Preparation method and application of spiro tetrahydro-gamma-carboline derivative

Abstract

The invention relates to a cGAS inhibitor as well as a preparation method and application thereof. Specifically, the compound disclosed by the invention has a structure as shown in a formula I, and the definitions of all groups and substituent groups are described in the specification. The invention also discloses a preparation method of the compound and application of the compound in preventing and / or treating inflammation and autoimmune diseases, neurodegenerative diseases, other diseases and the like. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the field of medicinal chemistry, and specifically designs a preparation method and application of a class of spirocyclic tetrahydro-γ-carboline derivatives. Background Art

[0002] The innate immune system is the body's first line of defense against pathogen infection and transmits signals to the adaptive immune system. These processes can be activated by pathogen pattern recognition receptors (PRRs) binding to pathogen-associated molecular patterns or damage-associated molecular patterns (DAMPs) of biological macromolecules. PRRs are mainly divided into four categories: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and intracellular nucleic acid receptors (Nature Reviews Immunology 2013, 13, 551-565).

[0003] Cyclic GMP-AMP synthase (cGAS) is a nucleoside transferase and a direct receptor for DNA (Nature 1963, 197, 564-566; Lancet 1963, 282, 113-116). It is located in the cytoplasm and can recognize dsDNA from pathogens, nuclei or mitochondria. cGAS binds to dsDNA and activates GTP and ATP to synthesize cyclic dinucleotide GMP-AMP (cGAMP). The second messenger cGAMP is transported to the endoplasmic reticulum to activate the stimulator of interferon genes (STING). Activated STING recruits and activates TANK-binding Kinase 1 (TBK1), which then phosphorylates interferon regulatory factor 3 (IRF3) and activates nuclear factor kappa-B (NF-κB), thereby inducing the mRNA expression of cytokines, tumor necrosis factor and type I interferon. Excessive activation of the cGAS-STING signaling pathway can cause a variety of serious autoinflammatory autoimmune diseases, which are characterized by excessive cytokine or interferon conduction, including Aicardi-Goutières syndrome (AGS), systemic lupus erythematosus (SLE), rheumatoid arthritis, atopic dermatitis, psoriasis, inflammatory bowel disease (IBD), and acute lung injury (ALI) (J Exp Med, 2016, 213, 2527-2538; Annu. Rev. Immunol. 2017, 35, 313-336).

[0004] Given that inhibiting the cGAS pathway may be a strategy for treating autoimmune diseases, a series of small molecule inhibitors targeting cGAS have been developed. In 2017, Pfizer reported that compound PF-06928215 had micromolar inhibitory activity against human cGAS, but no inhibitory activity at the cellular level (PLoS.ONE.2017,12,e0184843). In the same year, Ascano's team discovered that compound RU.521 had good inhibitory activity against mouse cGAS, but was ineffective against human cGAS (Nat Commun 2017,8,750). In 2019, Tuschi's team reported compound G140, which had good inhibitory activity against both human and mouse cGAS, but weak cellular activity (Nat. Commun.2019,10,2261).

[0005] Although a series of cGAS small molecule inhibitors have been reported, they have a single structural type, weak cellular activity, and a large difference in activity between human and mouse cells, which seriously limits the further research of cGAS small molecule drugs. Among the reported cGAS small molecule inhibitors, compound G140 has good inhibitory activity against both human and mouse sources. However, the compound has low inhibitory activity at the cellular level; the PK in the body is not ideal, the plasma exposure is low; and no disease model studies have been conducted in animals. At present, no cGAS small molecule inhibitors have entered clinical research. Therefore, it is of great significance to develop cGAS inhibitors with novel structures, high inhibitory activity, strong selectivity, good PK properties and efficacy in vivo for the treatment of autoimmune diseases, various inflammatory diseases, and tumors.

[0006] Summary of the invention

[0007] The purpose of the present invention is to provide a class of spirocyclic tetrahydro-γ-carboline derivatives, prodrugs, solvates, hydrates or pharmaceutically acceptable salts and pharmaceutical uses. The compound is a class of cGAS inhibitors and has a good inhibitory effect on cGAS.

[0008] In the first aspect of the present invention, there is provided a compound as shown in general formula I, or a pharmaceutically acceptable salt, racemate, R-isomer, S-isomer thereof,

[0009]

[0010] in,

[0011] Each n is independently 0, 1 or 2;

[0012] X is selected from the following group: O, NR 7 , C(R 7 ) 2 ;

[0013] R 1 , R 2 , R 3 Each independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, amino, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, C 1 -C 3 Haloalkyl, C 3 -C 6 Cycloalkyl;

[0014] R 4is selected from the group consisting of substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted 4-8 membered heterocyclic group; wherein the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, amino, cyano, one or more Ra substituted C l -C 6 Alkyl, one or more Ra substituted C l -C 6 haloalkyl, one or more Ra substituted C l -C 6 Alkoxy, one or more Ra substituted C l -C 6 Haloalkoxy, 3-6 membered cycloalkyl substituted by one or more Ra, 4-6 membered heterocyclyl substituted by one or more Ra; wherein Ra is selected from the group consisting of H, halogen, hydroxy, amino, cyano;

[0015] R 5 , R 6 Each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C l -C 6 alkyl;

[0016] Each R 7 Each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C l -C 6 alkyl.

[0017] In some embodiments, R 4 Selected from the group consisting of substituted or unsubstituted pyrazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted 1,2,3-triazolyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl.

[0018] In some embodiments, R 3 is hydrogen; R 1 , R 2 It is a halogen.

[0019] In some embodiments, R 5 Selected from the group consisting of hydrogen, hydroxyl, amino, C l -C 3 Alkyl; R 6 For hydrogen.

[0020] In some embodiments, X is selected from the group consisting of O, NH, CH 2 .

[0021] In some embodiments, the compound is selected from the group consisting of:

[0022]

[0023]

[0024]

[0025] In a second aspect of the present invention, a pharmaceutical composition is provided, characterized in that it comprises:

[0026] (1) a therapeutically effective amount of the compound according to the first aspect of the present invention;

[0027] Optional (2) a pharmaceutically acceptable carrier.

[0028] In the third aspect of the present invention, there is provided a use of the compound according to the first aspect of the present invention, characterized in that it is used for preparing a drug for preventing and / or treating diseases related to excessive activation of cGAS.

[0029] In some embodiments, the cGAS overactivation-related disease is selected from the group consisting of inflammation, autoimmune diseases, neurodegenerative diseases, non-alcoholic fatty liver disease and fibrosis, diabetes and its complications, ischemia-reperfusion injury, and stroke.

[0030] In some embodiments, the inflammation is selected from the group consisting of sepsis, acute pancreatitis, nephritis and renal fibrosis, urticaria, allergic rhinitis acute lung injury, pneumonia, contact dermatitis, respiratory allergy;

[0031] The autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, atopic dermatitis, psoriasis, inflammatory bowel disease, Aicardi-Goutieres syndrome, multiple sclerosis, scleroderma, Behcet's disease, ankylosing spondylitis;

[0032] The neurodegenerative disease is selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and epilepsy.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The HPLC spectrum of compound S9 is shown.

[0035] Figure 2 The HPLC spectrum of compound S33 is shown.

[0036] Figure 3 The HPLC spectrum of compound S34 is shown.

[0037] Figure 4 The expression level of interleukin-12 (IL-12) in serum of the acute lung injury mouse model of compound S33 is shown.

[0038] Figure 5 The expression level of tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of compound S33 in an acute lung injury mouse model is shown.

[0039] Figure 6 Cell infiltration in bronchoalveolar lavage fluid in the compound S33 acute lung injury mouse model is shown. DETAILED DESCRIPTION

[0040] After long-term and in-depth research, the inventors obtained a compound with excellent cGAS inhibitory activity at the cellular level through structural optimization (introducing a spirocyclic structure based on tetrahydrocarbazole). On this basis, the inventors completed the present invention.

[0041] the term

[0042] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0043] In the present invention, the term "halogen" refers to F, Cl, Br or I.

[0044] In the present invention, “C 1 -C 6 "C1-C4 alkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl, tert-pentyl, or the like. 1 -C 3 "Alkyl" has a similar meaning.

[0045] In the present invention, the term "C 2 -C 6 "Alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl. The term "C 2 -C 4 "Alkenyl" has a similar meaning.

[0046] In the present invention, the term "C 2 -C 6 "Alkynyl" refers to a straight or branched alkynyl group having 2 to 6 carbon atoms and containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl. The term "C 2 -C 4 "Alkynyl" has a similar meaning.

[0047] In the present invention, the term "C 3 -C 8 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The term "Cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl 3 -C 6 "Cycloalkyl" has a similar meaning.

[0048] In the present invention, the term "C 1 -C 6 "Alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy and butoxy. Preferably, C 1 -C 4 Alkoxy, more preferably C 1 -C 3 Alkoxy.

[0049] In the present invention, the term "heterocyclyl" or "heterocycloalkyl" refers to a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms selected from N, O and S, including (but not limited to) the following groups:

[0050] In the present invention, the term "aromatic ring" or "aryl group" has the same meaning, preferably "C 6 -C 10 The term "C 6 -C 10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms in the ring, such as phenyl, naphthyl, etc.

[0051] In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning, referring to a heteroaromatic group containing one to multiple heteroatoms. 3 -C 10 "Heteroaryl" refers to an aromatic heterocycle containing 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen and 3 to 10 carbon atoms. Non-limiting examples include: furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0052] In the present invention, the term "halo" means substituted with halogen.

[0053] In the present invention, the term "deuterated" means substituted with deuterium.

[0054] In the present invention, the term "substituted" means that one or more hydrogen atoms on a specific group are replaced by a specific substituent. The specific substituent is the substituent described above or the substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable position of the group, and the substituent may be the same or different at each position. It should be understood by those skilled in the art that the combination of substituents contemplated by the present invention is those that are stable or chemically feasible. The substituents include, but are not limited to, halogen, hydroxyl, carboxyl (-COOH), C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, 3- to 12-membered heterocyclyl, aryl, heteroaryl, C 1 -C 8 Aldehyde, C 2 -C 10 Acyl, C 2 -C 10 Ester group, amino group, C 1 -C 6 Alkoxy, C 1 -C 10 Sulfonyl, etc.

[0055] In the present invention, the term 1-6 refers to 1, 2, 3, 4, 5 or 6. Other similar terms independently have similar meanings. The term "plurality" refers to 2-6, such as 2, 3, 4, 5 or 6.

[0056] It should be understood that when a group is present in multiple different positions of a compound at the same time, its definition at each position is independent of each other and may be the same or different. That is, the term "selected from the following group:" has the same meaning as the term "each independently selected from the following group:".

[0057] Compound

[0058] The present invention obtains a new type of piperidine ENPP1 inhibitor containing quaternary carbon. The compounds maintain high inhibitory activity on ENPP1 molecular level and significantly improve the inhibitory activity on cellular ENPP1. Under the same batch test conditions as compound 1, most of the compounds IC 50 The value is less than 10nM.

[0059] Specifically, the present invention provides a compound, which is a compound represented by Formula I, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof,

[0060]

[0061] wherein each group is as defined above.

[0062] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; 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, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0063] Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0064] The present invention's various embodiments specifically describe the preparation methods of the compounds of formula I of the present invention, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention belongs.

[0065] Typically, the raw materials and reagents used in the preparation process of the compounds of the present invention can be purchased through commercial channels unless otherwise specified.

[0066] Pharmaceutical compositions and methods of administration

[0067] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compounds.

[0068] Since the compounds of the present invention have excellent anti-tumor activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate tumor-related diseases.

[0069] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically 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. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0070] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which 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 the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0071] The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

[0072] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0073] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0074] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0075] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0076] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0077] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0078] Compositions for parenteral injection may include 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 non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0079] Dosage forms for topical administration of the compounds of the invention 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 that may be required.

[0080] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as anti-tumor drugs).

[0081] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0082] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0083] Typically, the compound of formula I of the present invention can be prepared by a general synthetic route selected from the following group (the raw materials and reagents used can be purchased through commercial channels unless otherwise specified):

[0084] General synthetic route:

[0085]

[0086] Compared with the prior art, the present invention has the following main advantages:

[0087] (1) The compound has excellent cGAS inhibitory activity at the cellular level;

[0088] (2) The compound has good pharmacokinetic properties. Compared with the known compounds in the art, the oral and intravenous plasma exposure is significantly increased, the half-life is prolonged, and the clearance rate is reduced.

[0089] (3) The compound has good water solubility in PBS solution at pH = 7.4, which is higher than that of the compounds known in the art.

[0090] (4) The compound can be orally administered in mouse animal disease models of acute lung injury, ulcerative colitis and Crohn's disease, and has obvious in vivo efficacy, which is significantly different from the model group and is superior to the known compounds in the art.

[0091] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only for the purpose of explanation and are not intended to limit the scope and essence of the present invention.

[0092] 1 H-NMR was measured with Bruker 400MHz and 700MHz instruments; reagents were purchased from Domestic Reagent Co., Ltd. All solvents were redistilled before use, and the anhydrous solvents used were obtained by drying according to standard methods; unless otherwise specified, all reactions were carried out under nitrogen protection and followed by TLC, and post-treatment was washed with saturated sodium chloride aqueous solution and dried with anhydrous sodium sulfate; the purification of products was performed by silica gel (300-400 mesh) column chromatography unless otherwise specified.

[0093] Example 1: Synthesis of Compound S1

[0094]

[0095] Compound 1a (1 eq.) and tert-butyl 7-oxo-2-azaspiro[3.5]nonane-2-carboxylate (1.5 eq.) were dissolved in 1,4-dioxane solution, and concentrated sulfuric acid (20 eq.) was added, and the reaction was carried out at 115°C for 12 h. After the reaction, the pH was adjusted to 8 with NaOH (aq), and the intermediate 1b was obtained by filtration.

[0096]

[0097] Compound 1b (1 eq.) was dissolved in N,N-dimethylformamide, and acetoxyacetic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 1c was obtained by column chromatography.

[0098]

[0099] Compound 1c (1 eq.) was dissolved in 1,4-dioxane, and 1-methylpyrazole-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 1d.

[0100]

[0101] Compound 1d was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S1. 1 H NMR (700 MHz, CDCl 3 )δ8.46(s,1H),7.42(s,1H),7.23(s,1H),6.36(s,1H),3.97(s,3H),3.92(s,2H),3.82(d,J=9.7Hz,1H),3.74(d ,J=8.5Hz,1H),3.69(d,J=9.8Hz,1H),3.58(d,J=8.5Hz,1H),3.22(s,1H),2.82-2.71(m,4H),2.08-2.02(m,2H).

[0102] Example 2: Synthesis of Compound S2

[0103]

[0104] Compound 2a (1 eq.) and 8-oxo-2-azaspiro[4-]decane-2-carboxylic acid tert-butyl ester (1.5 eq.) were dissolved in 1,4-dioxane solution, and concentrated sulfuric acid (20 eq.) was added, and the reaction was carried out at 115°C for 12 h. After the reaction, the pH was adjusted to 8 with NaOH (aq), and the intermediate 2b was obtained by filtration.

[0105]

[0106] Compound 2b (1 eq.) was dissolved in N,N-dimethylformamide, and acetoxyacetic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 2c was obtained by column chromatography.

[0107]

[0108] Compound 2c (1 eq.) was dissolved in 1,4-dioxane, and 1-methylpyrazole-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 2d.

[0109]

[0110] Compound 2d was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S2. 1 H NMR (700 MHz, CDCl 3 )δ8.6(s,1H),7.38(d,J=2.3Hz,1H),7.20(d,J=2.9Hz,1H),6.32(t,J=2.5Hz ,1H),4.07-4.01(m,1H),3.96(d,J=7.0Hz,1H),3.93(s,3H),3.62-3.59(m,1H ),3.54-3.50(m,1H),3.35-3.31(m,1H),3.29-3.25(m,1H),3.05-2.99(m,1H) ,2.81-2.75(m,1H),2.74-2.67(m,1H),2.52-2.45(m,2H),1.82-1.71(m,4H).

[0111] Example 3: Synthesis of Compound S3

[0112]

[0113] Compound 3a (1 eq.) and 3-Boc-9-oxo-3-azaspiro[5.5]undecane (1.5 eq.) were dissolved in 1,4-dioxane solution, and concentrated sulfuric acid (20 eq.) was added, and the reaction was carried out at 115°C for 12 h. After the reaction, the pH was adjusted to 8 with NaOH (aq), and the intermediate 3b was obtained by filtration.

[0114]

[0115] Compound 3b (1 eq.) was dissolved in N,N-dimethylformamide, and acetoxyacetic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 3c was obtained by column chromatography.

[0116]

[0117] Compound 3c (1 eq.) was dissolved in 1,4-dioxane, and 1-methylpyrazole-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2(0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 3d.

[0118]

[0119] Compound 3d was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S3. 1 H NMR (700MHz, DMSO) δ11.32 (s, 1H), 7.75 (d, J = 2.1Hz, 1H), 7.10 (s, 1H), 6.44 ( d,J=2.1Hz,1H),4.43-4.41(m,1H),4.05(d,J=5.3Hz,2H),3.90(s,3H),3.60- 3.57(m,1H),3.36-3.28(m,1H),3.27-3.26(m,1H),3.17-3.12(m,1H),2.75- 2.72(m,2H),2.45(d,J=5.8Hz,2H),1.70(t,J=6.5Hz,2H),1.40-1.28(m,4H).

[0120] Example 4: Synthesis of Compound S4

[0121]

[0122] Compound 4a (1 eq.) and tert-butyl-2-oxo-8-azaspiro[4.5]decane-8-carboxylic acid ethyl ester (1.5 eq.) were dissolved in 1,4-dioxane solution, and concentrated sulfuric acid (20 eq.) was added, and the reaction was carried out at 110°C for 12 h. After the reaction, the pH was adjusted to 8 with NaOH (aq), and the intermediate 4b was obtained by filtration.

[0123]

[0124] Compound 4b (1 eq.) was dissolved in N,N-dimethylformamide, and acetoxyacetic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 4c was obtained by column chromatography.

[0125]

[0126] Compound 4c (1 eq.) was dissolved in 1,4-dioxane, and 1-methylpyrazole-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 4d.

[0127]

[0128] Compound 4d was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S4. 1 H NMR (700MHz, DMSO) δ11.42(s,1H),7.74(d,J=2.2Hz,1H),7.37(s,1H),6.63(d,J=2.2Hz,1H),4.48(t,J=5.4Hz,1H),4.10(d,J= 5.4Hz,2H),3.91(s,3H),3.63-3.59(m,1H),3.50-3.47(m,1H),3.38-3.34(m,2H),2.88(s,2H),2.78(s,2H),1.69-1.60(m,4H).

[0129] Example 5: Synthesis of Compound S5

[0130]

[0131] Compound 5a (1 eq.) was dissolved in 1,4-dioxane, and 4-pyrazoleboronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 5b.

[0132]

[0133] Compound 5b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S5. 1H NMR (700MHz, DMSO) δ12.94(s,1H),11.34(s,1H),7.88(s,1H),7.62(s,1H),6.97(s,1H),4.46-4.42(m,1H),3.98-3.85(m,2H),3.4 4-3.39(m,1H),3.31-3.25(m,1H),3.23-3.20(m,1H),3.03-3.29(m,1H),2.82-2.74(m,2H),2.39-2.33(m,2H),1.81-1.53(m,4H).

[0134] Example 6: Synthesis of Compound S6

[0135]

[0136] Compound 6a (1 eq.) was dissolved in 1,4-dioxane, and 4-pyrazoleboronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 6b.

[0137]

[0138] Compound 6b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S6. 1 H NMR (700MHz, Methanol) δ7.85 (s, 1H), 7.6 (s, 1H), 6.95 (d, J = 7.0Hz, 1H), 4.59-4.55 (m, 1H), 4.15-4.08 (m, 2H), 3.58-3.54 (m,2H),3.53-3.45(m,1H)3.42-3.38(m,1H),3.27-3.17(m,4H),2.89-2.80(m,2H),2.37-2.18(m,6H),1.90-1.73(m,4H).

[0139] Example 7: Synthesis of Compound S7

[0140]

[0141] Compound 7a (1 eq.) was dissolved in 1,4-dioxane, and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 7b.

[0142]

[0143] Compound 7b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S7. 1 H NMR (700MHz, DMSO) δ11.35(d,J=3.6Hz,1H),7.86(d,J=3.0Hz,1H),7.54(d,J= 6.9Hz,1H),6.95(s,1H),4.48-4.45(m,1H),3.99-3.92(m,2H),3.88(s,3H),3 .44-3.40(m,1H),3.32-3.30(m,1H),3.23(t,J=11.1Hz,1H),3.06-3.03(m,1H ),2.83-2.76(m,2H),2.42-2.36(m,2H),1.87-1.70(m,3H),1.70-1.56(m,1H).

[0144] Example 8: Synthesis of Compound S8

[0145]

[0146] Compound 8a (1 eq.) was dissolved in 1,4-dioxane, and 1-(difluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 8b.

[0147]

[0148] Compound 8b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S8. 1H NMR (700MHz, Methanol) δ8.14(d,J=3.9Hz,1H),7.79(d,J=6.4Hz,1H),7.53(td,J=59.7,8.7Hz,1H),7.00(d,J=4.4Hz,1H),4.13-3.99(m,2H),3.56 -3.47(m,1H),3.42-3.33(m,1H),3.29-3.25(m,1H),3.22-3.10(m,1H),2. 89-2.80(m,2H),2.37-2.32(m,2H),1.92-1.80(m,3H),1.79-1.65(m,1H).

[0149] Example 9: Synthesis of Compound S9

[0150]

[0151] Compound 9a (1 eq.) was dissolved in 1,4-dioxane, and 1-(difluoromethyl)pyrazole-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 9b.

[0152]

[0153] Compound 9b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S9. 1 H NMR (700MHz, Methanol) δ8.10 (dd, J=2.6, 1.5Hz, 1H), 7.53 (td, J=59.7, 18.6Hz, 1H), 7.17 (d, J=4.6Hz, 1H), 6.66 (dd, J=4.9, 2.6Hz, 1H), 4.14-4. 00(m,2H),3.58-3.49(m,1H),3.47-3.39(m,1H),3.30-3.15(m,2H),2.9 1-2.82(m,2H),2.52-2.43(m,2H),1.92-1.82(m,3H),1.81-1.70(m,1H).

[0154] Example 10: Synthesis of Compound S10

[0155]

[0156] Compound 10a (1 eq.) was dissolved in 1,4-dioxane, and 1-methyl-3-trifluoromethylpyrazole-5-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 10b.

[0157]

[0158] Compound 10b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S10. 1 H NMR (700MHz, Methanol) δ7.10 (d, J = 2.3Hz, 1H), 6.65 (s, 1H), 4.16-3.99 (m, 2H), 3.64 (s, 2H), 3 .58-3.38(m,2H),3.29-3.22(m,2H),2.91-2.84(m,2H),2.10-1.96(m,2H),1.92-1.62(m,4H).

[0159] Example 11: Synthesis of Compound S11

[0160]

[0161] Compound 11a (1 eq.) was dissolved in 1,4-dioxane, and 1-methyl-3-trifluoromethylpyrazole-5-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 11b.

[0162]

[0163] Compound 11b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S11. 1H NMR (700MHz, DMSO) δ11.50(s,1H),7.95(s,1H),7.21(s,1H),4.49-4.45(m,1H),4.20(d,J=7.3Hz,3H),4.01-3.84(m,2H),3.51-3. 41(m,1H),3.38-3.35(m,1H),3.21(d,J=10.3Hz,1H),3.12-3.07(m,1H),2.84-2.79(m,2H),2.53-2.38(m,2H),1.91-1.58(m,4H).

[0164] Example 12: Synthesis of Compound S12

[0165]

[0166] Compound 12a (1 eq.) was dissolved in 1,4-dioxane, and 2-fluoromethylpyridine-5-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0167] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 12b.

[0168]

[0169] Compound 12b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S12. 1 H NMR(700MHz, Methanol)δ8.75(d,J=13.3Hz,1H),8.07(d,J=7.6Hz,1H),7.91-7.89(m,1H),7.08(s,1H),4.21-3.98(m,2H),3.68 -3.38(m,2H),3.25-3.20(m,1H),3.16-3.04(m,1H),2.95-2.67(m,2H),2.15-2.08(m,2H),1.93-1.75(m,3H),1.74-1.57(m,1H).

[0170] Example 13: Synthesis of Compound S13

[0171]

[0172] Compound 13a (1 eq.) was dissolved in 1,4-dioxane, and 2-aminopyridine-5-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 13b

[0173]

[0174] Compound 13b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S13. 1 H NMR (700MHz, DMSO) δ11.38(s,1H),7.92(d,J=8.4Hz,1H),7.41-7.38(m,1H),6.90(s,1H),6.50(s,1H),6.04(s,2H),4.45(d,J=27.5Hz,1H),3.99- 3.84(m,2H),3.46-3.38(m,1H),3.28-3.17(m,2H),2.99-2.94(m,1H),2. 82-2.74(m,2H),2.25-2.17(m,2H),1.82-1.66(m,3H),1.63-1.43(m,1H).

[0175] Example 14: Synthesis of Compound S14

[0176]

[0177] Compound 14a (1 eq.) was dissolved in 1,4-dioxane, and 2-cyanopyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 14b.

[0178]

[0179] Compound 14b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S14. 1H NMR (700MHz, Methanol) δ8.74(t,J=4.8Hz,1H),7.97(s,1H),7.70(d,J=6.5Hz,1H),7.10(s,1H),4.13-3.99(m,2H),3.54-3.45(m,1H) ,3.36-3.33(m,1H),3.31-3.26(m,1H),3.17-3.07(m,1H),2.93-2.86(m,2H),2.22-2.15(m,2H),1.95-1.83(m,3H),1.78-1.59(m,1H).

[0180] Example 15: Synthesis of Compound S15

[0181]

[0182] Compound 15a (1 eq.) was dissolved in 1,4-dioxane, and 2-aminopyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 15b.

[0183]

[0184] Compound 15b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S15. 1 H NMR (700MHz, Methanol) δ7.91-7.89(m,1H),6.97(d,J=7.0Hz,1H),6.63-6.62(m,1H),6.58(d,J=6.8Hz,1H),4.15-3.96( m,2H),3.55-3.35(m,2H),3.27-3.06(m,2H),2.92-2.82(m,2H),2.35-2.24(m,2H),1.94-1.80(m,3H),1.78-1.63(m,1H).

[0185] Example 16: Synthesis of Compound S16

[0186]

[0187] Compound 16a (1 eq.) was dissolved in 1,4-dioxane, and 2-fluoropyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 16b.

[0188]

[0189] Compound 16b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S16. 1 H NMR (700MHz, Methanol) δ8.24(t,J=4.6Hz,1H),7.35(d,J=5.0Hz,1H),7.13(d,J=5.6Hz,1H),7.06(d,J=1.5Hz,1H),4.13-3.99(m,2H) ,3.54-3.35(m,2H),3.31-3.23(m,1H),3.18-3.07(m,1H),2.96-2.83(m,2H),2.23-2.18(m,2H),1.92-1.78(m,3H),1.75-1.62(m,1H).

[0190] Example 17: Synthesis of Compound S17

[0191]

[0192] Compound 17a (1 eq.) was dissolved in 1,4-dioxane, and 2-fluoromethoxypyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0193] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 17b.

[0194]

[0195] Compound 17b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S17. 1H NMR (700MHz, Methanol) δ8.23(t,J=4.6Hz,1H),7.60(td,J=73.2,9.4Hz,1H),7.19(d,J=5.0Hz,1H),7.01(d,J=3.6Hz,1H),6.97(d,J=11.2Hz,1H),4. 17-3.94(m,2H),3.62-3.34(m,2H),3.31-3.23(m,1H),3.16-3.04(m,1H),2 .88-2.78(m,2H),2.23-2.17(m,2H),1.89-1.76(m,3H),1.74-1.56(m,1H).

[0196] Example 18: Synthesis of Compound S18

[0197]

[0198] Compound 18a (1 eq.) was dissolved in 1,4-dioxane, and 2-fluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0199] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 18b.

[0200]

[0201] Compound 18b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S18. 1 H NMR (700MHz, Methanol) δ8.67-8.65 (m, 1H), 7.71 (d, J = 5.3Hz, 1H), 7.58-7.56 (m,1H),7.07(d,J=7.1Hz,1H),6.79(td,J=55.2,6.0Hz,1H),4.12-3.96(m,2H ),3.58-3.44(m,1H),3.34-3.30(m,1H),3.27-3.22(m,1H),3.16-3.04(m,1H) ,2.93-2.84(m,2H),2.18-2.14(m,2H),1.92-1.76(m,3H),1.74-1.55(m,1H).

[0202] Example 19: Synthesis of Compound S19

[0203]

[0204] Compound 19a (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0205] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 19b.

[0206]

[0207] Compound 19b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S19. 1 H NMR (700MHz, Methanol) δ8.74-8.72(m,1H),7.84(d,J=5.8Hz,1H),7.67-7.65(m,1H),7.07(d,J=4.6Hz,1H),4.12-3.96(m,2H),3. 53-3.44(m,1H),3.35-3.22(m,2H),3.13-3.03(m,1H),2.94-2.84(m,2H),2.18-2.11(m,2H),1.92-1.74(m,3H),1.73-1.57(m,1H).

[0208] Example 20: Synthesis of Compound S20

[0209]

[0210] Compound 20a (1 eq.) was dissolved in 1,4-dioxane, and 2,6-difluoropyridine-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0211] After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 20b.

[0212]

[0213] Compound 20b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S20.1 H NMR(700MHz,Methanol)δ8.00(s,1H),7.08(s,1H),7.00(s,1H),4.18-3.96(m,2H),3.56-3.43(m,1H),3.43- 3.36(m,1H),3.30-3.19(m,1H),3.18-2.97(m,1H),2.95-2.77(m,2H),2.25-2.02(m,2H),1.94-1.78(m,4H).

[0214] Example 21: Synthesis of Compound S21

[0215]

[0216] Compound 21a (1 eq.) was dissolved in 1,4-dioxane, and 2-cyano-3-fluoropyridine-5-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 21b.

[0217]

[0218] Compound 21b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S21. 1 H NMR (700MHz, Methanol) δ8.61(d,J=10.0Hz,1H),7.96(d,J=9.5Hz,1H),7.13(s,1H),4.58(s,1H),4.14-3.98(m,2H),3.56-3. 46(m,1H),3.42-3.37(m,1H),3.33-3.26(m,1H),3.16-3.08(m,1H),2.94-2.84(m,2H),2.25-2.16(m,2H),1.93-1.74(m,4H).

[0219] Example 22: Synthesis of Compound S22

[0220]

[0221] Compound 22a (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyrimidine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2(0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 22b.

[0222]

[0223] Compound 22b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S22. 1 H NMR (700MHz, Methanol) δ8.98(d,J=5.1Hz,1H),7.92(d,J=5.1Hz,1H),7.38(d,J=5.7Hz,1H),4.15-3.96(m,2H),3.57-3.36 (m,2H),3.34-3.25(m,1H),3.22-3.09(m,1H),2.96-2.88(m,2H),2.53-2.42(m,2H),1.95-1.84(m,3H),1.82-1.66(m,1H).

[0224] Example 23: Synthesis of Compound S23

[0225]

[0226] Compound 23a (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyrimidine-5-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0227] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 23b.

[0228]

[0229] Compound 23b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S23. 1H NMR(700MHz,Methanol)δ9.05(s,1H),9.04(s,1H),7.17(s,1H),4.13-3.96(m,2H),3.55-3.46(m,1H),3.43-3.35(m,1 H),3.33-3.24(m,1H),3.18-3.06(m,1H),2.95-2.83(m,2H),2.21-2.13(m,2H),1.95-1.83(m,3H),1.77-1.62(m,1H).

[0230] Example 24: Synthesis of Compound S24

[0231]

[0232] Compound 24a (1 eq.) was dissolved in 1,4-dioxane, and 1-(difluoromethyl)pyrazole-3-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 24b.

[0233]

[0234] Compound 24b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S24. 1 H NMR (700MHz, Methanol) δ8.10 (dd, J=2.6, 1.5Hz, 1H), 7.53 (td, J=59.7, 18.6Hz, 1H), 7.17 (d, J=4.6Hz, 1H), 6.66 (dd, J=4.9, 2.6Hz, 1H), 4.14-4. 00(m,2H),3.58-3.49(m,1H),3.47-3.39(m,1H),3.30-3.15(m,2H),2.9 1-2.82(m,2H),2.52-2.43(m,2H),1.92-1.82(m,3H),1.81-1.70(m,1H).

[0235] Example 25: Synthesis of Compound S25

[0236]

[0237] Compound 25a (1 eq.) was dissolved in 1,4-dioxane, and 2-cyanopyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 25b.

[0238]

[0239] Compound 25b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S25. 1 H NMR (700MHz, Methanol) δ8.74(t,J=4.8Hz,1H),7.97(s,1H),7.70(d,J=6.5Hz,1H),7.10(s,1H),4.13-3.99(m,2H),3.54-3.45(m,1H) ,3.36-3.33(m,1H),3.31-3.26(m,1H),3.17-3.07(m,1H),2.93-2.86(m,2H),2.22-2.15(m,2H),1.95-1.83(m,3H),1.78-1.59(m,1H).

[0240] Example 26: Synthesis of Compound S26

[0241]

[0242] Compound 26a (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0243] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 26b.

[0244]

[0245] Compound 26b was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S26. 1H NMR (700MHz, Methanol) δ8.74-8.73(m,1H),7.85(d,J=4.7Hz,1H),7.69(d,J=4.8Hz,1H),7.01-6.99(m,1H),4.12-3.96(m,2H),3.54-3.44( m,1H),3.36-3.32(m,1H),3.31-3.24(m,1H),3.16-3.03(m,1H),2.93- 2.83(m,2H),2.23-2.14(m,2H),1.94-1.78(m,3H),1.74-1.58(m,1H).

[0246] Example 27: Synthesis of Compound S27

[0247]

[0248] Compound 27a (1 eq.) was dissolved in N,N-dimethylformamide, and (R)-2-acetoxypropionic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 27b was obtained by column chromatography.

[0249]

[0250] Compound 27b (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0251] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 27c.

[0252]

[0253] Compound 27c was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S27. 1H NMR (700MHz, DMSO) δ11.69(s,1H),8.81(d,J=4.7Hz,1H),7.95(s,1H),7.78(d,J=4.9Hz,1H), 7.22(s,1H),4.80-4.70(m,1H),4.18-4.09(m,1H),3.61-3.44(m,1H),3.41-3.28(m,1H),3.21 -3.14(m,1H),3.12-2.96(m,1H),2.87-2.78(m,2H),2.12-1.97(m,2H),1.81-1.75(m,2H),1.6 9-1.56(m,1H),1.50-1.44(m,1H),1.10(dd,J=26.4,6.5Hz,1H),0.99(dd,J=25.0,6.5Hz,2H).

[0254] Example 28: Synthesis of Compound S28

[0255]

[0256] Compound 28a (1eeqq.) was dissolved in N,N-dimethylformamide, and (S)-2-acetoxypropionic acid (1.2eq.), EDCI (1.5eq.), HOBt (1.5eq.), and DIPEA (3.0eq.) were added in sequence, and the mixture was reacted for 12h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 28b was obtained by column chromatography.

[0257]

[0258] Compound 28b (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0259] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 28c.

[0260]

[0261] Compound 28c was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in sequence. The reaction was carried out at RT for 1 h. After the reaction was completed, the solvent was dried and separated by column chromatography to obtain compound S28. 1H NMR (700MHz, DMSO) δ11.69(s,1H),8.81(d,J=4.7Hz,1H),7.95(s,1H),7.78(d,J=4.9Hz,1H), 7.22(s,1H),4.80-4.70(m,1H),4.18-4.09(m,1H),3.61-3.44(m,1H),3.41-3.28(m,1H),3.21 -3.14(m,1H),3.12-2.96(m,1H),2.87-2.78(m,2H),2.12-1.97(m,2H),1.81-1.75(m,2H),1.6 9-1.56(m,1H),1.50-1.44(m,1H),1.10(dd,J=26.4,6.5Hz,1H),0.99(dd,J=25.0,6.5Hz,2H).

[0262] Example 29: Synthesis of Compound S29

[0263]

[0264] Compound 29a (1 eq.) was dissolved in N,N-dimethylformamide, and (R)-2-((tert-butyloxycarbonyl)amino)propionic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 29b was obtained by column chromatography.

[0265]

[0266] Compound 29b (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0267] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 29c.

[0268]

[0269] Compound 29c was dissolved in dichloromethane, trifluoroacetic acid (10 eq.) was added, and the reaction was carried out at RT for 1 h. The solvent was dried and separated by column chromatography to obtain compound S29. 1H NMR (400MHz, DMSO) δ11.69 (s, 1H), 8.81-8.79 (m, 1H), 7.93 (d, J = 14.0Hz, 1H), 7.78- 7.72(m,1H),7.22(d,J=5.2Hz,1H),4.04-3.68(s,2H),3.67-3.45(m,3H),3.44-3.30 (m,2H),3.18-3.02(m,2H),2.84-2.76(m,2H),2.11-1.96(m,2H),1.85-1.74(m,2H) ,1.71-1.56(m,1H),1.57-1.44(m,1H),0.92(d,J=6.7Hz,1H),0.86(d,J=6.7Hz,2H).

[0270] Example 30: Synthesis of Compound S30

[0271]

[0272] Compound 30a (1 eq.) was dissolved in N,N-dimethylformamide, and (S)-2-((tert-butyloxycarbonyl)amino)propionic acid (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 30b was obtained by column chromatography.

[0273]

[0274] Compound 30b (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0275] After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 30c.

[0276]

[0277] Compound 30c was dissolved in dichloromethane, trifluoroacetic acid (10 eq.) was added, and the reaction was carried out at RT for 1 h. The solvent was dried and separated by column chromatography to obtain compound S30. 1H NMR (400MHz, DMSO) δ11.69 (s, 1H), 8.81-8.79 (m, 1H), 7.93 (d, J = 14.0Hz, 1H), 7.78- 7.72(m,1H),7.22(d,J=5.2Hz,1H),4.04-3.68(s,2H),3.67-3.45(m,3H),3.44-3.30 (m,2H),3.18-3.02(m,2H),2.84-2.76(m,2H),2.11-1.96(m,2H),1.85-1.74(m,2H) ,1.71-1.56(m,1H),1.57-1.44(m,1H),0.92(d,J=6.7Hz,1H),0.86(d,J=6.7Hz,2H).

[0278] Example 31: Synthesis of Compound S31

[0279]

[0280] Compound 31a (1 eq.) was dissolved in N,N-dimethylformamide, and Boc-glycine (1.2 eq.), EDCI (1.5 eq.), HOBt (1.5 eq.), and DIPEA (3.0 eq.) were added in sequence, and the mixture was reacted for 12 h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the intermediate 31b was obtained by column chromatography.

[0281]

[0282] Compound 31b (1 eq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2 eq.), Pd(dppf)Cl 2 (0.01eq.), KOAc (2eq.), heated to 110°C, reacted for 12h.

[0283] After the reaction was completed, the solvent was dried and separated by column chromatography to obtain intermediate 31c.

[0284]

[0285] Compound 31c was dissolved in dichloromethane, trifluoroacetic acid (10 eq.) was added, and the reaction was carried out at RT for 1 h. The solvent was dried and separated by column chromatography to obtain compound S31. 1H NMR (400MHz, DMSO) δ11.67 (s, 1H), 8.83-8.81 (m, 1H), 7.94 (d, J = 4.8Hz, 1H ),7.79-7.77(m,1H),7.22(d,J=2.4Hz,1H),3.45-3.43(m,1H),3.22-3.16( m,3H),3.10(d,J=16.4Hz,1H),2.94(d,J=12.1Hz,1H),2.83-2.78(m,2H),2 .10-2.06(m,2H),1.82-1.76(m,2H),1.71-1.65(m,1H),1.61-1.44(m,1H).

[0286] Example 32: Synthesis of Compound S32

[0287]

[0288] Compound 32a (1eeq.) was dissolved in N,N-dimethylformamide, and (2R,3R)-2,3-bisacetoxypropionic acid (1.2eq.), EDCI (1.5eq.), HOBt (1.5eq.), and DIPEA (3.0eq.) were added in sequence, and the mixture was reacted for 12h at RT. After the reaction, the mixture was washed with water, extracted with ethyl acetate, the solvent was dried, and the mixture was analyzed by column chromatography to obtain compound 32b.

[0289]

[0290] Compound 32b (1eeqq.) was dissolved in 1,4-dioxane, and 2-trifluoromethylpyridine-4-boronic acid pinacol ester (1.2eq.), Pd(dppf)Cl 2 (0.01 eq.), KOAc (2 eq.), heated to 110°C, reacted for 12 h. After the reaction, the solvent was dried and separated by column chromatography to obtain intermediate 32c.

[0291]

[0292] Compound 32c was dissolved in tetrahydrofuran and aqueous solution, and lithium hydroxide monohydrate (2 eq.) was added in turn. The reaction was carried out at RT for 1 h. After the reaction was completed, the mixture was washed with water, extracted with ethyl acetate, and the solvent was dried to obtain an intermediate. The intermediate was dissolved in dichloromethane, trifluoroacetic acid (10 eq.) was added, and the mixture was separated by column chromatography to obtain compound S32. 1H NMR (700MHz, DMSO) δ11.69(s,1H),8.80(d,J=5.2Hz,1H),7.95(s,1H),7.77(d,J=5.1H z,1H),7.21(d,J=7.4Hz,1H),3.61-3.54(m,1H),3.48-3.36(m,2H),3.34-3.23(m,2H), 3.21-3.14(m,1H),3.11-3.06(m,1H),2.87-2.77(m,2H),2.14-2.01(m,4H),1.85-1.6 9(m,3H),1.64-1.47(m,1H),0.82(d,J=21.7,6.3Hz,1H),0.69(dd,J=40.6,6.2Hz,2H).

[0293] Biological Examples

[0294] Biological Example 1: Detection of cGAS inhibitory activity of some of the compounds in the examples in RAW-lucia cells

[0295] RAW-lucia cells are generated by stably integrating the interferon regulatory factor-induced luciferase reporter gene. Under the stimulation of exogenous signals such as dsDNA / cGAMP, the interferon regulatory factor signaling pathway can be activated to induce the expression of the downstream reporter gene luciferase. Luciferase catalyzes the oxidation of luciferin substrate to generate oxyluciferin. During the oxidation process, it emits bioluminescence, which can be measured by a multifunctional microplate reader. This value can indirectly reflect the inhibitory activity of the compound on cGAS. The specific experimental operation is as follows:

[0296] RAW-lucia cells in the logarithmic growth phase were cultured for 5*10 4 Cells were seeded per well in a 96-well plate, and different concentrations of compounds were added. The cells were pre-incubated in a 37°C incubator for 1 hour. The DMSO group was used as a negative control for the compound. Cells were transfected with 1 μg / ml HT-DNA (Sigma) and Lipofectamine 2000 (Invitrogen) complex and cultured for 24 hours. Transfection complex preparation method: 0.2 μg HT-DNA was added to 10 μl Opti-MEM (Gibco), 0.2 μl Lipofectamine 2000 was added to 10 μl Opti-MEM, after mixing, 20 μl of the complex was added to a 96-well plate; QUANTI-Luc was used. TM Luciferase activity was detected by using the reagent (InvivoGen). 20 μl of cell culture supernatant was added to a 96-well white plate, and then 50 μl of QUANTI-Luc TMReagents were used and the values ​​were read using a multifunctional microplate reader (BioTek, Winooski, VT).

[0297] Relative luciferase activity calculation method: cells treated with Lipofectamine 2000 were used as negative controls, cells treated with Lipofectamine 2000 and dsDNA complex were used as positive controls, relative luciferase activity = (RLU sample - RLU negative control) / (RLU positive control - RLU negative control), RLU represents the original luciferase reading. IC was obtained by fitting the compound concentration and relative luciferase activity using Graphpad prism9.3.1 software. 50 .

[0298] Table 1: Compounds tested for cGAS inhibitory activity in RAW-lucia cells

[0299]

[0300] The results showed that the IC values ​​of some compounds in the examples in RAW-lucia cells were 50 The range was 4.71-11.43 μM, which was better than the positive compound G140 (14.64 μM).

[0301] Biological Example 2: Pharmacokinetic study of the compound in rats

[0302] Methods: Twelve rats were divided into 4 groups and given S9 and G140 by oral gavage and tail vein injection, respectively. Blood samples of about 0.25 mL were collected from the jugular vein at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration in the intravenous injection group and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration in the oral gavage group. The concentrations of S9 and G140 in rat plasma samples were determined by LC-MS / MS, and the pharmacokinetic parameters were calculated using WinNolin software.

[0303]

[0304] Table 2: Pharmacokinetic study of compounds G140 and S9 in rats

[0305]

[0306] The results showed that compound S9 had significantly better pharmacokinetic properties than G140. Compared with G140, the oral and intravenous plasma exposures were significantly improved, the half-life was prolonged, and the clearance rate was reduced.

[0307] Biological Example 3: Chiral separation of compound S9

[0308]

[0309] Methods: High performance liquid chromatography was used with a chiral column IH (IH00CE-XK004); the injection volume was 5 μL; the mobile phase used was n-hexane / ethanol / diethylamine = 15 / 85 / 0.1 (V / V / V); the flow rate was 1.0 mL / min; and the wavelength was 254 nm.

[0310] HPLC spectra before and after separation are shown in Figure 1-Figure 3 .

[0311] Results: The chiral isomers were separated well, with the ee value of compound S33 being 96% and the ee value of compound S34 being 98%.

[0312] Biological Example 4: Detection of cGAS inhibitory activity of compounds S33 and S34 in THP1-Dual and RAW-lucia cells

[0313] The inhibitory activity of cGAS was detected in RAW-lucia cells using the same method as above.

[0314] Table 3: cGAS inhibitory activity of compounds S33, S34, and G140 in THP1-Dual and RAW-lucia cells

[0315] The results showed that compound S33 had a better inhibitory activity against cGAS in both human and mouse cells than the positive compound G140. In mouse cells, compound S33 had an inhibitory activity against cGAS that was more than 10 times higher than that of the positive compound G140.

[0316] Biological Example 5: Pharmacodynamic Evaluation of Compounds in Acute Lung Injury Animal Model

[0317] Eight-week-old male C57BL / 6 mice were used for the experiment and grouped according to body weight. A certain amount of compound powder was weighed and dissolved in DMSO, and then suspended in 0.2% hydroxypropyl methylcellulose to prepare an oral preparation (dose: 30 mg / kg). 30 minutes after administration, LPS (dose: 2 mg / kg) was instilled into the bronchi, and serum and bronchoalveolar lavage fluid were collected 5 hours after modeling. The concentration of inflammatory factors in serum and bronchoalveolar lavage fluid was detected by ELISA. (The normal group was neither induced by LPS nor given compounds; the model group was only induced by LPS without giving compounds; G140 and S33 were the drug-administered groups, and compounds were given 30 minutes before LPS induction).

[0318] Figure 4The results showed that after LPS modeling, the concentration of the inflammatory factor interleukin-12 in the mouse serum increased significantly. Compound S33 significantly inhibited the production of interleukin-12 in the serum of mice with acute lung injury, and its inhibitory effect was better than that of the positive compound G140.

[0319] Figure 5 The results showed that after LPS modeling, the concentration of inflammatory factor TNF-α in the bronchoalveolar lavage fluid of mice increased significantly. Compound S33 significantly inhibited the production of inflammatory factor TNF-α in the bronchoalveolar lavage fluid of mice with acute lung injury, and its inhibitory effect was better than that of the positive compound G140.

[0320] Figure 6 The results showed that after LPS modeling, the number of cells in bronchoalveolar lavage fluid increased significantly, indicating increased cell infiltration. After intervention with compound S33, the number of infiltrated cells decreased significantly, and was superior to the positive compound G140.

[0321] Figure 4-6 The results showed that compound S33 had good efficacy in the LPS-induced acute lung injury mouse model after oral administration, and its effect was better than that of the positive compound G140.

[0322] In summary, the novel spirocyclic tetrahydro-γ-carboline cGAS small molecule inhibitors provided by the present invention have a good inhibitory effect on the cGAS signaling pathway stimulated by dsDNA in THP1-Dual cells and RAW-lucia cells; have good pharmacokinetic properties in rats; in the LPS-induced acute lung injury model, can significantly inhibit the expression of inflammatory factor tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid and inflammatory factor interleukin-12 (IL-12) in serum, and reduce the number of cells in bronchoalveolar lavage fluid. Overactivation of the cGAS pathway is involved in inflammatory and autoimmune diseases, neurodegenerative diseases and other diseases, such as sepsis, acute pancreatitis, nephritis and renal fibrosis, grass hives, allergic rhinitis acute lung injury, pneumonia, contact dermatitis and respiratory allergies, systemic lupus erythematosus, rheumatoid arthritis, atopic dermatitis, psoriasis, inflammatory bowel disease, Aicardi-Goutieres syndrome, multiple sclerosis, scleroderma, Behcet's disease, ankylosing spondylitis, Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy, non-alcoholic fatty liver disease and fibrosis, diabetes and its complications, ischemia-reperfusion injury, and stroke. Therefore, the compound shown in Formula I provided by the present invention, or its isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof can provide a promising therapeutic strategy for treating inflammatory and autoimmune diseases.

[0323] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof, in, Each n is independently 0, 1 or 2; X is selected from the following group: O, NR 7 , C(R 7 )2; R 1 , R 2 , R 3 Each is independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, amino, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C3-C6 cycloalkyl; R 4 is selected from the group consisting of substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted 4-8 membered heterocyclic group; wherein the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, amino, cyano, one or more Ra substituted C l -C6 alkyl, one or more Ra substituted C l -C6 haloalkyl, one or more Ra substituted C l -C6 alkoxy, one or more Ra substituted C l -C6 haloalkoxy, 3-6 membered cycloalkyl substituted by one or more Ra, 4-6 membered heterocyclyl substituted by one or more Ra; wherein Ra is selected from the group consisting of H, halogen, hydroxy, amino, cyano; R 5 , R 6 Each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C l -C6 alkyl; Each R 7 Each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C l -C6 alkyl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof, characterized in that: R 4 Selected from the group consisting of substituted or unsubstituted pyrazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted 1,2,3-triazolyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl.

3. The compound according to claim 1, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof, wherein: R 3 is hydrogen; R 1 , R 2 It is a halogen.

4. The compound according to claim 1, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof, wherein: R 5 Selected from the group consisting of hydrogen, hydroxyl, amino, C l -C3 alkyl; R 6 For hydrogen.

5. The compound according to claim 1, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof, wherein: X is selected from the group consisting of O, NH, and CH2.

6. The compound according to claim 1, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof, wherein: The compound is selected from the following group:

7. A pharmaceutical composition, characterized in that Include: (1) a therapeutically effective amount of a compound according to any one of claims 1 to 6; and optionally (2) a pharmaceutically acceptable carrier.

8. A use of the compound according to claim 1, characterized in that: Used for preparing drugs for preventing and / or treating diseases related to over-activation of cGAS.

9. The use according to claim 8, characterized in that The diseases associated with cGAS overactivation are selected from the following groups: inflammation, autoimmune diseases, neurodegenerative diseases, non-alcoholic fatty liver disease and fibrosis, diabetes and its complications, ischemia-reperfusion injury, and stroke.

10. The use according to claim 9, characterized in that The inflammation is selected from the group consisting of sepsis, acute pancreatitis, nephritis and renal fibrosis, urticaria, allergic rhinitis, acute lung injury, pneumonia, contact dermatitis, and respiratory allergy; The autoimmune disease is selected from the following group: systemic lupus erythematosus, rheumatoid arthritis, atopic dermatitis, psoriasis, inflammatory bowel disease, Aicardi-Goutieres syndrome, multiple sclerosis, scleroderma, Behcet's disease, ankylosing spondylitis; the neurodegenerative disease is selected from the following group: Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy.

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