CN118619928A - Nitrogen oxyalkyl chain substituted benzimidazole dimer and preparation method and application thereof - Google Patents

Abstract

The invention relates to a benzimidazole dimer substituted by a nitroxide alkyl chain, a preparation method and application thereof. Specifically, the compound has a structure shown in a formula I, wherein the definition of each group and substituent is described in the specification. The invention also discloses a preparation method of the compound and application of the compound in preventing and/or treating diseases such as tumors.

Description

Nitrogen oxyalkyl chain substituted benzimidazole dimer and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a nitrogen-oxygen alkyl chain substituted benzimidazole dimer, and a preparation method and application thereof.

Background

Interferon gene stimulating factor STING (STimulator of INterferon Genes) is an important protein molecule of human body involved in antiviral innate immune response, and is a multi-domain transmembrane protein with molecular weight of 42kDa and located in endoplasmic reticulum membrane. It consists of 379 amino acids, contains a transmembrane region (TM 1-4, aa 1-154), a cyclic dinucleotide-binding region (CBD, aa 155-341) and a carbon segment tail (CTT, aa 342-379). STING is widely distributed in endothelial cells, epithelial cells and a series of hematopoietic cells, such as T cells, macrophages, dendritic cells, etc., and is a key regulatory protein for the production of type I interferon. During viral or bacterial infection, exogenous DNA from a pathogenic bacterium can be recognized by the host cytoplasmic nucleic acid receptor cGAS (cyclic Guanosine Monophosphate (GMP) -Adenosine Monophosphate (AMP) synthase), activating cGAS enzymatic functions. cGAS synthesizes the second messenger molecule cGAMP (2 ',3' -CYCLIC GMP-AMP) using ATP and GTP. The interaction of cGAMP and endoplasmic reticulum localization protein STING results in a conformational change in STING, thereby recruiting and activating downstream TBK1 kinase and IRF3 transcription factors. IRF3 enters the nucleus to promote the expression of Type I interferon (Type I IFN), and initiates a natural immune response to resist invasion of pathogenic bacteria. cGAS-STING is a natural immune important signaling pathway in the body, and its abnormal activation or inhibition is associated with a variety of diseases including viral infection, autoimmune diseases, and tumors. Among them, anti-tumor immune response is receiving a great deal of attention. Activating cGAS-STING channel, promoting I-type interferon release, activating APC cell, activating T cell to generate adaptive anti-tumor immune response, inducing immune memory, and achieving persistent tumor killing effect. Thus, targeting activation of cGAS-STING signaling pathway is expected to be the most promising new target for immunotherapy, especially in combination with PD-1/PD-L1 antibodies for treatment of "cold" tumors without T-cell infiltration, relative to immune checkpoint inhibitors targeting immunosuppressive molecules.

Although there are a number of STING small molecule agonists that have entered clinical trials, clinical progression is slow. Among them, ADU-S100 (stage II) developed by Aduro and MK1454 (stage I) developed by merck, both of which are cyclic dinucleotide analogs and are administered by intratumoral injection. The medicine has the defects of large molecular weight, poor cell membrane permeability, negative electricity containing phosphate groups in the structure, extremely easy hydrolysis of phosphate bonds, extremely poor PK properties and the like, and severely limits the clinical use of the compound. In 2018, GSK corporation reported the first benzimidazole STING agonist (Nature 2018,564,439-443) that could be administered intravenously, however, this class of compounds still has serious metabolic problems.

Therefore, there is a need to further develop novel, highly potent and safe STING small molecule agonists that induce the production of type I interferon IFN- β.

Disclosure of Invention

The invention aims to provide a compound shown in a formula I, a preparation method thereof and application thereof in preventing and/or treating diseases such as tumors.

In a first aspect of the invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt, tautomer thereof,

Wherein,

W is selected from the group consisting of: CH ₂, NH, O;

n ₁ is selected from the group consisting of: 0.1, 2 and 3;

n ₂ is selected from the group consisting of: 0.1, 2 and 3;

Ring C is selected from the group consisting of substituted or unsubstituted: a 5-, 6-or 7-membered monocyclic heterocycloalkyl group containing 1 or 2N, a 7-, 8-, 9-or 10-membered spirocycloalkyl group containing 2N, an 8-, 9-or 10-membered fused-ring heterocycloalkyl group containing 2N, a 7-or 8-membered bridged-ring heterocycloalkyl group containing 2N, said substitution being substituted by 1,2, 3 or 4R ₁;

R ₁ are each independently selected from the group consisting of: C1-C6 alkyl, halogen, hydroxy, -COOH, - (C=O) -O-C1-C6 alkyl, - (C=O) -C1-C6 alkyl, -NH-C1-C6 alkyl, haloC 1-C6 alkyl, hydroxy-substituted C1-C6 alkyl;

or 2R ₁ attached to the same atom together with the atom form a C3-C6 cycloalkyl or a 4-5 membered heterocycloalkyl containing 1O;

Z is selected from the group consisting of: absence, =o, -CH ₂ -NH- (c=o) -;

X is O or NH or is absent;

Y is NH;

R is selected from the group consisting of: H. - (C=O) -O-C1-C6 alkyl,

In another preferred embodiment, W is O.

In another preferred embodiment, n ₁ is 1.

In another preferred embodiment, n ₂ is 1.

In another preferred embodiment, the 5, 6 or 7 membered monocyclic heterocycloalkyl containing 1 or 2N is selected from the group consisting of:

in another preferred embodiment, the 7, 8, 9 or 10 membered spirocyclic heterocycloalkyl containing 2N is selected from the group consisting of:

in another preferred embodiment, the 8, 9 or 10 membered, heterocyclic alkyl group containing 2N is In another preferred embodiment, the 7 or 8 membered bridged heterocycloalkyl containing 2N is selected from the group consisting of:

In another preferred embodiment, Z is selected from the group consisting of: absent, =o, -CH ₂ -NH- (c=o) -.

In another preferred embodiment, Z is =o.

In another preferred embodiment, X is O or NH or is absent.

In another preferred embodiment, X is O or NH.

In another preferred embodiment, X is absent.

In another preferred embodiment, R is selected from the group consisting of: H. - (c=o) -O-tert-butyl,

In another preferred embodiment, the compound is selected from the group consisting of:

in a second aspect of the invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of a compound of the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer thereof.

In a third aspect of the invention there is provided the use of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer thereof, for the manufacture of a medicament which is a STING agonist.

In another preferred embodiment, the medicament is for use selected from the group consisting of:

1) For preventing and/or treating a disease selected from the group consisting of: tumor and infectious diseases;

2) As an immune composition or vaccine adjuvant.

In another preferred embodiment, the disease is STING related disease.

In another preferred embodiment, the disease is a disease of low STING expression.

In another preferred embodiment, the tumor is selected from the group consisting of: breast cancer, ovarian cancer, liver cancer, melanoma, prostate cancer, colon cancer, colorectal cancer, gastric cancer, pancreatic cancer, bile duct cancer, head and neck cancer, brain glioma, endometrial tumor, lung cancer.

In another preferred embodiment, the infectious disease is selected from the group consisting of: viral infection, bacterial infection, fungal infection.

In another preferred embodiment, the viral infection is an infection with a virus selected from the group consisting of: new coronaviruses, ebola viruses, influenza viruses, simian bean viruses, herpesviruses, dengue viruses.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows fold activation of STING signals in THP1-dual cells by different concentrations of compound S1 and positive drug diABZI.

FIG. 2 shows the dose-response curves of compound S1 and positive drug diABZI on THP1-dual cell STING agonist activity.

FIG. 3 shows fold activation of STING signals in THP1-dual cells by different concentrations of compound S29 and positive drug diABZI.

FIG. 4 shows the dose-response curves of compound S29 and positive drug diABZI on THP1-dual cell STING agonist activity.

Figure 5 shows the in vivo anti-tumor efficacy of compound S2 in a CT26 mouse colorectal cancer model.

Detailed Description

The present inventors have conducted long and intensive studies to unexpectedly prepare a compound of formula I having excellent STING agonistic activity through structural optimization. On this basis, the inventors completed the present invention.

Terminology

In the present invention, unless otherwise indicated, terms used have the ordinary meanings known to those skilled in the art.

In the present invention, the term "halogen" refers to F, cl, br or I.

In the present invention, "C1-C6 alkyl" means a straight-chain or branched alkyl group comprising 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, neopentyl, t-pentyl, or the like.

In the present invention, the term "C2-C6 alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms containing one double bond, including without limitation ethenyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl and the like.

In the present invention, the term "C2-C6 alkynyl" refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms containing one triple bond, and includes, without limitation, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like.

In the present invention, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The term "C3-C6 cycloalkyl" has similar meaning.

In the present invention, the term "halo" refers to substitution with halogen.

In the present invention, the term 1-6 refers to 1,2, 3, 4, 5 or 6. Other similar terms each independently have similar meanings.

It will be understood that when a group is present in a compound in a plurality of different positions at the same time, the definition of each position is independent of the other and may be the same or different. That is, the term "selected from the group consisting of: the "and the term" are each independently selected from the group consisting of: "has the same meaning.

Compounds of formula (I)

The invention provides a compound shown in a formula I, or pharmaceutically acceptable salts and tautomers thereof,

Wherein each group is as defined above.

In another preferred embodiment, any of W, n ₁、n₂ and C, Z, X, Y, R in the compounds described are each independently a group corresponding to a particular compound described herein.

As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and the like; amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

Another preferred class of salts are salts of the compounds of the invention with bases, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butylamine, ethylenediamine, hydroxyethylamine, dihydroxyethylamine, and triethylamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

It will be appreciated that the examples of the present invention specifically describe the preparation of the compounds of formula I of the present invention, but these specific methods do not constitute any limitation on the present invention. The compounds of the present invention may also be conveniently prepared by optionally combining the various synthetic methods described in this specification or known in the art, such combinations being readily apparent to those skilled in the art to which the present invention pertains.

It will be appreciated that the starting materials and reagents used in the preparation process of the compounds of the invention are commercially available unless otherwise indicated.

Pharmaceutical compositions and methods of administration

The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound, or a pharmaceutically acceptable salt, tautomer thereof.

Because the compound of the present invention has excellent antitumor activity, the compound of the present invention and various crystalline forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient are useful for the treatment, prevention and alleviation of diseases associated with tumors.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 10-1000mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The pharmaceutical composition is injection, capsule, tablet, pill, powder or granule.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., antineoplastic agents).

The methods of treatment of the present invention may be administered alone or in combination with other therapeutic means or therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 50 to 1000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Compared with the prior art, the invention has the following main advantages:

(1) The compound has remarkably more excellent STING agonistic activity;

(2) The compound has remarkably more excellent water solubility, pharmacokinetics performance and safety performance;

(3) The compound has excellent anti-tumor effect.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

1. Preparation example

The following preparation examples are illustrative of the preparation of a portion of the compounds of formula I of the present invention.

1. Synthesis of Compound S1 and Compound S2

Synthesis of Compounds 1-2:

Compounds 1-1 (2 g,8.67 mmol) were suspended in dichloromethane (28 mL), cooled to 0deg.C after N ₂ protection, and a solution of boron tribromide in dichloromethane (1M, 52 mL) was slowly added and stirred overnight at 40deg.C. After the reaction was completed, the reaction solution was slowly added dropwise to about 300mL of ice water, stirred for 30 minutes after the completion of the addition, and the solid was suction-filtered and dried to obtain Compound 1-2 [ Synthesis of Compound 1-1 referred to Nature 2018,564,439-443]

Synthesis of Compounds 1-3:

Compound 1-2 (500 mg,2.31 mmol), fragment 1 (428 mg,2.77 mmol) and potassium carbonate (418 mg,3.00 mmol) were dissolved in N, N-dimethylformamide (4 mL). Stirred at 70℃overnight. After the reaction is completed, the reaction solution is directly stirred and put on a column, and the compounds 1-3 are obtained after column purification.

Synthesis of Compounds 1-5:

Compounds 1 to 4 (379 mg,1.35 mmol) and 1 to 3 (500 mg,1.12 mmol) were weighed into a microwave reactor, 4mL of isopropanol was added thereto, and N, N-diisopropylethylamine (1 mL) was added dropwise with stirring. The reaction was carried out at 130℃for 14 hours with the aid of microwaves. After the reaction is completed, the reaction solution is spin-dried, dissolved by using a dichloromethane/methanol mixed solvent, and then is stirred with silica gel and is put on a column, and the compounds 1-5 are obtained through column purification. [ Synthesis of Compounds 1-4, reference Nature2018,564,439-443]

Synthesis of Compounds 1-6:

Compounds 1-5 (450 mg,0.65 mmol) were dissolved in methanol (4 mL) and dichloromethane (4 mL), sodium dithionite (1.7 g,9.82 mmol) was dissolved in water (8 mL) and then slowly added dropwise to the above system, followed by stirring at room temperature for 10 min. After the reaction was complete, sodium bicarbonate (1.6 g,19.65 mmol) was added and stirring was continued for 15 minutes. Removing insoluble substances from the reaction solution by suction filtration, dissolving the filtrate by methanol after spin drying, removing insoluble substances by suction filtration again, loading the filtrate on a column by mixing neutral alumina, and purifying by the column to obtain the compounds 1-6.

Synthesis of Compounds 1-8:

Compounds 1-6 (530 mg,0.84 mmol) were dissolved in N, N-dimethylformamide (5 mL). 1, 4-Dioxahexacyclic solution of Compounds 1-7 (0.4M, 5.3 mL) was added dropwise at 0deg.C. After stirring at 0℃for 15 minutes, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (405 mg,2.11 mmol) was added, followed by slow dropwise addition of triethylamine (0.6 mL,4.22 mmol), and the addition was warmed to room temperature for 2 hours. After the reaction is completed, the reaction solution is directly stirred and put on a column, and the compounds 1-8 are obtained through column purification. [ Synthesis of Compounds 1-7 Ref CN109071514]

Synthesis of Compounds 1-9:

compounds 1-8 (750 mg,0.79 mmol) were dissolved in methanol (8 mL) and dichloromethane (8 mL), and a1, 4-dioxane solution of hydrogen chloride (4M, 3 mL) was added and stirred at room temperature for 1 hour. After the reaction is completed, spin-drying is carried out to obtain the compounds 1-9.

Synthesis of compound S1:

Compounds 1 to 9 (550 mg,0.64 mmol) and t-butoxycarbonylaminooxyacetic acid (186 mg,0.97 mmol) were dissolved in N, N-dimethylformamide (5 mL), cooled to 0℃and N, N-diisopropylethylamine (0.45 mL,2.59 mmol) was added dropwise with stirring, stirring was continued for 30 minutes, HATU (370 mg,0.97 mmol) was added and the addition was allowed to stand at room temperature for 2 hours. After the reaction is completed, the reaction solution is completely spin-dried, and is beaten by ethanol petroleum ether, and the solid is filtered out and dried to obtain the compound S1.¹H NMR(400MHz,DMSO-d6)δ12.83(s,2H),10.07(s,1H),7.97(s,2H),7.64(d,J＝9.3Hz,2H),7.35(s,2H),7.28(d,J＝14.0Hz,2H),6.55(s,1H),6.51(d,J＝3.0Hz,1H),5.81(s,2H),4.91(d,J＝12.5Hz,4H),4.53(s,4H),4.37(s,2H),3.93(s,2H),3.69(s,3H),2.25(s,6H),2.11(dd,J＝8.7,3.0Hz,4H),1.65(s,2H),1.40(d,J＝3.1Hz,9H),1.33-1.21(m,6H).

Synthesis of compound S2:

Compound S1 (694 mg,0.68 mmol) was dissolved in methanol (7 mL) and dichloromethane (7 mL), and a 1, 4-dioxane solution (3 mL) of 4M hydrogen chloride was added and stirred at room temperature for 1 hour. After the reaction is completed, spin drying is carried out to obtain the compound S2.¹H NMR(400MHz,DMSO-d6)δ11.03(s,2H),8.02(d,J＝27.9Hz,2H),7.66(d,J＝2.7Hz,2H),7.36(s,4H),7.34(s,2H),6.51(s,2H),5.89-5.63(m,2H),4.93(s,4H),4.90(s,4H),4.51(d,J＝7.5Hz,4H),4.03(s,2H),3.73(s,3H),3.36(d,J＝13.0Hz,2H),3.16(t,J＝1.5Hz,4H),2.10(s,6H),2.02(s,4H),1.31–1.20(m,6H).LCMS(m/z):922.4[M+H]⁺

2. Synthesis of Compound S3 and Compound S4

Synthesis of fragment 2:

Compound 2-1 (1.08 g,5.39 mmol) and compound 2-2 (1.02 g,6.47 mmol) were dissolved in acetone (10 mL), and an aqueous solution of sodium hydroxide (5M, 1.61 mL) was slowly added dropwise and stirred overnight at room temperature. After the reaction is completed, the solvent is dried by rotation, the solvent is dissolved by dichloromethane and washed twice, the organic layer is dried by saturated saline solution and anhydrous sodium sulfate, then the mixture is stirred and put on a column, and the fragment 2 is obtained after the mixture is purified by the column.

Synthesis of Compound 2-3:

The synthesis of compounds 2-3 is identical to that of compounds 1-3, except that fragment 2 is substituted for fragment 1.

Synthesis of Compounds 2-5:

the synthesis of compounds 2-5 is identical to that of compounds 1-5.

Synthesis of Compounds 2-6:

The synthesis of compounds 2-6 is identical to that of compounds 1-6, except that 2-5 is substituted for 1-5.

Synthesis of Compounds 2-7:

The synthesis of compounds 2-7 is identical to that of compounds 1-7, except that compounds 1-6 are replaced with 2-6.

Synthesis of Compounds 2-8:

The synthesis of compounds 2-8 is identical to that of compounds 1-8, except that 2-7 is substituted for 1-7.

Synthesis of Compounds 2-9:

The synthesis of compounds 2-9 is identical to that of compounds 1-9, except that 2-8 is substituted for 1-8.

Synthesis of compound S3:

The synthesis of compound S3 is the same as that of compound S1, except that 1-9 is replaced with 2-9 .¹H NMR(400MHz,DMSO-d6)δ12.81(s,2H),10.08(d,J＝22.1Hz,1H),7.99(d,J＝9.3Hz,2H),7.65(d,J＝4.5Hz,2H),7.33(s,2H),7.30(s,2H),6.56-6.48(m,2H),5.83(dd,J＝19.8,14.1Hz,2H),4.91(dd,J＝12.9,4.8Hz,4H),4.52(q,J＝8.0Hz,4H),4.42(s,1H),4.39(s,1H),4.26(s,2H),4.03–3.93(m,2H),3.72(d,J＝5.7Hz,3H),3.46(s,2H),3.41(s,2H),2.11(d,J＝6.8Hz,6H),1.77(s,4H),1.40(s,9H),1.38(s,4H),1.27(q,J＝7.2Hz,6H).

Synthesis of compound S4:

The synthesis of compound S4 is identical to that of compound S2, except that S3 is substituted for S1 .¹H NMR(400MHz,DMSO-d6)δ11.75(s,2H),9.97(s,2H),9.50(s,2H),8.05(d,J＝20.0Hz,2H),7.67(dd,J＝6.2,1.2Hz,2H),7.39(s,2H),7.36(s,2H),5.90-5.65(m,2H),5.06-4.88(m,4H),4.50(q,J＝7.1Hz,4H),4.05(s,2H),3.74(s,3H),3.70-3.59(m,3H),3.53(s,3H),3.41(s,1H),3.30(s,1H),3.17(d,J＝9.0Hz,4H),2.50(p,J＝1.8Hz,6H),2.16(s,2H),2.09-1.98(m,2H),1.25(td,J＝7.1,1.4Hz,6H).

3. Synthesis of Compound S5 and Compound S6

Synthesis of fragment 3:

the synthesis of fragment 3 is identical to that of fragment 2, except that 3-1 is substituted for 2-1.

Synthesis of Compound 3-3:

the synthesis of compound 3-3 is identical to that of compound 1-3, except that fragment 3 is substituted for fragment 1. Synthesis of Compounds 3-5:

The synthesis of compounds 3-5 is identical to that of compounds 1-5.

Synthesis of Compounds 3-6:

the synthesis of compounds 3-6 is identical to that of compounds 1-6, except that 3-5 is substituted for 1-5.

Synthesis of Compounds 3-7:

the synthesis of compounds 3-7 is identical to that of compounds 1-7, except that 3-6 is substituted for 1-6.

Synthesis of Compounds 3-8:

the synthesis of compounds 3-8 is identical to that of compounds 1-8, except that 3-7 is substituted for 1-7.

Synthesis of Compounds 3-9:

the synthesis of compounds 3-9 is identical to that of compounds 1-9, except that 3-8 is substituted for 1-8.

Synthesis of compound S5:

The synthesis of compound S5 is the same as that of compound S1, except that 3-9 is substituted for 1-9 .¹H NMR(400MHz,DMSO-d6)δ10.08(s,2H),8.56(s,1H),8.35(s,2H),8.07(d,J＝17.1Hz,2H),7.65(s,2H),7.26(d,J＝12.0Hz,2H),6.52(d,J＝11.8Hz,2H),5.82(s,2H),4.90(d,J＝19.6Hz,4H),4.51(s,4H),4.32(s,2H),4.00(s,2H),3.72(s,3H),3.47(s,2H),3.37(s,4H),2.86(s,4H),2.10(d,J＝8.6Hz,6H),1.83(d,J＝24.6Hz,6H),1.46-1.34(m,9H),1.26(q,J＝7.2Hz,6H).

Synthesis of compound S6:

The synthesis of compound S6 is identical to that of compound S2, except that S5 is substituted for S1 .¹H NMR(400MHz,DMSO-d6)δ11.58(d,J＝31.1Hz,2H),9.93(s,2H),9.52(s,2H),8.05(d,J＝26.3Hz,2H),7.66(d,J＝4.2Hz,2H),7.36(s,2H),6.51(s,2H),5.88-5.62(m,2H),4.92(d,J＝18.6Hz,3H),4.50(d,J＝7.5Hz,4H),4.07(s,2H),3.75(d,J＝5.8Hz,2H),3.69-3.58(m,1H),3.50(s,1H),3.36(d,J＝20.0Hz,2H),3.21(s,4H),3.10–2.94(m,1H),2.72(s,1H),2.10(s,6H),1.98(s,4H),1.25(q,J＝7.2,6.0Hz,6H).

4. Synthesis of Compound S7 and Compound S8

Synthesis of fragment 4:

The synthesis of fragment 4 is identical to that of fragment 2, except that fragment 2-1 is replaced with 4-1.

Synthesis of Compound 4-3:

The synthesis of compound 4-3 is identical to that of compound 1-3, except that fragment 4 is substituted for fragment 1. Synthesis of Compounds 4-5:

the synthesis of compounds 4-5 is identical to that of compounds 1-5.

Synthesis of Compounds 4-6:

the synthesis of compound 4-6 is identical to that of compound 1-6, except that 1-5 is replaced with 4-5.

Synthesis of Compounds 4-7:

the synthesis of compounds 4-7 is identical to that of compounds 1-7, except that compounds 1-6 are replaced with 4-6.

Synthesis of Compounds 4-8:

The synthesis of compounds 4-8 is identical to that of compounds 1-8, except that 4-7 is substituted for 1-7.

Synthesis of Compounds 4-9:

The synthesis of compounds 4-9 is identical to that of compounds 1-9, except that 4-8 is substituted for 1-8.

Synthesis of compound S7:

The synthesis of compound S7 is the same as that of compound S1, except that 4-9 is substituted for 1-9 .¹H NMR(400MHz,DMSO-d6)δ12.78(s,2H),10.06(d,J＝17.9Hz,1H),7.87(d,J＝7.8Hz,2H),7.56(d,J＝6.4Hz,2H),7.31(s,2H),7.28(s,2H),6.5-6.46(m,2H),5.79(dd,J＝17.4,13.7Hz,2H),4.89(dd,J＝10.8,3.4Hz,4H),4.52(q,J＝7.8Hz,4H),4.37(s,2H),4.31(s,2H),4.24(s,2H),4.05-3.95(m,4H),3.72(d,J＝7.5Hz,3H),3.54(s,2H),3.45(s,2H),2.09(d,J＝5.7Hz,6H),1.74(s,4H),1.41(s,9H),1.39(s,4H),1.26(q,J＝7.2Hz,6H).

Synthesis of compound S8:

The synthesis of compound S8 is identical to that of compound S2, except that S7 is substituted for S1 .¹H NMR(400MHz,DMSO-d6)δ11.67(s,2H),9.89(s,2H),9.48(s,2H),8.15(d,J＝17.6Hz,2H),7.74(dd,J＝3.4,5.6Hz,2H),7.42(s,2H),7.34(s,2H),5.78-5.63(m,2H),5.03–4.78(m,4H),4.46(q,J＝6.8Hz,4H),4.15(s,2H),3.71(s,3H),3.69-3.57(m,4H),3.49(s,4H),3.40(s,2H),3.33(s,2H),3.15(d,J＝7.6Hz,4H),2.50(d,J＝1.7Hz,6H),2.14(s,4H),2.11-1.99(m,2H),1.25(q,J＝6.9,1.3Hz,6H).

5. Synthesis of Compound S9

Synthesis of fragment 5:

Fragment 5 was synthesized identically to fragment 2, except that 5-1 was substituted for 2-1.

Synthesis of Compound 5-3:

The synthesis of compound 5-3 is identical to that of compound 1-3, except that fragment 5 is substituted for fragment 1. Synthesis of Compounds 5-5:

The synthesis of compounds 5-5 is identical to that of compounds 1-5.

Synthesis of Compounds 5-6:

The synthesis of compounds 5-6 is identical to that of compounds 1-6, except that 5-5 is substituted for 1-5.

Synthesis of Compounds 5-7:

the synthesis of compounds 5-7 is identical to that of compounds 1-7, except that compounds 1-6 are replaced with 5-6.

Synthesis of Compounds 5-8:

The synthesis of compounds 5-8 is identical to that of compounds 1-8, except that compounds 1-7 are replaced with 5-7.

Synthesis of Compounds 5-9:

The synthesis of compounds 5-9 is identical to that of compounds 1-9, except that 5-8 is substituted for 1-8.

Synthesis of Compounds 5-10:

The synthesis of compound 5-10 is identical to that of compound S1, except that 1-9 is replaced with 5-9.

Synthesis of compound S9:

the synthesis of compound S9 is identical to that of compound S2, except that S1 is replaced with 5-10. LCMS (m/z): 922.4[ M+H ] ⁺

6. Synthesis of Compound S10

Synthesis of fragment 6:

The synthesis of fragment 6 is identical to that of fragment 2, except that fragment 2-1 is replaced with 6-1.

Synthesis of Compound 6-3:

the synthesis of compound 6-3 is identical to that of compound 1-3, except that fragment 6 is substituted for fragment 1. Synthesis of Compound 6-5:

The synthesis of compound 6-5 is identical to that of compound 1-5.

Synthesis of Compound 6-6:

the synthesis of compound 6-6 is identical to that of compound 1-6, except that 1-5 is replaced with 6-5.

Synthesis of Compounds 6-7:

The synthesis of compound 6-7 is identical to that of compound 1-7, except that 1-6 is replaced with 6-6.

Synthesis of Compounds 6-8:

the synthesis of compounds 6-8 is identical to that of compounds 1-8, except that compounds 1-7 are replaced with 6-7.

Synthesis of Compounds 6-9:

the synthesis of compounds 6-9 is identical to that of compounds 1-9, except that compounds 1-8 are replaced with 6-8.

Synthesis of Compounds 6-10:

The synthesis of compound 6-10 is identical to that of compound S1, except that 1-9 is replaced with 6-9.

Synthesis of compound S10:

The synthesis of compound S10 is identical to that of compound S2, except that S1 is replaced with 6-10. LCMS (m/z): 934.4[ M+H ] ⁺

7. Synthesis of Compound S11

Synthesis of fragment 7:

the synthesis of fragment 7 was identical to that of fragment 2, except that 7-1 was used in place of 2-1.

Synthesis of Compound 7-3:

The synthesis of compound 7-3 is identical to that of compound 1-3, except that fragment 7 is substituted for fragment 1. Synthesis of Compound 7-5:

the synthesis of compounds 7-5 was identical to that of compounds 1-5.

Synthesis of Compounds 7-6:

The synthesis of compound 7-6 is identical to that of compound 1-6, except that 7-5 is substituted for 1-5.

Synthesis of Compounds 7-7:

The synthesis of compound 7-7 is identical to that of compound 1-7, except that 1-6 is replaced with 7-6.

Synthesis of Compounds 7-8:

The synthesis of compounds 7-8 is identical to that of compounds 1-8, except that 7-7 is substituted for 1-7.

Synthesis of Compounds 7-9:

The synthesis of compounds 7-9 is identical to that of compounds 1-9, except that 7-8 is substituted for 1-8.

Synthesis of Compounds 7-10:

the synthesis of compound 7-10 is identical to that of compound S1, except that 1-9 is replaced with 7-9.

Synthesis of compound S11:

The synthesis of compound S11 is identical to that of compound S2, except that S1 is replaced with 7-10. LCMS (m/z): 944.4[ M+H ] ⁺

8. Synthesis of Compound S12

Synthesis of fragment 8:

the synthesis of fragment 8 is identical to that of fragment 2, except that fragment 2-1 is replaced with 8-1.

Synthesis of Compound 8-3:

The synthesis of compound 8-3 is identical to that of compound 1-3, except that fragment 8 is substituted for fragment 1. Synthesis of Compound 8-5:

the synthesis of compound 8-5 is identical to that of compound 1-5.

Synthesis of Compounds 8-6:

The synthesis of compound 8-6 is identical to that of compound 1-6, except that 1-5 is replaced with 8-5.

Synthesis of Compounds 8-7:

the synthesis of compound 8-7 is identical to that of compound 1-7, except that 1-6 is replaced with 8-6.

Synthesis of Compounds 8-8:

The synthesis of compound 8-8 is identical to that of compound 1-8, except that 1-7 is replaced with 8-7.

Synthesis of Compounds 8-9:

the synthesis of compounds 8-9 is identical to that of compounds 1-9, except that 8-8 is substituted for 1-8.

Synthesis of Compounds 8-10:

the synthesis of compound 8-10 is identical to that of compound S1, except that 1-9 is replaced with 8-9.

Synthesis of compound S12:

the synthesis of compound S12 is identical to that of compound S2, except that S1 is replaced with 8-10. LCMS (m/z): 966.4[ M+H ] ⁺

9. Synthesis of Compound S13

Synthesis of fragment 9:

The synthesis of fragment 9 was identical to that of fragment 2, except that 9-1 was used in place of 2-1.

Synthesis of Compound 9-3:

The synthesis of compound 9-3 is identical to that of compound 1-3, except that fragment 9 is substituted for fragment 1. Synthesis of Compound 9-5:

The synthesis of compound 9-5 is identical to that of compound 1-5.

Synthesis of Compound 9-6:

The synthesis of compound 9-6 is identical to that of compound 1-6, except that 9-5 is substituted for 1-5.

Synthesis of Compounds 9-7:

The synthesis of compound 9-7 is identical to that of compound 1-7, except that 9-6 is substituted for 1-6.

Synthesis of Compounds 9-8:

The synthesis of compound 9-8 is identical to that of compound 1-8, except that 9-7 is substituted for 1-7.

Synthesis of Compound 9-9:

The synthesis of compound 9-9 is identical to that of compound 1-9, except that 9-8 is substituted for 1-8.

Synthesis of Compounds 9-10:

the synthesis of compound 9-10 is the same as that of compound S1, except that 9-9 is substituted for 1-9.

Synthesis of compound S13:

The synthesis of compound S13 is identical to that of compound S2, except that S1 is replaced with 9-10. LCMS (m/z): 950.4[ M+H ] ⁺

10. Synthesis of Compound S14

Synthesis of fragment 10:

the synthesis of fragment 10 was identical to that of fragment 2, except that 10-1 was used in place of 2-1.

Synthesis of Compound 10-3:

the synthesis of compound 10-3 is identical to that of compound 1-3, except that fragment 10 is substituted for fragment 1. Synthesis of Compound 10-5:

the synthesis of compound 10-5 is identical to that of compound 1-5.

Synthesis of Compound 10-6:

The synthesis of compound 10-6 is identical to that of compound 1-6, except that 10-5 is substituted for 1-5.

Synthesis of Compound 10-7:

The synthesis of compound 10-7 is identical to that of compound 1-7, except that 10-6 is substituted for 1-6.

Synthesis of Compound 10-8:

The synthesis of compound 10-8 is identical to that of compound 1-8, except that 10-7 is substituted for 1-7.

Synthesis of Compound 10-9:

The synthesis of compound 10-9 is identical to that of compound 1-9, except that 10-8 is substituted for 1-8.

Synthesis of Compound 10-10:

The synthesis of compound 10-10 is identical to that of compound S1, except that 10-9 is substituted for 1-9.

Synthesis of compound S14:

The synthesis of compound S14 is identical to that of compound S2, except that S1 is replaced with 10-10. LCMS (m/z): 938.4[ M+H ] ⁺

11. Synthesis of Compound S15

Synthesis of fragment 11:

The synthesis of fragment 11 is identical to that of fragment 2, except that 11-1 is substituted for 2-1.

Synthesis of Compound 11-3:

The synthesis of compound 11-3 is identical to that of compound 1-3, except that fragment 11 is substituted for fragment 1. Synthesis of Compound 11-5:

The synthesis of compound 11-5 is identical to that of compound 1-5.

Synthesis of Compound 11-6:

the synthesis of compound 11-6 is identical to that of compound 1-6, except that 11-5 is substituted for 1-5.

Synthesis of Compound 11-7:

The synthesis of compound 11-7 is identical to that of compound 1-7, except that 11-6 is substituted for 1-6.

Synthesis of Compound 11-8:

The synthesis of compound 11-8 is identical to that of compound 1-8, except that 11-7 is substituted for 1-7.

Synthesis of Compounds 11-9:

The synthesis of compound 11-9 is identical to that of compound 1-9, except that 11-8 is substituted for 1-8.

Synthesis of Compounds 11-10:

The synthesis of compound 11-10 is the same as that of compound S1, except that 11-9 is substituted for 1-9.

Synthesis of compound S15:

The synthesis of compound S15 is identical to that of compound S2, except that S1 is replaced with 11-10. LCMS (m/z): 922.4[ M+H ] ⁺

12. Synthesis of Compound S16

Synthesis of fragment 12:

The synthesis of fragment 12 is identical to that of fragment 2, except that fragment 2-1 is replaced with 12-1.

Synthesis of Compound 12-3:

The synthesis of compound 12-3 is identical to that of compound 1-3, except that fragment 12 is substituted for fragment 1. Synthesis of Compound 12-5:

the synthesis of compound 12-5 is identical to that of compound 1-5.

Synthesis of Compound 12-6:

The synthesis of compound 12-6 is identical to that of compound 1-6, except that 12-5 is substituted for 1-5.

Synthesis of Compound 12-7:

The synthesis of compound 12-7 is identical to that of compound 1-7, except that 12-6 is substituted for 1-6.

Synthesis of Compound 12-8:

The synthesis of compound 12-8 is identical to that of compound 1-8, except that 12-7 is substituted for 1-7.

Synthesis of Compound 12-9:

The synthesis of compound 12-9 is identical to that of compound 1-9, except that 12-8 is substituted for 1-8.

Synthesis of Compounds 12-10:

The synthesis of compound 12-10 is the same as that of compound S1, except that 12-9 is substituted for 1-9.

Synthesis of compound S16:

The synthesis of compound S16 is identical to that of compound S2, except that S1 is replaced with 12-10. LCMS (m/z): 894.4[ M+H ] ⁺

13. Synthesis of Compound S17

Synthesis of fragment 13:

the synthesis of fragment 13 was identical to that of fragment 2, except that fragment 13-1 was used in place of 2-1.

Synthesis of Compound 13-3:

the synthesis of compound 13-3 is identical to that of compound 1-3, except that fragment 13 is substituted for fragment 1. Synthesis of Compound 13-5:

the synthesis of compound 13-5 is identical to that of compound 1-5.

Synthesis of Compound 13-6:

the synthesis of compound 13-6 is identical to that of compound 1-6, except that 13-5 is substituted for 1-5.

Synthesis of Compound 13-7:

the synthesis of compound 13-7 is identical to that of compound 1-7, except that 13-6 is substituted for 1-6.

Synthesis of Compound 13-8:

the synthesis of compound 13-8 is identical to that of compound 1-8, except that 13-7 is substituted for 1-7.

Synthesis of Compound 13-9:

The synthesis of compound 13-9 is identical to that of compound 1-9, except that 13-8 is substituted for 1-8.

Synthesis of Compounds 13-10:

The synthesis of compound 13-10 is the same as that of compound S1, except that 13-9 is substituted for 1-9.

Synthesis of compound S17:

The synthesis of compound S17 is identical to that of compound S2, except that S1 is replaced with 13-10. LCMS (m/z): 934.4[ M+H ] ⁺

14. Synthesis of Compound S18

Synthesis of fragment 14:

The synthesis of fragment 14 is identical to that of fragment 2, except that 14-1 is substituted for 2-1.

Synthesis of Compound 14-3:

the synthesis of compound 14-3 is identical to that of compound 1-3, except that fragment 14 is substituted for fragment 1. Synthesis of Compound 14-5:

The synthesis of compound 14-5 is identical to that of compound 1-5.

Synthesis of Compound 14-6:

The synthesis of compound 14-6 is identical to that of compound 1-6, except that 14-5 is substituted for 1-5.

Synthesis of Compound 14-7:

the synthesis of compound 14-7 is identical to that of compound 1-7, except that compound 1-6 is replaced with 14-6.

Synthesis of Compound 14-8:

The synthesis of compound 14-8 is identical to that of compound 1-8, except that 14-7 is substituted for 1-7.

Synthesis of Compound 14-9:

The synthesis of compound 14-9 is identical to that of compound 1-9, except that 14-8 is substituted for 1-8.

Synthesis of Compounds 14-10:

The synthesis of compound 14-10 is the same as that of compound S1, except that 14-9 is substituted for 1-9.

Synthesis of compound S18:

the synthesis of compound S18 is identical to that of compound S2, except that S1 is replaced with 14-10. LCMS (m/z): 934.4[ M+H ] ⁺

15. Synthesis of Compound S19

Synthesis of fragment 15:

the synthesis of fragment 15 is identical to that of fragment 2, except that fragment 2-1 is replaced with 15-1.

Synthesis of Compound 15-3:

The synthesis of compound 15-3 is identical to that of compound 1-3, except that fragment 15 is substituted for fragment 1. Synthesis of Compound 15-5:

The synthesis of compound 15-5 is identical to that of compound 1-5.

Synthesis of Compound 15-6:

The synthesis of compound 15-6 is identical to that of compound 1-6, except that 1-5 is replaced with 15-5.

Synthesis of Compound 15-7:

The synthesis of compound 15-7 is identical to that of compound 1-7, except that 1-6 is replaced with 15-6.

Synthesis of Compound 15-8:

The synthesis of compound 15-8 is identical to that of compound 1-8, except that 1-7 is replaced with 15-7.

Synthesis of Compounds 15-9:

the synthesis of compound 15-9 is identical to that of compound 1-9, except that 15-8 is substituted for 1-8.

Synthesis of Compounds 15-10:

The synthesis of compound 15-10 is the same as that of compound S1, except that 1-9 is replaced with 15-9.

Synthesis of compound S19:

The synthesis of compound S19 is identical to that of compound S2, except that S1 is replaced with 15-10. LCMS (m/z): 920.4[ M+H ] ⁺

16. Synthesis of Compound S20

Synthesis of fragment 16:

the synthesis of fragment 16 is identical to that of fragment 2, except that fragment 2-1 is replaced with 16-1.

Synthesis of Compound 16-3:

The synthesis of compound 16-3 is identical to that of compound 1-3, except that fragment 16 is substituted for fragment 1. Synthesis of Compound 16-5:

the synthesis of compound 16-5 is identical to that of compound 1-5.

Synthesis of Compound 16-6:

The synthesis of compound 16-6 is identical to that of compound 1-6, except that 16-5 is substituted for 1-5.

Synthesis of Compound 16-7:

The synthesis of compound 16-7 is identical to that of compound 1-7, except that 16-6 is substituted for 1-6.

Synthesis of Compound 16-8:

The synthesis of compound 16-8 is identical to that of compound 1-8, except that 16-7 is substituted for 1-7.

Synthesis of Compound 16-9:

The synthesis of compound 16-9 is identical to that of compound 1-9, except that 16-8 is substituted for 1-8.

Synthesis of Compound 16-10:

the synthesis of compound 16-10 is the same as that of compound S1, except that 16-9 is substituted for 1-9.

Synthesis of compound S20:

The synthesis of compound S20 is identical to that of compound S2, except that S1 is replaced with 16-10. LCMS (m/z): 920.4[ M+H ] ⁺

17. Synthesis of Compound S21

Synthesis of fragment 17:

the synthesis of fragment 17 is identical to that of fragment 2, except that fragment 17-1 is substituted for 2-1.

Synthesis of Compound 17-3:

the synthesis of compound 17-3 is identical to that of compound 1-3, except that fragment 17 is substituted for fragment 1. Synthesis of Compound 17-5:

The synthesis of compound 17-5 is identical to that of compound 1-5.

Synthesis of Compound 17-6:

The synthesis of compound 17-6 is identical to that of compound 1-6, except that 1-5 is replaced with 17-5.

Synthesis of Compound 17-7:

The synthesis of compound 17-7 is identical to that of compound 1-7, except that compound 1-6 is replaced with 17-6.

Synthesis of Compound 17-8:

The synthesis of compound 17-8 is identical to that of compound 1-8, except that 1-7 is replaced with 17-7.

Synthesis of Compound 17-9:

The synthesis of compound 17-9 is identical to that of compound 1-9, except that 1-8 is replaced with 17-8.

Synthesis of Compounds 17-10:

The synthesis of compound 17-10 is identical to that of compound S1, except that 1-9 is replaced with 17-9.

Synthesis of compound S21:

The synthesis of compound S21 is identical to that of compound S2, except that S1 is replaced with 17-10. LCMS (m/z): 934.4[ M+H ] ⁺

18. Synthesis of Compound S22

Compound S2 (40 mg,0.043 mmol) and Linker-1 (14 mg,0.065 mmol) N, N-dimethylformamide (1 mL) were dissolved, N-diisopropylethylamine (30. Mu.L) was added dropwise at 0℃and after stirring for 30 minutes, HATU (25 mg,0.065 mmol) was added and the addition was allowed to warm to room temperature for 2 hours. After the reaction is completed, the reaction solution is completely spin-dried, and then is pulped by ethanol petroleum ether, and the solid is filtered out and dried to obtain the compound S22.LCMS (m/z): 1115.5[ M+H ] ⁺

19. Synthesis of Compound S23

The synthesis of compound S23 was identical to that of S22, except that Linker-1 was replaced with Linker-2. LCMS (m/z): 1320.6[ M+H ] ⁺

20. Synthesis of Compound S24

The synthesis of compound S24 was identical to that of S22, except that Linker-3 was used instead of Linker-1. LCMS (m/z): 1496.7[ M+H ] ⁺

21. Synthesis of Compound S25

The synthesis of compound S25 was identical to that of S22, except that Linker-4 was used instead of Linker-1. LCMS (m/z): 1332.6[ M+H ] ⁺

22. Synthesis of Compound S26

Compound S2 (20 mg,0.019 mmol) and Linker-5 (29 mg,0.039 mmol) were dissolved, N-dimethylformamide (1 mL) was added dropwise at 0deg.C, N-diisopropylethylamine (34 μL), and after stirring was continued for 30 minutes, hoBt (3 mg,0.019 mmol) was added and the reaction was allowed to proceed to room temperature for 2 hours. After the reaction is completed, the reaction solution is completely spin-dried, and then is beaten by dichloromethane petroleum ether, and the solid is filtered out and dried to obtain the compound S26.LCMS (m/z): 1520.7[ M+H ] ⁺

23. Synthesis of Compounds S27 and S28

In addition to usingReplacement ofThe synthesis of compounds S27 and S28 is otherwise identical to that of S1 and S2. LC-MS (m/z): 1021.5[ M+H ] ⁺ (Compound S28); 921.5[ M+H ] ⁺ (Compound S27).

24. Synthesis of Compound S29

Synthesis of Compound 29-1:

In addition to using Replacement ofThe synthesis of compound 29-1 was identical to that of S1.

Synthesis of Compound 29-2:

the synthesis of compound 29-2 is identical to that of S2.

Synthesis of compound S29:

The synthesis of compound 29-1 is identical to that of S23, except that S2 is replaced with compound 29-2. LCMS (m/z): 1304.6[ M+H ] ⁺

2. Cell screening experiments for activating human interferon gene stimulating protein (STING) and promoting IFN-beta expression by compounds

The detection method and the principle are as follows: THP1-dual cells of human origin, which contain an ISG-containing reporter system inside them. The reporter system can induce activation of the ISG promoter and produce luciferase that is present in the cell supernatant and can be detected and quantified by the luciferase detection reagent QUANTI-Luc ^TM. When the cells are added with the compound, if STING is activated, expression of ISG is promoted, thereby promoting increased secretion of luciferase downstream.

Reagent, consumable and instrument: in the experiment, THP1-dual cells are purchased from InvivoGen company, DMEM culture solution is purchased from Thermo FISHER SCIENTIFIC company, FBS is purchased from Gibco company in Australia, luciferase detection reagent QUANTI-Luc ^TM is purchased from InvivoGen company, and an enzyme-labeled instrument is a multifunctional enzyme-labeled instrument manufactured by Envision company

The test method comprises the following steps:

1. Adding a compound: after cells were attached to the 96-well cell culture plate, 20. Mu.L of a compound diluted with physiological saline was added to each well at a concentration of 10nM, and a positive control compound was diABZI (structural formula ) At a concentration of 10nM, the control group was physiological saline without 1% Dimethylsulfoxide (DMSO) and 3 replicate wells each. Incubation was performed for 24 hours.

2. Adding cells: THP1-dual cell count, cell concentration was adjusted to 5X 10 ⁵/mL, and 180. Mu.L of cells were added to each well for incubation. Thus, the final volume of each test well was 200. Mu.L, the DMSO content was 0.1%, and the test concentration of the compound was 10nM. The positive control compound was detected at a final concentration of diABZI at 10nM, incubated for 24 hours; 180. Mu.L of culture medium was added to the blank.

3. Detecting the color reaction: after 24 hours, 20. Mu.L of the culture broth was taken from each well into a new bottom-transmitting 96-well plate, and the fluorescence value was measured immediately (protected from light) by adding QUANTI-Luc ^TM. Mu.L of luciferase assay reagent.

4. Screening concentration of the compounds: 10nM.

5. Analysis of results: fold Change= Compound Luminescence/Control Luminescence

6. Assessment of results: when the activation multiple (Fold change) is more than or equal to 10, the method is effective. Wherein A is more than or equal to 200B is 150-200C is 50-100.

Experimental results:

as shown in table 1, the test compounds showed strong STING agonist activity at a concentration of 10nM, with the activity of most compounds being significantly better than the positive control drug diABZI.

Furthermore, we examined the agonistic activity of STING at concentrations of 1, 10, 100, 1000 and 10000nM for representative compound S1. As shown in fig. 1, compound S1 activated STING signaling dose-dependently, and STING agonistic activity was superior to that of positive drug diABZI at each concentration.

As shown in FIG. 2, the EC50 and Emax values of compound S1 for human STING agonism were 2.73nM and 269.6-fold activation, respectively, which are significantly better than positive drug diABZI, and the EC50 and Emax were 11.61nM and 210.2-fold activation, respectively.

As shown in fig. 3, compound S29 activated STING signaling dose-dependently, and STING agonistic activity was superior to that of positive drug diABZI at each concentration.

As shown in fig. 4, compound S29 activated humanized STING with EC50 and Emax values of 1.52nM and 310.0 fold activation, respectively, significantly better than positive drug diABZI.

TABLE 1 ability of partial Compounds to promote expression of type I interferon at 10nM concentration activating human interferon gene stimulatory proteins in THP1-dual cells

3. In vivo efficacy of compound S2 in CT26 mouse colorectal cancer model

The experimental method comprises the following steps: 12 female Balb/C mice of 4-6 weeks of age, each injected with CT-26 cells (2.5X 10 ⁵) until the tumor volume reached 50-100mm ³. Mice were randomly divided into 2 groups of 6 mice each, which were intraperitoneally injected with compound S2 (2 mg/kg) 3 times on days 1, 4, 7, respectively. Animals were randomly grouped and body weight was recorded, and each mouse in the group was labeled after grouping; the mouse body weight and tumor volume were measured every two days, and the state of the mouse was observed and recorded at any time.

Figure 5 shows the in vivo anti-tumor efficacy of compound S2 in a CT26 mouse colorectal cancer model.

As can be seen from fig. 5, the compound S2 described in the present invention is effective in inhibiting tumor growth in a CT26 mouse colorectal cancer model. 2mg/kg (three times) is injected into the abdominal cavity, and the tumor growth inhibition rate reaches 75.4% 11 days after the administration, and has no obvious effect on the weight of mice.

4. Pharmacokinetic (PK) property study of Compound S29

The experimental method comprises the following steps: male SD rats 6, 223-254g, were randomly divided into 2 groups of 3, and S29 or diABZI were intravenously administered at a dose of 1mg/kg. The medicine is fasted overnight before administration, and is fed uniformly for 4 hours after administration and is free to drink water. 60. Mu.L of blood samples were collected from rats via jugular vein into miniature K2EDTA tubes at 5min,0.25,0.5,1.0,2.0,4.0,8.0 and 24h, centrifuged at 8000rpm for 6min, plasma was isolated and frozen in a refrigerator at-20 ℃. The concentrations of S29 and diABZI in rat plasma were determined by LC-MS/MS method and pharmacokinetic parameters after administration were calculated using the non-compartmental model of WinNonlinm8.2.0 software (Pharsight, USA).

Experimental results: as shown in table 2.

TABLE 2 pharmacokinetic profile of Compounds S29 and diABZI in rats

The above data show that the PK profile of compound S29 is significantly improved with respect to diABZI, with a half-life (T1/2) of 2.45 hours, and plasma exposure (AUC) above 1000h ng/mL, 2-fold that of positive compound diABZI.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. A compound shown in a formula I, or pharmaceutically acceptable salts and tautomers thereof,

Wherein,

W is selected from the group consisting of: CH ₂, NH, O;

n ₁ is selected from the group consisting of: 0.1, 2 and 3;

n ₂ is selected from the group consisting of: 0.1, 2 and 3;

Ring C is selected from the group consisting of substituted or unsubstituted: a 5-, 6-or 7-membered monocyclic heterocycloalkyl group containing 1 or 2N, a 7-, 8-, 9-or 10-membered spirocycloalkyl group containing 2N, an 8-, 9-or 10-membered fused-ring heterocycloalkyl group containing 2N, a 7-or 8-membered bridged-ring heterocycloalkyl group containing 2N, said substitution being substituted by 1,2, 3 or 4R ₁;

R ₁ are each independently selected from the group consisting of: C1-C6 alkyl, halogen, hydroxy, -COOH, - (C=O) -O-C1-C6 alkyl, - (C=O) -C1-C6 alkyl, -NH-C1-C6 alkyl, haloC 1-C6 alkyl, hydroxy-substituted C1-C6 alkyl;

or 2R ₁ attached to the same atom together with the atom form a C3-C6 cycloalkyl or a 4-5 membered heterocycloalkyl containing 1O;

Z is selected from the group consisting of: absence, =o, -CH ₂ -NH- (c=o) -;

X is O or NH or is absent;

Y is NH;

R is selected from the group consisting of: H. - (C=O) -O-C1-C6 alkyl,

2. The compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof, wherein the 5,6, or 7 membered monocyclic heterocycloalkyl containing 1 or 2N is selected from the group consisting of:

3. the compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof, wherein the 7, 8, 9, or 10 membered spirocyclic heterocycloalkyl containing 2N is selected from the group consisting of:

4. The compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof, wherein the 8, 9, or 10 membered, fused heterocycloalkyl containing 2N is

5. The compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof, wherein the 7 or 8 membered bridged heterocycloalkyl containing 2N is selected from the group consisting of:

6. The compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof, wherein R is selected from the group consisting of: H. - (c=o) -O-tert-butyl,

7. The compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof, wherein the compound is selected from the group consisting of:

8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of a compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof.

9. Use of a compound according to claim 1, or a pharmaceutically acceptable salt, tautomer thereof, for the manufacture of a medicament which is a STING agonist.

10. The use according to claim 9, wherein the medicament is for a use selected from the group consisting of:

1) For preventing and/or treating a disease selected from the group consisting of: tumor and infectious diseases;

2) As an immune composition or vaccine adjuvant.