Hydroxybenzimidazole dimer, and preparation method therefor and use thereof

Abstract

The present invention relates to a hydroxybenzimidazole dimer, and a preparation method therefor and a use thereof. Specifically, the compound in the present invention has a structure represented by formula I, wherein the definitions of groups and substituents are as described in the description. Also disclosed in the present invention are a preparation method for the compound and a use of the compound in prevention and / or treatment of STING-related diseases.

Description

Hydroxybenzimidazole dimers, methods of making and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to hydroxybenzimidazole dimers, methods of making and uses thereof. BACKGROUND

[0002] STimulator of INterferon Genes (STING) is an important protein molecule involved in the body's antiviral innate immune response. It is a 42 kDa multi-domain transmembrane protein located on the endoplasmic reticulum membrane. It consists of 379 amino acids, containing a transmembrane region (TM1-4, aa 1-154), a cyclic dinucleotide binding domain (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 pathogenic bacteria can be recognized by the host cytoplasmic nucleic acid receptor cGAS (cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase) to activate the cGAS enzyme activity function. cGAS uses ATP and GTP to synthesize the second messenger molecule cGAMP (2', 3'-cyclic GMP-AMP). cGAMP and endoplasmic reticulum-localized protein STING interact, leading to changes in STING conformation, thereby recruiting and activating downstream TBK1 kinase and IRF3 transcription factor. IRF3 enters the nucleus to promote the expression of type I interferon (Type I IFN) and initiate the natural immune response to resist pathogenic bacteria invasion. cGAS-STING is an important signaling pathway of the body's innate immunity, and its abnormal activation or inhibition is related to a variety of diseases, including viral infection, autoimmune diseases and tumors, etc. Among them, its anti-tumor immune response is receiving widespread attention. Activating the cGAS-STING pathway promotes the release of type I interferon, activates APC cells, and then activates T cells to produce adaptive anti-tumor immune response and induce immune memory, achieving a persistent tumor killing effect. Therefore, compared with immune checkpoint inhibitors targeting immunosuppressive molecules, targeting the activation of the cGAS-STING signaling pathway is expected to become the most promising new target for immunotherapy, especially in combination with PD-1 / PD-L1 antibodies for the treatment of "cold" tumors without T cell infiltration.

[0003] Although there are many STING small molecule agonists entering clinical trial research, the clinical progress is slow. Among them, ADU-S100 (phase II) developed by Aduro Company and MK1454 (phase I) developed by Merck Company are both cyclic dinucleotide analogues and the administration mode is intratumoral injection. Such drugs have the disadvantages of large molecular weight, poor cell membrane permeability, containing negative phosphate groups in the structure, and the phosphate ester bond is easily hydrolyzed, and the PK property is very poor, which seriously limits the use of such compounds in clinical. In 2018, GSK reported the first intravenously injectable benzimidazole STING agonist (Nature 2018, 564, 439-443.). However, this compound still has serious metabolic problems.

[0004] Therefore, it is necessary to further develop new efficient and safe STING small molecule agonists to induce the production of type I interferon IFN-β. SUMMARY

[0005] The purpose of the present application is to provide a compound represented by formula I and a preparation method thereof and the use thereof in the prevention and / or treatment of STING related diseases.

[0006] In a first aspect of the present application, a compound of formula I or a pharmaceutically acceptable salt thereof is provided,

[0007] wherein,

[0008] n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5;

[0009] n2 is selected from the group consisting of 0, 1, 2, 3, 4.

[0010] In another preferred embodiment, n1 is selected from the group consisting of 0, 1, 2, 3, 4;

[0011] n2 is selected from the group consisting of 1, 2, 3, 4.

[0012] In another preferred embodiment, n1 is selected from the group consisting of 0, 1, 2, 3;

[0013] n2 is selected from the group consisting of 1, 2, 3.

[0014] In another preferred embodiment, n1 is selected from the group consisting of 1, 2, 3.

[0015] In another preferred embodiment, n2 is selected from the group consisting of 1, 2.

[0016] In another preferred embodiment, n1 is selected from the group consisting of 1, 2, 3;

[0017] n2 is selected from the group consisting of 1, 2.

[0018] In another preferred embodiment, the compound is selected from the group consisting of:

[0019] In a second aspect of the present application, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of the compound of the first aspect of the present application or a pharmaceutically acceptable salt thereof.

[0020] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, or a combination thereof.

[0021] In another preferred embodiment, the pharmaceutical composition is for use in preventing and / or treating a STING-related disease.

[0022] In a third aspect of the present application, there is provided use of the compound of the first aspect of the present application or a pharmaceutically acceptable salt thereof for the manufacture of a medicament, which is a STING agonist.

[0023] In a fourth aspect of the present application, there is provided use of the compound of the first aspect of the present application or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in:

[0024] 1) preventing and / or treating a STING-related disease;

[0025] 2) use as an immune composition or vaccine adjuvant.

[0026] In another preferred embodiment, the STING-related disease is a STING high expression disease.

[0027] In another preferred embodiment, the STING-related disease is selected from the group consisting of a tumor, an infectious disease, a mental disease.

[0028] In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, ovarian cancer, liver cancer, melanoma, prostate cancer, colon cancer, rectal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, head and neck cancer, brain glioma, endometrial tumor, lung cancer and esophageal cancer.

[0029] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, gastric cancer, pancreatic cancer and esophageal cancer.

[0030] In another preferred embodiment, the lung cancer is non-small cell lung cancer.

[0031] In another preferred embodiment, the infectious disease is selected from the group consisting of viral infection, bacterial infection, fungal infection.

[0032] In another preferred embodiment, the viral infection is an infection of a virus selected from the group consisting of: SARS-CoV-2, Ebola virus, influenza virus, simian virus 40, herpes virus, dengue virus.

[0033] In another preferred embodiment, the bacterial infection is selected from the group consisting of: pneumococcal infection, salmonella infection, Mycobacterium tuberculosis infection, Staphylococcus aureus infection, Pseudomonas aeruginosa infection.

[0034] In another preferred embodiment, the fungal infection is an infection of a fungus selected from the group consisting of: Candida albicans, Aspergillus, Cryptococcus, Mucor, Histoplasma.

[0035] In another preferred embodiment, the mental disease is selected from the group consisting of: depression, post-traumatic stress disorder, autism.

[0036] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the result of the agonistic activity of compound C2 on STING at concentrations of 1, 10, 100, 1000 and 10000 nM.

[0038] Figure 2 shows the EC50 and Emax values of compound C2 for human STING agonism.

[0039] Figure 3 shows the EC50 and Emax values of compound C1 for human STING agonism. DETAILED DESCRIPTION

[0040] The present inventors, through long-term and in-depth research, have unexpectedly prepared a compound of formula I with excellent STING agonistic activity and pharmacokinetic performance through structural optimization. On this basis, the present inventors have completed the present application.

[0041] Compound

[0042] The present application provides a compound of formula I or a pharmaceutically acceptable salt thereof,

[0043] wherein each group is as defined above.

[0044] In another preferred embodiment, in the compound, n1 and n2 are each independently the corresponding group in the specific compound described in the present application.

[0045] As used herein, the term "pharmaceutically acceptable salt" refers to those salts of the compounds of the present application which are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts are those formed from the compounds of the present application and an acid. Suitable acids for salt formation include, but are not limited to, hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, and the like inorganic acids; formic, acetic, trifluoroacetic, propionic, oxalic, malonic, succinic, fumaric, maleic, lactic, malic, tartaric, citric, picric, benzoic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, and the like organic acids; and proline, phenylalanine, aspartic acid, glutamic acid, and the like amino acids.

[0046] Another preferred class of salts are those formed from the compounds of the present application and a base, 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 alkylammonium salts and other pharmaceutically acceptable amine salts), such as methylammonium, ethylammonium, propylammonium, dimethylammonium, trimethylammonium, diethylammonium, triethylammonium, t-butylammonium, ethylenediammonium, hydroxyethylammonium, dihydroxyethylammonium, trihydroxyethylammonium, and the amine salts formed from morpholine, piperazine, lysine, respectively.

[0047] It is understood that the various embodiments of the present application specifically describe methods of making the compounds of the present application of Formula I, but these specific methods do not limit the present application in any way. The compounds of the present application can also be readily made by combining various synthetic methods described in the present specification or known in the art, as would be readily apparent to one skilled in the art.

[0048] It is understood that the starting materials and reagents used in the preparation of the compounds of the present application in the schemes herein described are commercially available unless otherwise specified.

[0049] Pharmaceutical compositions and methods of administration

[0050] The present application also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of the compound or a pharmaceutically acceptable salt thereof.

[0051] Because the compounds of the present application have excellent anti-tumor activity, the compounds of the present application and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present application as the main active ingredient can be used to treat, prevent, and alleviate diseases associated with tumors.

[0052] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0053] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0054] The pharmaceutical composition is an injection, capsule, tablet, pill, powder, or granule.

[0055] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0056] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such excipients as (a) fillers or extenders, e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) moisturizing agents, e.g., glycerol and sorbitol; (h) respiration accelerators, e.g., high-oleic acid cottonseed oil; and (i) lubricants, e.g., magnesium stearate, calcium stearate, stearic acid, glyceryl monostearate, sodium stearyl fumarate, and talc. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.

[0057] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, e.g., enteric coatings and other materials well known in the art. They can optionally contain opacifying agents, and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0058] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and the like, as well as mixtures thereof.

[0059] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0060] Suspensions, in addition to the active compounds, can contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar-agar, as well as mixtures thereof.

[0061] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0062] Dosage forms for topical administration of a compound of this application include ointments, powders, sprays, and inhalers. The active component is admixed with a carrier, which can be a sterile, physiologically acceptable carrier, and any buffers, preservatives, or propellants as can be required.

[0063] A compound of this application can be administered alone or in combination with other pharmaceutically acceptable compounds, such as anti-tumor drugs.

[0064] The therapeutic methods of this application can be administered alone or in combination with other therapeutic procedures or therapeutic agents.

[0065] When a pharmaceutical composition is used, a safe and effective amount of a compound of this application is administered to a mammal (e.g., a human) in need of treatment, wherein the amount is administered in a dose that is pharmaceutically considered an effective dose, and for a 60 kg body weight human, the daily dose is usually 1-2000 mg, preferably 50-1000 mg. Of course, the specific dose will also take into account a route of administration, the health condition of the patient, and the like, which are within the skill of the skilled practitioner.

[0066] Compared with the prior art, the present application has the following main advantages:

[0067] (1) The compound of the present application has significantly more excellent STING agonistic activity;

[0068] (2) The compound of the present application has significantly more excellent pharmacokinetic performance;

[0069] (3) The compound of the present application has excellent safety.

[0070] The present application is further illustrated by the following specific examples. It is to be understood that these examples are merely illustrative of the present application and do not in any way limit the scope of the application. The experimental procedures in the following examples, unless otherwise indicated, were carried out under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise specified.

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application belongs. Also, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The preferred materials and methods are presented by way of example only.

[0072] I. Preparation Examples

[0073] The following preparation examples exemplarily prepared some of the compounds of Formula I of the present application.

[0074] 1. Synthesis of compound C1

[0075] Synthesis of fragment 1-3:

[0076] Compound 1 (1.22 g, 5.39 mmol) and compound 3 (1.02 g, 6.47 mmol) were dissolved in acetone (10 mL), and sodium hydroxide aqueous solution (5 M, 1.61 mL) was added dropwise slowly. The reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was evaporated, and the residue was dissolved in dichloromethane and washed with water twice. The organic layer was dried over saturated brine and anhydrous sodium sulfate, and then loaded onto a column. Compound 1-3 was obtained by column purification.

[0077] Synthesis of compound 1-5:

[0078] Compound 1-4 (500 mg, 2.31 mmol), fragment 1-3 (839 mg, 2.77 mmol), and potassium carbonate (415 mg, 3.00 mmol) were dissolved in N,N-dimethylformamide (4 mL). The reaction was stirred at 70°C overnight. After the reaction was completed, the reaction solution was directly loaded onto a column. Compound 1-5 was obtained by column purification.

[0079] Synthesis of compound 1-7:

[0080] Compound 1-6 (379 mg, 1.35 mmol) and compound 1-5 (541 mg, 1.12 mmol) were weighed in a microwave reactor, and isopropanol (4 mL) was added. N,N-diisopropylethylamine (1 mL) was added dropwise with stirring. The reaction was performed at 130°C for 14 hours with microwave assistance. After the reaction was completed, the reaction solution was evaporated, and the residue was dissolved in a mixture of dichloromethane / methanol and loaded onto a column. Compound 1-7 was obtained by column purification. [Synthesis of compound 1-6 is described in Nature 2018, 564, 439-443]

[0081] Synthesis of compound 1-8:

[0082] Compound 1-7 (472 mg, 0.65 mmol) was dissolved in methanol (4 mL) and dichloromethane (4 mL), sodium dithionite (1.7 g, 9.82 mmol) was dissolved in water (8 mL) and added slowly into the above system, stirred at room temperature for 10 minutes. After the reaction was completed, sodium bicarbonate (1.6 g, 19.65 mmol) was added and stirred for 15 minutes. The reaction solution was filtered to remove the insoluble matter, the filtrate was rotary evaporated, dissolved in methanol, filtered again to remove the insoluble matter, and the filtrate was stirred with neutral alumina and column-purified to obtain compound 1-8.

[0083] Synthesis of compound 1-10:

[0084] Compound 1-8 (831 mg, 0.84 mmol) was dissolved in N,N-dimethylformamide (5 mL). Compound 1-9 (0.4 M, 5.3 mL) in 1,4-dioxane was added dropwise at 0°C. After stirring at 0°C 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 then the temperature was raised to room temperature and reacted for 2 hours. After the reaction was completed, the reaction solution was directly stirred and column-purified to obtain compound 1-10. [Synthesis of compound 1-9, refer to CN109071514]

[0085] Synthesis of compound 1-11:

[0086] Compound 1-10 (702 mg, 0.79 mmol) was dissolved in methanol (8 mL) and dichloromethane (8 mL), and hydrogen chloride solution (4 M, 3 mL) was added, and stirred at room temperature for 1 hour. After the reaction was completed, it was rotary evaporated to obtain compound 1-11.

[0087] Synthesis of compound C1:

[0088] Compound 1-11 (570 mg, 0.64 mmol) and fragment 1 (74 mg, 0.97 mmol) were dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (0.45 mL, 2.59 mmol) was added dropwise under stirring at 0°C, and then HATU (370 mg, 0.97 mmol) was added, the temperature was raised to room temperature and reacted for 2 hours. After the reaction was completed, the reaction solution was directly stirred and column-purified to obtain compound C1.

[0089] 1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 2H), 8.31 (s, 2H), 8.03 (d, J = 15.6 Hz, 2H), 7.59 (s, 2H), 7.17 (d, J = 11.8 Hz, 2H), 6.45 (d, J = 10.9 Hz, 2H), 5.76 (s, 2H), 4.87 (d, J = 7.3 Hz, 4H), 4.71 (s, 1H), 4.50 (s, 4H), 4.35 (s, 2H), 4.04 (s, 2H), 3.67 (s, 3H), 3.45 (s, 2H), 3.41 (s, 4H), 2.85 (s, 4H), 2.09 (d, J = 7.8 Hz, 6H), 1.78 (d, J = 17.5 Hz, 6H), 1.31 (q, J = 7.2 Hz, 6H). MS (ESI, [M+H] + )m / z 947.4.

[0090] 2. Synthesis of compound C2

[0091] The synthesis of compound C2 is identical to that of compound CI except that fragment 2 replaces fragment 1.

[0092] 1 H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 2H), 8.28 (s, 2H), 8.01 (d, J = 2.6 Hz, 2H), 7.48 (s, 2H), 7.23 (d, J = 8.9 Hz, 2H), 6.21 (d, J = 2.7 Hz, 2H), 5.73 (s, 2H), 4.87 (s, 2H), 4.81 (d, J = 5.4 Hz, 4H), 4.69 (s, 1H), 4.34 (s, 4H), 4.31 (s, 2H), 4.07 (s, 2H), 3.61 (s, 3H), 3.46 (s, 2H), 3.40 (s, 4H), 2.82 (s, 4H), 2.06 (d, J = 7.8 Hz, 6H), 1.73 (d, J = 2.8 Hz, 6H), 1.29 (q, J = 3.5 Hz, 6H). MS (ESI, [M+H] + )m / z 961.5

[0093] 3. Synthesis of compound C3

[0094] The synthesis of compound C3 is identical to that of compound CI except that fragment 3 replaces fragment 1.

[0095] 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 2H), 8.31 (s, 2H), 8.15 (d, J = 21.9 Hz, 2H), 7.38 (s, 2H), 7.31 (d, J = 6.4 Hz, 2H), 6.53 (d, J = 9.1 Hz, 2H), 5.69 (s, 2H), 4.89 (s, 2H), 4.73 (d, J = 2.8 Hz, 4H), 4.64 (s, 1H), 4.37 (s, 4H), 4.29 (s, 2H), 4.12 (s, 2H), 3.59 (s, 3H), 3.43 (s, 2H), 3.39 (s, 4H), 3.15 (s, 2H), 2.79 (s, 4H), 2.03 (d, J = 3.9 Hz, 6H), 1.69 (d, J = 2.1 Hz, 6H), 1.21 (q, J = 2.7 Hz, 6H). MS (ESI, [M+H] + m / z 975.5

[0096] 4. Synthesis of compound C4

[0097] Synthesis of fragment 1-2:

[0098] The synthesis of fragment 1-2 is the same as that of fragment 1-3 except that compound 2 replaces compound 3.

[0099] Synthesis of compound 2-5:

[0100] The synthesis of compound 2-5 is the same as that of compound 1-5 except that fragment 1-2 replaces fragment 1-3.

[0101] Synthesis of compound 2-7:

[0102] The synthesis of compound 2-7 is the same as that of compound 1-7 except that compound 2-5 replaces compound 1-5.

[0103] Synthesis of compound 2-8:

[0104] The synthesis of compound 2-8 is the same as that of compound 1-8 except that compound 2-7 replaces compound 1-7.

[0105] Synthesis of compound 2-10:

[0106] The synthesis of compound 2-10 is the same as that of compound 1-10 except that compound 2-8 replaces compound 1-8.

[0107] Synthesis of compound 2-11:

[0108] The synthesis of compound 2-11 is the same as that of compound 1-11 except that compound 2-10 replaces compound 1-10.

[0109] Synthesis of compound C4:

[0110] The synthesis of compound C4 is the same as that of compound CI except that compound 2-11 replaces compound 1-11.

[0111] 1 H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 2H), 8.27 (s, 2H), 8.07 (d, J = 4.7 Hz, 2H), 7.57 (s, 2H), 7.12 (d, J = 2.5 Hz, 2H), 6.41 (d, J = 6.9 Hz, 2H), 5.68 (s, 2H), 4.83 (d, J = 7.3 Hz, 4H), 4.78 (s, 1H), 4.59 (s, 4H), 4.31 (s, 2H), 4.08 (s, 2H), 3.63 (s, 3H), 3.39 (s, 2H), 3.24 (s, 2H), 2.79 (s, 4H), 2.07 (d, J = 7.8 Hz, 6H), 1.71 (d, J = 3.2 Hz, 6H), 1.35 (q, J = 2.4 Hz, 6H). MS (ESI, [M+H] + )m / z 933.4

[0112] 5. Synthesis of compound C5

[0113] Synthesis of fragment 1-5:

[0114] The synthesis of fragment 1-5 is the same as that of fragment 1-3 except that compound 5 replaces compound 3.

[0115] Synthesis of compound 3-5:

[0116] The synthesis of compound 3-5 is the same as that of compound 1-5 except that fragment 1-5 replaces fragment 1-3.

[0117] Synthesis of compound 3-7:

[0118] The synthesis of compound 3-7 is the same as that of compound 1-7 except that compound 3-5 replaces compound 1-5.

[0119] Synthesis of compound 3-8:

[0120] The synthesis of compound 3-8 is the same as that of compound 1-8 except that compound 3-7 replaces compound 1-7.

[0121] Synthesis of compound 3-10:

[0122] The synthesis of compound 3-10 is identical to that of compound 1-10 except that compound 3-8 replaces compound 1-8.

[0123] Synthesis of compound 3-11:

[0124] The synthesis of compound 3-11 is identical to that of compound 1-11 except that compound 3-10 replaces compound 1-10.

[0125] Synthesis of compound C5:

[0126] The synthesis of compound C5 is identical to that of compound C1 except that compound 3-11 replaces compound 1-11.

[0127] 1 H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 2H), 8.53 (s, 2H), 8.19 (d, J = 12.5 Hz, 2H), 7.34 (s, 2H), 7.26 (d, J = 4.6 Hz, 2H), 6.41 (d, J = 1.4 Hz, 2H), 5.79 (s, 2H), 4.79 (d, J = 5.3 Hz, 4H), 4.68 (s, 1H), 4.34 (s, 4H), 4.19 (s, 2H), 4.02 (s, 2H), 3.67 (s, 3H), 3.39 (s, 2H), 3.07 (s, 4H), 2.76 (s, 4H), 2.45 (s, 4H), 2.21 (d, J = 3.7 Hz, 6H), 1.75 (d, J = 1.7 Hz, 6H), 1.29 (q, J = 4.7 Hz, 6H). MS (ESI, [M+H] + )m / z 975.5

[0128] 6、Synthesis of compound C6

[0129] The synthesis of compound C6 is identical to that of compound C5 except that fragment 2 replaces fragment 1.

[0130] 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 2H), 8.53 (s, 2H), 8.19 (d, J = 12.5 Hz, 2H), 7.34 (s, 2H), 7.26 (d, J = 4.6 Hz, 2H), 6.41 (d, J = 1.4 Hz, 2H), 5.79 (s, 2H), 4.79 (d, J = 5.3 Hz, 4H), 4.68 (s, 1H), 4.34 (s, 4H), 4.19 (s, 2H), 4.02 (s, 2H), 3.76 (s, 2H), 3.67 (s, 3H), 3.39 (s, 2H), 3.07 (s, 4H), 2.76 (s, 4H), 2.45 (s, 4H), 2.21 (d, J = 3.7 Hz, 6H), 1.75 (d, J = 1.7 Hz, 6H), 1.29 (q, J = 4.7 Hz, 6H). MS (ESI, [M+H] + m / z 989.5

[0131] 7. Synthesis of compound C7

[0132] The synthesis of compound C7 is identical to that of compound C4 except that fragment 2 replaces fragment 1.

[0133] 1 H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 2H), 8.26 (s, 2H), 8.12 (d, J = 2.9 Hz, 2H), 7.49 (s, 2H), 7.31 (d, J = 3.2 Hz, 2H), 6.76 (d, J = 6.2 Hz, 2H), 5.71 (s, 2H), 4.91 (d, J = 5.3 Hz, 4H), 4.69 (s, 1H), 4.61 (s, 4H), 4.31 (s, 2H), 4.14 (s, 2H), 3.71 (s, 3H), 3.41 (s, 2H), 3.39 (s, 4H), 2.76 (s, 4H), 2.15 (d, J = 5.2 Hz, 6H), 1.73 (d, J = 7.3 Hz, 6H), 1.34 (q, J = 5.1 Hz, 6H). MS (ESI, [M+H] + m / z 947.5

[0134] 8. Synthesis of compound C8

[0135] The synthesis of compound C8 is identical to that of compound C4 except that fragment 3 replaces fragment 1.

[0136] 1H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 2H), 8.21 (s, 2H), 8.17 (d, J = 5.6 Hz, 2H), 7.43 (s, 2H), 7.29 (d, J = 3.2 Hz, 2H), 6.54 (d, J = 6.8 Hz, 2H), 5.69 (s, 2H), 4.78 (d, J = 6.9 Hz, 4H), 4.65 (s, 1H), 4.59 (s, 4H), 4.37 (s, 2H), 4.02 (s, 2H), 3.67 (s, 3H), 3.37 (s, 2H), 3.34 (s, 4H), 3.29 (s, 2H), 2.65 (s, 4H), 2.11 (d, J = 3.5 Hz, 6H), 1.75 (d, J = 2.9 Hz, 6H), 1.31 (q, J = 4.3 Hz, 6H). MS (ESI, [M+H] + m / z 961.5

[0137] II. Cell screening assay to test the ability of compounds to activate human STING and promote IFN-β expression

[0138] Detection method and principle: Human THP1-dual cells, which have ISG-containing reporter systems inside. The reporter system can induce the activation of the ISG promoter and produce luciferase, which exists in the cell supernatant and can be detected and quantified by the luciferase detection reagent QUANTI-Luc. When the cells are added with compounds, if STING is activated, the expression of ISG is promoted, and the secretion of downstream luciferase is increased.

[0139] Reagents, consumables and instruments: THP1-dual cells in the experiment are purchased from InvivoGen company, DMEM culture solution is purchased from Thermo Fisher Scientific company, FBS is purchased from Australian Gibco company, luciferase detection reagent QUANTI-Luc is purchased from InvivoGen company, and the enzyme-labeled instrument is a multifunctional enzyme-labeled instrument of Envision company

[0140] Test method:

[0141] 1. Add compounds: after the cells in the 96-well cell culture plate are attached, add 20 μL of the compound diluted with normal saline to each well, and the concentration of the compound is 10 nM. The positive control compounds are diABZI (structure formula: ) and S6 (structure formula Chinese patent application number: 2024102724800), and the concentration is 10 nM. The control group is not added with 1% dimethyl sulfoxide (DMSO) in normal saline, and each group has 3 repeated wells. Incubate for 24 hours and then detect.

[0142] 2. Add cells: Count THP1-dual cells and adjust the cell concentration to 5 × 10⁻⁶. 5 / mL, 180μL of cells were added to each well for incubation. Therefore, the final volume of each test well was 200μL, the DMSO content was 0.1%, and the test concentration of the compound was 10nM. The positive control compounds were diABZI and S6 at a final concentration of 10nM, respectively. After incubation for 24 hours, the results were detected. A blank control group was prepared with 180μL of culture medium.

[0143] 3. Detection of colorimetric reaction: After 24 hours, transfer 20 μL of culture medium from each well to a new bottom-transparent 96-well plate, and add the luciferase detection reagent QUANTI-Luc. TM 50 μL, measure fluorescence value immediately (protect from light).

[0144] 4. Screening concentration of the compound: 10 nM.

[0145] 5. Results Analysis: Fold Change = Compound Luminescence / Control Luminescence.

[0146] 6. Result evaluation: Valid when the activation multiple (Fold change) ≥ 10. Among them, A ≥ 250, 200 ≤ B < 250, 150 ≤ C < 200, and 50 ≤ D < 100.

[0147] Experimental results:

[0148] As shown in Table 1, the tested compounds exhibited strong STING agonist activity at a concentration of 10 nM, with most of the compounds showing significantly better activity than the positive control drugs diABZI and S6.

[0149] Furthermore, we investigated the STING agonistic activity of representative compounds C1 and C2 at concentrations of 1, 10, 100, 1000, and 10000 nM. As shown in Figure 1, compound C2 dose-dependently activated the STING signal, and its STING agonistic activity at all concentrations was superior to that of the positive control drug S6.

[0150] As shown in Figure 2, compound C2 exhibits significantly better EC50 and Emax activation values ​​for human STING agonists than compound S6, with EC50 values ​​of 8.657 nM and Emax values ​​of 300.2. As shown in Figure 3, compound C1 also shows significantly better EC50 and Emax activation values ​​for human STING agonists than compound S6, with EC50 values ​​of 2.080 nM and Emax values ​​of 356.4.

[0151] Table 1. Ability of compounds to activate interferon-stimulated genes in THP1-dual cells at a concentration of 10 nM

[0152] III. Study of the pharmacokinetic (PK) properties of compounds C1 and C2

[0153] Experimental method: 6 male SD rats, weighing 223-254 g, were randomly divided into 2 groups, 3 rats in each group, and were administered C1, C2 or S6 intravenously at a dose of 1 mg / kg. The rats were fasted overnight before administration, and were fed uniformly 4 h after administration, and were allowed to drink freely. Blood samples of 60 μL were collected from the rats by way of the jugular vein at 5 min, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 24 h, and were centrifuged in micro-K2EDTA tubes at 8000 rpm for 6 min, and the plasma was separated and frozen in a -20°C refrigerator. The concentrations of C1, C2 and S6 in the rat plasma were determined by LC-MS / MS, and the pharmacokinetic parameters after administration were calculated by non-compartment model using WinNonlin m8.2.0 software (Pharsight Corporation, USA). Experimental results: as shown in Table 2.

[0154] Table 2. Comparison of the pharmacokinetic properties of compounds C1, C2 and S6 in rats

[0155] The above data show that, relative to S6, the PK properties of compounds C1 and C2 are significantly improved, with a half-life (T1 / 2) of 3.0 h, which is nearly 1-fold longer than S6, and a plasma exposure (AUC) of more than 1825 h*ng / mL, which is nearly 2-fold that of compound S6.

[0156] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present application upon reading and understanding the above lecture of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. A compound of Formula I or a pharmaceutically acceptable salt thereof, wherein, n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5; n2 is selected from the group consisting of 0, 1, 2, 3, 4.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein, n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5; n2 is selected from the group consisting of 1, 2, 3, 4.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein, n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5; n2 is selected from the group consisting of 1, 2, 3, 4.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein n1 is selected from the group consisting of 1, 2, 3.

5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein n2 is selected from the group consisting of 1, 2.

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

7. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

8. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, For the preparation of a medicament which is a STING agonist.

9. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, For the preparation of a medicament for use selected from the group consisting of: 1) for the prevention and / or treatment of a STING related disease; 2) as an immune composition or vaccine adjuvant.

10. Use according to claim 9, characterized in that, The STING related disease is selected from the group consisting of a tumor, an infectious disease, a mental disease.

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