, found 827.1164.

Example 2

SF1 murine macrophage activation evaluation

Murine macrophage J774A.1 was used as the evaluation cell line. The stimulation of the compounds was evaluated by flow analysis using SF1 at a stimulation concentration of 10. mu.M and an incubation time of 14h, followed by staining the markers with the PE-anti CD86 (activation marker) antibody. Dithio cdg (dithio cdg), currently in the form of dithio CDN for clinical trials, was set as a control group. Given the relatively poor transmembrane effect of cyclic dinucleotides, we measured the activation effect in the presence and absence of transfection reagents.

The results are shown in FIG. 1, in the left panel of FIG. 1, the abscissa represents the fluorescence intensity and the ordinate represents the number of cells; in the right panel of FIG. 1, the abscissa represents the subject and the ordinate represents the mean fluorescence intensity of CD86-PE, wherein dithio CDG + lipo represents the pretreatment of dithio CDG with a transfection reagent and SF1+ lipo represents the pretreatment of SF1 with a transfection reagent. The results show that SF1 can effectively activate antigen presenting cells, the stimulation effect is more obvious under the help of the transmembrane promotion of a transfection reagent, and the activity of SF1 is better than that of dithio CDG under both conditions.

Example 3

In vivo and in vitro BM1 compared with SF1 stimulation

To compare the activity of the linker-linked structures, the prepared compounds were evaluated at the cell level and the mouse level. The cell layer adopts mouse-derived macrophage J774A.1 as an evaluation cell line. SF1 and BM1 stimulation concentration 10. mu.M, transfection reagent pretreatment, incubation time 14h, after which the compounds were evaluated for stimulation effect by flow analysis using a PE-anti CD86 (activation marker) antibody staining marker.

The results are shown in FIG. 2, which shows that the compound of formula SF1 is more effective than the compound of formula BM1 at the cellular level.

To further evaluate BM1, we performed in vivo evaluations using female C57BL/6 mice. Mice were immunized intraperitoneally with a compound of formula SF1, a compound of formula BM1 (20 nmol). After 20 hours, spleens of mice were ground and dispersed into single cells, stained and flow-labeled with anti-CD11c (dendritic cell marker), anti-F4/80 (macrophage marker) and anti-CD86 antibody.

The results are shown in fig. 3, and indicate that under in vivo conditions, the compound of formula BM1 and the compound of formula SF1 have equivalent stimulatory effects, indicating that the ligation methods and sites can be applied to various covalent applications of cyclic dinucleotides.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A method for attaching a linker arm to a compound of formula SF, wherein the linker arm is attached to S in the phosphorothioate of the compound of formula SF,

2. the method according to claim 1, characterized in that it comprises: reacting the S group of the compound of formula SF with a halide by nucleophilic substitution to produce the linker arm product.

3. The method of claim 2, wherein the halide is an alpha halide, optionally the alpha halide is a benzyl halide or a haloacetyl.

4. The method of claim 3, wherein B is selected from the group consisting of compounds of formula SF₁Or B₂Each independently is a base; x is selected from-H, -OH, -F; y is selected from-OH and-SH;

optionally, the base is selected from natural base or non-natural base, and the natural base is selected from base A, base G, base C, base T and base U.

5. The method according to claim 1, wherein the compound of formula SF is prepared by the following steps:

n is 2, and Z is selected from silicon hydroxyl and F.

6. The method according to claim 5, wherein in the step (4), the cyclizing reagent is 5, 5-dimethyl-2-chloro-1, 3, 2-dioxaphosphorinanyl phosphate;

the oxidant is at least one selected from iodine or 3H-1, 2-benzodithiol-3-one 1, 1-dioxide.

7. The method according to claim 5, wherein the deprotection reaction is performed on the compound represented by the formula S1, pyridine-trifluoroacetate, tert-butylamine and dichloroacetic acid in the step (1), and the deprotection reaction is performed at room temperature;

optionally, the phosphorylation reaction in step (2) is carried out under anhydrous conditions;

optionally, the oxidation and deprotection reactions in step (3) are carried out at room temperature;

optionally, the nucleophilic substitution and oxidation reaction in step (4) is carried out at room temperature;

optionally, the nucleophilic substitution reaction in step (5) is performed under room temperature conditions;

optionally, the deprotection reaction in step (6) is carried out under an oil bath at 50 ℃.

8. A compound obtained by attaching a linker arm to a compound of formula SF by the method of any one of claims 1 to 7.

9. A pharmaceutical composition, comprising:

a compound; and

pharmaceutically acceptable adjuvants, carriers, excipients, solvents or their combination,

the compound of claim 8.

10. Use of a compound according to claim 8 in the manufacture of a medicament for the treatment of an immune disorder;

optionally, the medicament is for the treatment of tumors or for antiviral, antibacterial;

optionally, the medicament is for activating an interferon gene stimulating protein.

11. A pharmaceutical combination, comprising:

a compound of claim 8; and

at least one drug for treating an immune disorder;

optionally, the at least one drug for treating an immune disease is selected from at least one of an immune checkpoint blocking antibody, an immunostimulant, a vaccine, a chimeric antigen receptor T cell, radiotherapy, and a chemotherapeutic drug.