CN115197207A - Tetrahydro-gamma-carboline derivatives, and pharmaceutical composition and application thereof - Google Patents

Abstract

The invention relates to tetrahydro-gamma-carboline derivatives which have cGAS (glutathione S-transferase) inhibiting activity. Specifically, the tetrahydro-gamma-carboline derivative provided by the invention has a structure shown in a formula I, wherein the definition of each group and substituent is described in the specification. The compound, the isomer, the prodrug, the solvate, the hydrate or the pharmaceutically acceptable salt thereof provided by the invention can be used for preparing a medicine used as a cGAS inhibitor, and the medicine can be used for resisting inflammation, resisting infection and preventing or treating autoimmune diseases.

Description

Tetrahydro-gamma-carboline derivatives, and pharmaceutical composition and application thereof

Technical Field

The invention relates to tetrahydro-gamma-carboline (TH gamma Cs) compounds, derivatives thereof, pharmaceutical compositions and pharmaceutical uses thereof.

Background

Pattern Recognition Receptors (PRRs) are widely present on the surface of natural immune cells, on the membrane of intracellular compartments, in the cytoplasm and in the blood, and can generate moderate immune responses against pathogens or self-tissue damage. The body monitors extracellular danger signals and non-self components of the cytoplasm primarily through a series of PRRs, such as Lipopolysaccharide (LPS), cyclic dinucleotides, etc. (Nature Reviews Immunology 2013,13, 551-565).

Cyclic GMP-AMP synthase (cGAS) is a nucleotidyl transferase and also the major sensor of dsDNA in most cells, perceiving dsDNA from pathogens, nuclei or mitochondria (Nature 1963,197, 564-566. After the dsDNA is recognized, the cGAS-STING signaling pathway is activated, phosphorylating interferon regulatory factor 3 (IRF3) and nuclear factor-. Kappa.B (NF-. Kappa.B), respectively. In one aspect, phosphorylated IRF3 translocates into the nucleus, activating transcription of a gene encoding type I interferon (IFNinterferon, IFN-I). On the other hand, activated NF-. Kappa.B translocates to the nucleus, initiating transcription of proinflammatory cytokines interleukin-6 (interleukin 6, IL-6), tumor Necrosis Factor (TNF), and IFN-I genes. Excessive activation of the cGAS-STING pathway is involved in inflammatory and autoimmune diseases such as sepsis, acute pancreatitis, rheumatoid arthritis, systemic lupus erythematosus and Aicardi-Goutieres syndrome (J Exp Med,2016,213,2527-2538, annu, rev, immune.2017, 35, 313-336.

Inhibition of cGAS by small molecule inhibitors may provide a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases. However, small molecule inhibitors that specifically inhibit cGAS and are safe and effective are rare. Ru.521 is a small molecule inhibitor having only murine cGAS inhibitory activity but lacking human cGAS inhibitory activity (Nat Commun 2017,8, 750); then screening a compound library of human-derived cGAS inhibitors by a luciferase assay to finally obtain compounds G140 and G150 (Nat Commun 2019,10, 2261); pf06928215, compound PF 06928215, was also found to be capable of potently inhibiting human cGAS enzyme activity at the molecular level, but not inhibitory activity in cells (PLoS ONE 2017,12 (9): e 0184843).

Therefore, the development of novel and potent cGAS inhibitors for the treatment of inflammatory and autoimmune diseases is of great interest.

Disclosure of Invention

The invention aims to provide a compound shown in a formula I, a derivative, a pharmaceutical composition and pharmaceutical application thereof.

In a first aspect, the present invention provides a compound represented by formula I, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof:

wherein R is ₁ Is hydrogen, C1-C3 alkyl, or C1-C3 alkyl substituted with a group selected from halogen, amino, cyano, hydroxy or alkoxy;

R ₂ and R ₃ Each independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C6 aryl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C6 aryl, C5-C6 heteroaryl substituted by halogen, amino, cyano, hydroxy, alkoxy or-COORd;

or, R ₂ And R ₃ Are connected to form a three-to six-membered N-containing or O-containing heterocyclic group;

or, R ₂ And R ₃ One and R ₈ Are linked to form a three-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen, saturated or unsaturated five-to six-membered heterocyclic group, or saturated or unsaturated five-to six-membered heterocyclic group substituted by halogen, amino, cyano, C1-C3 alkyl or C1-C3 haloalkyl;

R ₅ 、R ₆ and R ₇ Each independently is hydrogen, halogen, cyano, hydroxy, alkoxy, C1-C3 alkyl, -CH = CH2, -C ≡ CH, C3-C4 cycloalkyl, or C1-C3 alkyl, C3-C4 cycloalkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

R ₈ is hydrogen, C1-C3 alkyl, or C1-C3 alkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a three-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, cyano, hydroxy, alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with a group selected from the group consisting of halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, and heteroaryl.

Preferably, in the compound shown in the general formula I, or isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof,

R ₁ is hydrogen or C1-C3 alkyl;

R ₂ and R ₃ Each independently hydrogen, C1-C3 alkyl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C3 alkyl, C5-C6 heteroaryl substituted by halogen, amino, hydroxy, alkoxy or-COORd;

or, R ₂ And R ₃ Linked to form a three-to six-membered heterocyclic group containing 1-2 heteroatoms selected from the group consisting of: o, N;

or, R ₂ And R ₃ One and R ₈ Are linked to form a five-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen, unsaturated five-to six-membered heterocyclic group, or unsaturated five-to six-membered heterocyclic group substituted by halogen, amino or C1-C3 alkyl;

R ₅ 、R ₆ and R ₇ Each independently hydrogen or halogen;

R ₈ is hydrogen, C1-C3 alkyl;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a five-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, hydroxy, alkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with a group selected from the group consisting of halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, and heteroaryl.

Further preferably, in the compound represented by the general formula I, or an isomer, a prodrug, a solvate, a hydrate or a pharmaceutically acceptable salt thereof,

R ₁ is hydrogen;

R ₂ and R ₃ Each independently hydrogen, C1-C3 alkyl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C3 alkyl, C5-C6 heteroaryl substituted with halogen, hydroxy or-COORd;

or, R ₂ And R ₃ Linked to form a three-to six-membered heterocyclic group containing 1-2 heteroatoms selected from the group consisting of: o, N;

or, R ₂ And R ₃ One and R ₈ Are linked to form a five-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen or

R ₅ 、R ₆ And R ₇ Each independently hydrogen or halogen;

R ₈ is hydrogen;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a five-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, hydroxy, alkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

Still further preferably, the compounds include, but are not limited to, compounds S1 to S30 listed in Table 1.

The second aspect of the invention provides a use of the compound shown in the general formula I, or an isomer, a prodrug, a solvate, a hydrate or a pharmaceutically acceptable salt thereof in preparing a medicament serving as a cGAS inhibitor.

Wherein the cGAS inhibitor is used for the treatment and/or prevention of clinical diseases and for laboratory diagnosis and/or detection.

In a third aspect, the present invention provides the use of a compound of formula I, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the anti-inflammatory, anti-infective, or prevention and/or treatment of an autoimmune disease.

Wherein, the anti-inflammatory, anti-infection, prevention and/or treatment of autoimmune diseases comprise antivirus, prevention and/or treatment of bacterial infection autoimmune diseases, prevention and/or treatment of cardiovascular diseases, prevention and/or treatment of neurodegenerative diseases, prevention and/or treatment of inflammatory bowel diseases, prevention and/or treatment of diabetes, prevention and/or treatment of arthritis, anti-tumor immunity and the like.

A fourth aspect of the present invention provides a pharmaceutical composition for use as a cGAS inhibitor, comprising: (i) One or more therapeutically and/or prophylactically effective amounts of a compound of claim 1, or an isomer, prodrug, solvate, hydrate thereof, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

A fifth aspect of the present invention provides a pharmaceutical composition for anti-inflammatory, anti-infective, preventing and/or treating an autoimmune disease, comprising: (i) One or more therapeutically and/or prophylactically effective amounts of a compound of claim 1, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

Drawings

FIG. 1 is a graph showing the results of measuring the solubility of the positive compound G140, the compounds S7 (HCl salt) and S17 (HCl salt) according to the example of the present invention in water;

FIG. 2 shows the Western blotting of the effect of compound S7 of the present invention on dsDNA-stimulated phosphorylation of the cGAS signaling pathway in THP-1 cells;

FIG. 3 shows the western blotting of the effect of compound S17 of the present invention on dsDNA-stimulated phosphorylation of the cGAS signaling pathway in THP-1 cells;

FIG. 4 is a graph showing the results of the inhibition of TNF-. Alpha.release activity of tumor necrosis factor in LPS-induced acute inflammatory mouse animal models by compounds S7 and S17 according to the example of the present invention;

FIG. 5 is a graph showing the results of inhibition of the release activity of TNF IL-6 by compounds S7 and S17 according to the examples of the present invention in an LPS-induced acute inflammatory mouse animal model;

FIG. 6 is a graph showing the results of the inhibition of TNF IL-12 release activity of compounds S7 and S17 according to the example of the present invention in a mouse animal model of LPS-induced acute inflammation.

Detailed Description

The invention provides a compound shown in a formula I, a derivative, a pharmaceutical composition and pharmaceutical application thereof. Through long-term and intensive research, the inventor discovers a tetrahydro-gamma-carboline (TH gamma Cs) derivative with better water solubility, which can effectively inhibit cGAS, thereby effectively resisting inflammation and infection and treating autoimmune diseases.

Compound (I)

According to a first aspect of the present invention, some embodiments of the present invention provide a compound of formula I, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof:

wherein R is ₁ Is hydrogen, C1-C3 alkyl, or C1-C3 alkyl substituted by a group selected from various halogens, amino, cyano, hydroxy, or alkoxy groups;

R ₂ and R ₃ Each independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C6 aryl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C6 aryl, C5-C6 heteroaryl substituted by halogen, amino, cyano, hydroxy, alkoxy or-COORd;

or, R ₂ And R ₃ Are connected to form a three-to six-membered N-containing or O-containing heterocyclic group;

or, R ₂ And R ₃ One and R ₈ Are linked to form a three-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen, saturated or unsaturated five-to six-membered heterocyclic group, or saturated or unsaturated five-to six-membered heterocyclic group substituted by halogen, amino, cyano, C1-C3 alkyl or C1-C3 haloalkyl;

R ₅ 、R ₆ and R ₇ Each independently hydrogen, halogen, cyano, hydroxy, alkoxy, C1-C3 alkyl, -CH = CH2, -C ≡ CH, C3-C4 cycloalkyl, or C1-C3 alkyl, C3-C4 cycloalkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

R ₈ is hydrogen, C1-C3 alkyl, or C1-C3 alkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a three-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, cyano, hydroxy, alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with a group selected from the group consisting of halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

Preferably, in the compound shown in the general formula I, or isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof,

R ₁ is hydrogen or C1-C3 alkyl;

R ₂ and R ₃ Each independently hydrogen, C1-C3 alkyl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C3 alkyl, C5-C6 heteroaryl substituted with halogen, amino, hydroxy, alkoxy, or-COORd;

or, R ₂ And R ₃ Linked to form a three-to six-membered heterocyclic group containing 1-2 heteroatoms selected from the group consisting of: o, N;

or, R ₂ And R ₃ One and R ₈ Are linked to form a five-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen, unsaturated five-to six-membered heterocyclic group, or unsaturated five-to six-membered heterocyclic group substituted by halogen, amino or C1-C3 alkyl;

R ₅ 、R ₆ and R ₇ Each independently hydrogen or halogen;

R ₈ is hydrogen, C1-C3 alkyl;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a five-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, hydroxy, alkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with a group selected from the group consisting of halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

Further preferably, in the compound shown in the general formula I, or an isomer, a prodrug, a solvate, a hydrate or a pharmaceutically acceptable salt thereof,

R ₁ is hydrogen;

R ₂ and R ₃ Each independently hydrogen, C1-C3 alkyl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C3 alkyl, C5-C6 heteroaryl substituted with halogen, hydroxy or-COORd;

or, R ₂ And R ₃ Linked to form a three-to six-membered heterocyclic group containing 1-2 heteroatoms selected from: o, N;

or, R ₂ And R ₃ One and R ₈ Are connected to form a five-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen or

R ₅ 、R ₆ And R ₇ Each independently hydrogen or halogen;

R ₈ is hydrogen;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a five-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, hydroxy, alkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

In some preferred examples, the compounds include, but are not limited to, the compounds listed in table 1 below:

TABLE 1

Salt form

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, which is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with 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, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc.

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

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

The term "prodrug" includes a class of compounds which are biologically active or inactive in nature and which, when administered by an appropriate method, undergo a metabolic or chemical reaction in the body to convert the compound to formula I, or a salt or solution of a compound of formula I. The prodrugs include, but are not limited to, carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amide, carbamate, azo, phosphoramide, glucoside, ether, acetal forms of the compounds.

Process for the preparation of compounds

The process for the preparation of the compounds of formula I according to the invention is described in more detail below, without restricting the invention in any way. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

Typically, the compounds of formula I of the present invention may be prepared by a general synthetic route selected from the following group (wherein the starting materials and reagents used are commercially available without specific reference):

general synthetic route one:

wherein the definitions of the groups are the same as in the summary of the invention section of the present specification.

Pharmaceutical use

According to a second aspect of the present invention, some embodiments of the present invention provide the use of a compound of formula I, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use as a cGAS inhibitor; wherein the cGAS inhibitor is used for the treatment and/or prevention of clinical diseases and for laboratory diagnosis and/or detection.

According to a third aspect of the present invention, some embodiments of the present invention provide the use of a compound of formula I, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for anti-inflammatory, anti-infective, or preventing and/or treating an autoimmune disorder. Wherein, the anti-inflammatory, anti-infection, prevention and/or treatment of autoimmune diseases comprise antivirus, prevention and/or treatment of bacterial infection autoimmune diseases, prevention and/or treatment of cardiovascular diseases, prevention and/or treatment of neurodegenerative diseases, prevention and/or treatment of inflammatory bowel diseases, prevention and/or treatment of diabetes, prevention and/or treatment of arthritis, anti-tumor immunity and the like.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising:

(i) One or more therapeutically effective amounts of said compound, or its isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof; and

(ii) A pharmaceutically acceptable carrier.

Since the compounds of the present invention are effective in inhibiting cGAS, pharmaceutical compositions comprising the compounds of the present invention, or isomers, prodrugs, solvates, hydrates, or pharmaceutically acceptable salts thereof, are useful in anti-inflammatory, anti-infective, and treating autoimmune diseases.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range 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. The therapeutically effective amount is determined according to the age, condition, course of treatment, etc. of the subject to be treated.

The "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are sugars (e.g., glucose, sucrose, lactose, etc.), starches (e.g., corn starch, potato starch, etc.), celluloses and derivatives thereof (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is 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, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) Disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary amine compounds; (g) Wetting agents, such as cetyl alcohol and glycerol 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 capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such a composition may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

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

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

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

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

Dosage forms for topical administration of the compounds of the present 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 which may be required if necessary.

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the scope of the invention. For the following examples, standard procedures and purification methods known to those skilled in the art may be used. Unless otherwise specified, the starting materials are generally available from commercial sources. Commercial solvents and reagents were generally used without further purification, anhydrous solvents were processed by standard methods, and other reagents were commercially available as analytical grade. Unless otherwise stated, all temperatures are expressed in degrees Celsius (Celsius) and room or ambient temperature means 20 to 25 degrees Celsius. The structure of the compound is determined by nuclear magnetic resonance spectroscopy (NMR). The nuclear magnetic resonance hydrogen spectral shift (δ) is given in parts per million (ppm). Hydrogen nuclear magnetic resonance spectroscopy was performed using a Mercury-300MHz nuclear magnetic resonance spectrometer and deuterated chloroform (CDCl) ₃ ) Deuterated methanol (CD) ₃ OD) as solvent and Tetramethylsilane (TMS) as internal standard. The chromatographic column generally uses 200-300 mesh silica gel as a carrier.

1. Compound preparation examples section

The following preparations exemplarily prepare a part of the compounds of formula I of the present invention, each of which is represented by S1 to S30, respectively.

1. Synthesis of Compound S1

Step 1: concentrated sulfuric acid (20 eq) was added to a 1, 4-dioxane solution containing compound 1a (1 eq) and piperidin-4-one hydrochloride (1.5 eq) and the reaction was carried out at 115 ℃ for 12 hours. The pH was then adjusted to 8 with NaOH (a.q.) and filtered to give intermediate 1b.

Step 2: to a solution of Boc-glycine (1.2 eq) in N, N-dimethylformamide was added compound 1b (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S1 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.70(s,1H),7.14(d,J＝23.4Hz,1H),6.89(dd,J＝10.5,5.8Hz,1H),5.58(d,J＝25.2Hz,1H),4.75(s,1H),4.53(s,1H),4.10(s,2H),4.01(t,J＝5.8Hz,1H),3.72(t,J＝5.8Hz,1H),2.87(d,J＝5.6Hz,2H),1.45(d,J＝2.8Hz,9H).

2. Synthesis of Compound S2

Step 1: to a dichloromethane solution containing compound S1 (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S2 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.25(dd,J＝19.9,1.7Hz,1H),6.86(d,J＝10.9Hz,1H),4.73(s,1H),4.60(s,1H),3.99(t,J＝5.8Hz,1H),3.79(t,J＝5.7Hz,1H),3.60(d,J＝9.2Hz,2H),2.93(t,J＝5.9Hz,1H),2.86(t,J＝6.0Hz,1H).

3. Synthesis of Compound S3

Step 1: concentrated sulfuric acid (20 eq) was added to a 1, 4-dioxane solution containing compound 3a (1 eq) and piperidin-4-one hydrochloride (1.5 eq) and the reaction was carried out at 115 ℃ for 12 hours. The pH was then adjusted to 8 with NaOH (a.q.) and filtered to give intermediate 1b.

Step 2: to a solution of Boc-glycine (1.2 eq) in N, N-dimethylformamide was added compound 1b (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S3 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.34(d,J＝7.7Hz,1H),7.15–7.02(m,2H),5.57(d,J＝13.4Hz,1H),4.79(s,1H),4.57(s,1H),4.11(d,J＝4.4Hz,2H),4.03(t,J＝5.9Hz,1H),3.75(t,J＝5.8Hz,1H),2.98–2.84(m,2H),1.46(d,J＝3.4Hz,9H).

4. Synthesis of Compound S4

And step 3: to a dichloromethane solution containing compound S3 (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S4 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.18(dd,J＝10.9,8.4Hz,1H),7.00(ddd,J＝8.3,6.5,2.0Hz,1H),4.75(s,1H),4.62(s,1H),4.00(t,J＝5.8Hz,1H),3.80(t,J＝5.7Hz,1H),3.60(d,J＝9.7Hz,2H),2.93(t,J＝5.8Hz,1H),2.86(t,J＝5.9Hz,1H).

5. Synthesis of Compound S5

Step 1: NBS (1.2 eq) was added to a sulfuric acid solution containing compound 5a (1 eq) dissolved therein at 15 ℃ and the reaction was carried out at 65 ℃ for 3 hours. Then quenching by ice water, extracting by EA, and concentrating to obtain an intermediate 5b.

Step 2: to an ethanol solution containing intermediate 5b (1 eq) was added compound NH in sequence ₄ Cl (2 eq), iron powder (10 eq), the reaction was stirred at 60 ℃ for 4 hours. Then the solvent is dried by spinning, and the intermediate 5c is obtained by direct column chromatography separation.

And step 3: adding NaNO dropwise into hydrochloric acid solution dissolved with intermediate 5c (1 eq) at-5 deg.C ₂ The solution (1.5 eq) was stirred for 1 hour. Then adding SnCl ₂ (2.5 eq), stirred at 0 ℃ for 2 hours. Filtering to obtain an intermediate 5d.

And 4, step 4: concentrated sulfuric acid (20 eq) was added to a 1, 4-dioxane solution containing intermediate 5d (1 eq) and piperidin-4-one hydrochloride (1.5 eq) and the reaction was carried out at 115 ℃ for 12 hours. The pH was then adjusted to 8 with NaOH (a.q.) and filtered to give intermediate 5e.

And 5: to a solution of N, N-dimethylformamide in which N-acetylglycine (1.2 eq) was dissolved, compound 5e (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq) were added in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S5 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.28(d,J＝14.4Hz,1H),7.33(s,1H),5.54(s,1H),5.10(s,1H),4.89(s,1H),4.12(dd,J＝14.7,4.4Hz,2H),4.01(t,J＝5.8Hz,1H),3.74(t,J＝5.7Hz,1H),2.89(dt,J＝17.1,5.2Hz,2H),1.56(s,3H).

6. Synthesis of Compound S6

Step 1: to a solution of the compound S5 (1 eq) and 1-methylpyrazole-3-boronic acid pinacol ester (2 eq) in 1, 4-dioxane was added KOAc (3 eq) and Pd (dppf) Cl ₂ ·CH ₂ Cl ₂ (0.2 eq), the reaction was stirred at 100 ℃ for 6 hours. Then the solvent is dried by spinning, and the compound S6 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.41(d,1H),7.43(dd,J＝11.6Hz,1H),7.29(d,J＝9.4Hz,1H),6.69–6.62(m,1H),6.45(dd,J＝16.2Hz,1H),4.72(d,J＝4.4Hz,2H),4.14(d,J＝3.9Hz,2H),4.10(s,2.4H),3.99(s,0.6H),4.00–3.97(t,1.4H),3.72(t,J＝5.8Hz,0.6H),2.91(t,J＝5.9Hz,2H),2.04(d,3H).

7. Synthesis of Compound S7

Step 1: to a solution of Boc-glycine (1.2 eq) in N, N-dimethylformamide was added compound 5e (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 7a is obtained by direct column chromatography separation.

Step 2: to a solution of intermediate 7a (1 eq) and 1-methylpyrazole-3-boronic acid pinacol ester (2 eq) in 1, 4-dioxane was added KOAc (3 eq) and Pd (dppf) Cl ₂ ·CH2Cl ₂ (0.2 eq), the reaction was stirred at 100 ℃ for 6 hours. Then the solvent is dried by spinning, and the intermediate 7b is obtained by direct column chromatography separation.

And step 3: dissolving in waterTo a dichloromethane solution containing intermediate 7b (1 eq) was added trifluoroacetic acid (20 eq), and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S7 is obtained by column chromatography separation. ¹ H NMR(600MHz,DMSO-d ₆ )δ11.57(d,J＝29.2Hz,1H),7.80(dd,J＝4.0,2.2Hz,1H),7.25(d,J＝27.8Hz,1H),6.59(dd,J＝35.0,2.3Hz,1H),4.59(d,J＝7.1Hz,2H),3.95(d,J＝22.6Hz,3H),3.83(t,J＝5.9Hz,1H),3.68(t,J＝5.8Hz,1H),3.42(s,1H),3.33(d,J＝6.3Hz,3H),2.89(t,J＝5.8Hz,1H),2.82(t,J＝5.9Hz,1H).

8. Synthesis of Compound S8

Step 1: to a solution of Boc-sarcosine (1.2 eq) in N, N-dimethylformamide was added compound 5e (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 8a is obtained by direct column chromatography separation.

Step 2: to a solution of intermediate 8a (1 eq) and 1-methylpyrazole-3-boronic acid pinacol ester (2 eq) in 1, 4-dioxane was added KOAc (3 eq) and Pd (dppf) Cl ₂ ·CH ₂ Cl ₂ (0.2 eq), the reaction was stirred at 100 ℃ for 6 hours. Then the solvent is dried by spinning, and the intermediate 8b is obtained by direct column chromatography separation.

And step 3: to a dichloromethane solution containing intermediate 8b (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S8 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.67(dd,J＝9.9Hzc,1H),7.19(d,1H),6.48(dd,J＝19.0Hz,1H),4.57(t,J＝1.6Hz,1H),4.54(t,J＝1.5Hz,1H),4.00(d,3H),3.94(t,J＝5.9Hz,1H),3.77(t,J＝5.8Hz,1H),3.74(s,1H),3.61(s,1H),2.98(t,J＝5.9Hz,1H),2.90(t,J＝5.9Hz,1H),2.50(d,3H).

9. Synthesis of Compound S9

Step 1: to a methanol solution containing the compound S7 (1 eq) dissolved therein were added 40% aqueous formaldehyde (2.2 eq), HOAc (1 eq) and NaBH in this order ₃ CN (1.5 eq). The reaction was stirred at room temperature overnight. Then saturated NaHCO ₃ Solution quenching and EA extraction. Then the compound S9 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.68(dd,J＝11.8Hz,1H),7.18(d,1H),6.47(dd,J＝12.1Hz,1H),4.58(s,1H),4.52(s,1H),4.00(d,3H),3.92(t,J＝6.0Hz,1H),3.88(t,J＝5.7Hz,1H),3.29(s,1H),3.15(s,1H),3.00–2.95(m,1H),2.88(d,J＝6.1Hz,1H),2.29(s,3H),2.22(s,3H).

10. Synthesis of Compound S10

Step 1: to a solution of Boc-L-proline (1.2 eq) in N, N-dimethylformamide was added compound 5e (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 10a is obtained by direct column chromatography separation.

Step 2: to a solution of intermediate 10a (1 eq) and 1-methylpyrazole-3-boronic acid pinacol ester (2 eq) in 1, 4-dioxane was added KOAc (3 eq) and Pd (dppf) Cl ₂ ^. CH ₂ Cl ₂ (0.2 eq), the reaction was stirred at 100 ℃ for 6 hours. Then the solvent is dried by spinning, and the intermediate 10b is obtained by direct column chromatography separation.

And step 3: to a dichloromethane solution containing intermediate 10b (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S10 is obtained by column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ7.69(d,J＝15.3Hz,1H),7.19(d,1H),6.48(d,J＝16.8Hz,1H),4.71–4.47(m,2H),4.06(m,0.6H),4.00(d,3H),3.98–3.80(m,2.4H),3.35(s,1H),3.17–3.09(m,1H),2.98(m,1H),2.91(m,1H),2.79(m,1H),2.33–1.99(m,1H),1.89–1.49(m,3H).

11. Synthesis of Compound S11

Step 1: to a solution of 4-morpholineacetic acid hydrochloride (1.2 eq) in N, N-dimethylformamide was added compound 5e (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (4.2 eq) in that order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 11a is obtained by direct column chromatography separation.

Step 2: to a solution of intermediate 11a (1 eq) and 1-methylpyrazole-3-boronic acid pinacol ester (2 eq) in 1, 4-dioxane was added KOAc (3 eq) and Pd (dppf) Cl ₂ ^. CH ₂ Cl ₂ (0.2 eq), the reaction was stirred at 100 ℃ for 6 hours. Then the solvent is dried by spinning, and the compound S11 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.68(d,J＝10.3Hz,1H),7.18(d,1H),6.47(d,J＝15.3Hz,1H),4.64(s,1H),4.52(s,1H),3.99(d,J＝6.5Hz,3H),3.95–3.89(m,2H),3.73–3.64(m,2H),3.51–3.41(m,2H),3.27(s,1H),3.14(s,1H),3.05–2.99(m,1H),2.89(t,J＝5.6Hz,1H),2.49(s,2H),2.32(s,2H).

12. Synthesis of Compound S12

Step 1: to a solution of N, N-dimethylformamide containing N-Boc-4, 4-difluoro-L-proline (1.2 eq) dissolved therein were added the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 12a is obtained by direct column chromatography separation.

Step 2: to a dichloromethane solution containing intermediate 12a (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then the solvent is dried by rotation, extracted by EA and saturated NaHCO ₃ And (5) washing. Then the compound S12 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.64(dd,J＝9.5Hz,1H),7.16(d,1H),6.45(dd,J＝20.3Hz,1H),4.55(d,J＝11.9Hz,2H),4.17(d,1H),4.08(s,1H),3.99(d,3H),3.95–3.89(m,2H),3.78(t,J＝5.8Hz,1H),3.24–3.07(m,2H),2.96(t,J＝5.8Hz,1H),2.87(t,J＝5.9Hz,1H),2.61–2.48(m,1H),2.39–2.26(m,1H).

13. Synthesis of Compound S13

Step 1: to a solution of N, N-dimethylformamide containing N-Boc-cis-4-hydroxy-L-proline (1.2 eq) dissolved therein were added in this order the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq), and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S13 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.64(d,J＝22.5Hz,1H),7.56–7.27(m,2H),7.23(d,J＝15.4Hz,1H),6.52–6.36(m,1H),5.32–4.83(m,1H),4.79–4.57(m,2H),4.43–4.27(m,2H),4.19–4.10(m,2H),4.01(d,J＝30.8Hz,3H),3.94(s,1H),3.72–3.42(m,3H),2.94–2.84(m,2H),2.21(dq,J＝36.2,14.9,13.6Hz,2H),1.44(d,J＝3.4Hz,9H).

14. Synthesis of Compound S14

Step 1: to a dichloromethane solution containing compound S13 (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S14 is obtained by column chromatography separation. ¹ H NMR(500MHz,Methanol-d ₄ )δ7.66(dd,J＝11.5,2.2Hz,1H),7.18(d,J＝19.9Hz,1H),6.47(dd,J＝25.3,2.3Hz,1H),4.57(d,J＝17.2Hz,2H),4.33(td,J＝5.2,2.7Hz,1H),4.26–4.16(m,1H),4.16–4.06(m,1H),4.01(d,J＝30.3Hz,3H),3.93(t,J＝5.9Hz,1H),3.91–3.87(m,1H),3.81(t,J＝5.7Hz,1H),3.10(dt,J＝12.1,6.2Hz,1H),3.03–2.94(m,2H),2.89(t,J＝5.9Hz,1H),2.38(ddd,J＝13.5,9.8,5.5Hz,1H),1.95(dq,J＝11.5,6.6,4.9Hz,1H).

15. Synthesis of Compound S15

Step 1: to a solution of N, N-dimethylformamide containing N-Boc-2-aminoisobutyric acid (1.2 eq) dissolved therein were added in this order the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq), and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S15 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.65(d,1H),7.41(dd,J＝14.3Hz,1H),7.26(d,1H),6.43(dd,J＝16.6Hz,1H),5.04(s,1H),4.68(d,J＝7.7Hz,2H),4.12(d,2H),4.03(d,J＝46.2Hz,3H),3.95(d,J＝5.9Hz,1H),3.68(t,J＝5.8Hz,1H),2.88(t,J＝5.9Hz,2H),1.86(s,1H),1.50(d,6H),1.41(d,9H).

16. Synthesis of Compound S16

Step 1: dissolving in waterTo a dichloromethane solution containing compound S15 (1 eq) was added trifluoroacetic acid (20 eq), and the mixture was stirred at room temperature for 2 hours. Then the solvent is dried by rotation, extracted by EA and saturated NaHCO ₃ And (6) washing. Then the compound S16 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.67(dd,J＝10.4Hz,1H),7.19(d,1H),6.48(dd,J＝20.6Hz,1H),4.59(s,1H),4.56(s,1H),4.16(s,1H),4.07(s,1H),4.01(d,3H),3.94(t,J＝5.9Hz,1H),3.82(t,J＝5.8Hz,1H),2.99(t,J＝5.8Hz,1H),2.90(t,J＝5.9Hz,1H),1.32(d,6H).

17. Synthesis of Compound S17

Step 1: to a solution of Boc-glycine (1.2 eq) in N, N-dimethylformamide was added successively the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) and stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 17a is obtained by direct column chromatography separation.

And 2, step: to a dichloromethane solution containing intermediate 17a (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S17 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.66(dd,J＝9.4,1H),7.18(d,1H),6.47(dd,J＝20.9Hz,1H),4.59(s,1H),4.55(s,1H),4.21(s,1H),4.13(s,1H),4.00(d,3H),3.92(t,J＝5.8Hz,1H),3.80(t,J＝5.8Hz,1H),3.44(d,1H),3.35(s,1H),2.98(t,J＝5.9Hz,1H),2.89(t,J＝6.0Hz,1H).

18. Synthesis of Compound S18

Step 1: to a solution of 2-furylacetic acid (1.2 eq) in N, N-dimethylformamide was added successivelyCompound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), DIPEA (3 eq) was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S18 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.66(dd,J＝9.4,1H),7.18(d,1H),6.47(dd,J＝20.9Hz,1H),4.59(s,1H),4.55(s,1H),4.21(s,1H),4.13(s,1H),4.00(d,3H),3.92(t,J＝5.8Hz,1H),3.80(t,J＝5.8Hz,1H),3.44(d,1H),3.35(s,1H),2.98(t,J＝5.9Hz,1H),2.89(t,J＝6.0Hz,1H).

19. Synthesis of Compound S19

Step 1: to a solution of Boc-L-serine (1.2 eq) in N, N-dimethylformamide was added the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 19a is obtained by direct column chromatography separation.

And 2, step: to a dichloromethane solution containing intermediate 19a (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then the solvent is dried by rotation, extracted by EA and saturated NaHCO ₃ And (6) washing. Then the compound S19 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.66(dd,J＝9.7Hz,1H),7.18(d,1H),6.46(dd,J＝21.3Hz,1H),4.56(d,J＝13.8Hz,2H),4.28–4.18(m,1H),4.17–4.07(m,1H),4.00(d,3H),3.92(t,J＝5.9Hz,1H),3.80(t,J＝5.8Hz,1H),3.74(d,J＝5.2Hz,2H),3.63–3.56(m,1H),2.99(t,J＝5.9Hz,1H),2.89(t,J＝5.8Hz,1H).

20. Synthesis of Compound S20

Step 1: to a solution of N, N-dimethylformamide in which N-Boc-L-histidine (1.2 eq) was dissolved, the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq) were added in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 20a is obtained by direct column chromatography separation.

Step 2: to a dichloromethane solution containing intermediate 20a (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then spin-drying the solvent, EA extraction, saturated NaHCO ₃ And (6) washing. Then the compound S20 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.70–7.64(m,2H),7.19(d,J＝16.1Hz,1H),6.96(s,1H),6.48(dd,J＝20.3Hz,1H),4.58(d,J＝10.6Hz,2H),4.22(s,1H),4.12(s,1H),4.00(d,3H),3.94(t,J＝6.0Hz,1H),3.90(t,J＝6.4Hz,1H),3.82(t,J＝5.9Hz,1H),3.11(m,1H),3.02–2.94(m,2H),2.92–2.88(m,1H).

21. Synthesis of Compound S21

Step 1: to a solution of Boc-1-aminocyclopropylcarboxylic acid (1.2 eq) in N, N-dimethylformamide were added in this order the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), DIPEA (3 eq), and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 21a is obtained by direct column chromatography separation.

Step 2: to a dichloromethane solution containing intermediate 21a (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then the solvent is dried by rotation, extracted by EA and saturated NaHCO ₃ And (6) washing. Then the compound S21 is obtained by column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.49(d,1H),8.45(s,1H),7.42(dd,J＝9.8Hz,1H),7.29(s,1H),6.44(dd,J＝13.9Hz,1H),4.70(d,J＝10.1Hz,2H),4.17–4.13(m,2H),4.07(s,2H),4.02–3.97(m,2.4H),3.73(t,J＝5.8Hz,0.6H),2.91(t,J＝5.8Hz,2H),1.44–1.39(m,2H),0.88–0.82(m,2H).

22. Synthesis of Compound S22

Step 1: to a solution of compound 22a (1.2 eq) in dimethylformamide was added compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S22 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.27(d,1H),7.76(d,J＝14.1Hz,1H),7.49–7.42(m,1H),7.31(d,J＝11.2Hz,1H),7.18–7.06(m,4H),6.47(dd,J＝22.0Hz,1H),4.77(s,2H),4.25(d,J＝4.1Hz,2H),4.13(s,2.4H),4.02(t,J＝6.0Hz,1.6H),4.00(s,0.6H),3.77(t,J＝5.8Hz,0.4H),3.00–2.92(m,2H),2.34(d,3H).

23. Synthesis of Compound S23

Step 1: to a solution of 4-Boc-1-piperazineacetic acid (1.2 eq) in N, N-dimethylformamide was added the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), DIPEA (3 eq) in that order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the intermediate 23a is obtained by direct column chromatography separation.

Step 2: to a dichloromethane solution containing intermediate 23a (1 eq) was added trifluoroacetic acid (20 eq) and the mixture was stirred at room temperature for 2 hours. Then the solvent is dried by rotation, extracted by EA and saturated NaHCO ₃ And (5) washing. Then the compound S23 is obtained by column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.67(dd,1H),7.19(d,J＝17.4Hz,1H),6.49(dd,J＝19.4Hz,1H),4.60(s,1H),4.56(s,1H),4.22(s,1H),4.13(s,1H),4.01(d,3H),3.95(t,J＝5.9Hz,1H),3.82(t,J＝5.8Hz,1H),3.04(d,J＝4.2Hz,2H),3.00(t,J＝5.8Hz,1H),2.92–2.88(m,5H),2.58–2.51(m,4H).

24. Synthesis of Compound S24

Step 1: to a solution of 3-methyl-5-isoxazoleacetic acid (1.2 eq) in N, N-dimethylformamide was added in this order the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), DIPEA (3 eq) and stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S24 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.42(d,1H),7.43(dd,J＝10.8Hz,1H),7.28(d,J＝9.9Hz,1H),6.99–6.90(m,1H),6.45(dd,J＝17.8Hz,1H),6.08(d,1H),4.71(d,2H),4.15(d,2H),4.08(s,2H),3.99–3.96(m,2H),3.80–3.69(m,3H),2.93–2.88(m,2H),2.27(d,3H).

25. Synthesis of Compound S25

Step 1: to a solution of 3- (5- (trifluoromethyl) -1,2, 4-oxadiazol-3-yl) benzoic acid (1.2 eq) in N, N-dimethylformamide was added in this order the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S25 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.77(d,1H),8.50(d,J＝9.7Hz,1H),8.18(t,J＝7.3Hz,1H),7.98(d,J＝7.5Hz,1H),7.57–7.51(m,2H),7.41(dd,J＝25.7Hz,1H),7.22(d,1H),6.42(dd,J＝26.9Hz,1H),4.72(d,J＝15.3Hz,2H),4.33(dd,J＝10.9,4.0Hz,2H),4.12(s,2H),3.97(t,J＝5.7Hz,1.4H),3.95(s,1H),3.70(t,J＝5.8Hz,0.6H),2.87(t,J＝5.8Hz,2H).

26. Synthesis of Compound S26

Step 1: to a solution of 5- (2-thienyl) -3-isoxazolic acid (1.2 eq) in N, N-dimethyl formamideTo the amide solution were added the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq), and DIPEA (3 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S26 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.24(d,1H),7.88(d,J＝17.4Hz,1H),7.56–7.47(m,2H),7.42(s,1H),7.29(d,J＝12.4Hz,1H),7.13(t,J＝4.4Hz,1H),6.78(d,J＝2.6Hz,1H),6.50–6.40(m,1H),4.75(s,2H),4.32(d,J＝4.6Hz,2H),4.09(d,J＝29.8Hz,3H),4.02(t,J＝5.6Hz,2H),3.99(s,1H),3.78(t,J＝5.5Hz,1H),3.00–2.90(m,2H),2.79(s,1H).

27. Synthesis of Compound S27

Step 1: to a solution of shikimic acid (1.2 eq) in N, N-dimethylformamide was added the compound S6 (1 eq), EDCI (1.5 eq), HOBt (1.5 eq) and DIPEA (3 eq) in that order and stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S27 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.66(dd,J＝12.8,2.2Hz,1H),7.18(d,1H),6.47(dd,J＝21.5Hz,2H),4.60(s,1H),4.55(s,1H),4.34(t,J＝4.1Hz,1H),4.21(s,1H),4.12(s,1H),4.06–3.94(m,5H),3.93(t,J＝4.5Hz,1H),3.83(t,J＝5.7Hz,1H),3.64(dd,J＝7.8Hz,1H),2.98(t,J＝5.7Hz,1H),2.89(t,J＝5.9Hz,1H),2.78–2.72(m,1H),2.19(dd,J＝17.8Hz,1H).

28. Synthesis of Compound S28

Step 1: et was added to a solution of the compound S6 (1 eq) in N, N-dimethylformamide ₃ N (1.5 eq), after cooling to 0 ℃ 5, 5-dimethyl-2-chloro-1, 3, 2-dioxaphosphorinanyl phosphate (1.2 eq) was added dropwise and stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S28 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.85(d,J＝92.3Hz,1H),7.41(d,J＝2.1Hz,1H),7.21(s,1H),6.42(dd,J＝7.7,2.0Hz,1H),4.60(d,J＝43.3Hz,2H),4.22–4.17(m,1H),4.13(t,J＝9.9Hz,2H),4.01(d,J＝8.0Hz,3H),3.96–3.93(m,1H),3.89(s,1H),3.87(s,2H),3.84(d,J＝14.1Hz,2H),3.60–2.85(t,J＝5.7Hz,2H),1.08(d,J＝23.4Hz,3H),0.96(d,J＝5.5Hz,3H).

29. Synthesis of Compound S29

Step 1: et was added to a solution of the compound S6 (1 eq) in N, N-dimethylformamide ₃ N (1.5 eq), cooled to 0 ℃ and then added dropwise with vinyl chlorophosphate (1.2 eq) and stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S29 is obtained by direct column chromatography separation. ¹ H NMR(400MHz,Chloroform-d)δ8.62(d,J＝61.0Hz,1H),7.42(dd,J＝11.7,2.2Hz,1H),7.27(d,J＝10.3Hz,1H),6.43(dd,J＝16.1,2.3Hz,1H),4.66(d,J＝34.7Hz,2H),4.45–4.36(m,2H),4.29(d,J＝12.2Hz,2H),4.12(q,J＝7.2Hz,1H),4.04(s,2H),3.98(s,1H),3.96(s,1H),3.96–3.92(m,2H),2.89(s,2H),2.05(s,1H).

30. Synthesis of Compound S30

Step 1: to a solution of the compound S6 (1 eq) in N, N-dimethylformamide were added the compound DIPEA (2 eq) and methyl 2-chloropyrimidine-5-carboxylate (1.2 eq) in this order, and the mixture was stirred at room temperature for 6 hours. Then the solvent is dried by spinning, and the compound S30 is obtained by direct column chromatography separation. 1H NMR (400mhz, dmso-d 6) δ 8.83 (d, J =8.7hz, 2h), 8.29 (d, J =14.5hz, 1h), 7.45 (dd, J =25.5,2.2hz, 1h), 7.30 (d, J =14.3hz, 1h), 6.71 (dt, J =23.1,4.0hz, 1h), 6.47 (dd, J =29.2,2.2hz, 1h), 4.79 (d, J =18.9hz, 2h), 4.37 (dd, J =15.5,4.2hz, 2h), 4.20 (s, 2H), 4.03 (t, J =5.8hz, 2h), 3.94 (d, J =41.4hz, 3h), 3.84-3.80 (m, 1H), 3.05-2.86 (m, 86H).

2. Biological Experimental examples section

1. Detection of murine cGAS inhibitory Activity of a portion of the exemplified Compounds in RAW-Lucia ISG cells

The RAW-Lucia ISG cells used in the experiment are derived from rat-derived RAW 264.7 macrophages, and can stably express reporter gene (interferon stimulation response element-luciferase) plasmids. Under the stimulation of exogenous signals such as dsDNA, the downstream Interferon Regulatory Factor (IRF) signal path of cGAS can be activated, and the expression of downstream reporter gene luciferase is induced. Luciferase catalyzes a luciferin (luciferin) substrate to be oxidized to generate oxyluciferin, and in the oxidation process, bioluminescence is emitted, and the fluorescence value can be measured by a multifunctional enzyme-labeling instrument. In the experiment, dsDNA is adopted to induce IRF signal path activation, and a compound with better cGAS inhibitory activity is screened out, and the specific experimental operation is as follows:

RAW-Lucia ISG cells (Invivogen) in logarithmic growth phase 1 × 10 ⁵ One well was seeded in 96-well plates, different concentrations of compound were added and preincubated for 1h in 37 ℃ incubator. DMSO group as negative control for compounds; cells were transfected with 1. Mu.g/mL dsDNA (Sigma) and Lipofectamine2000 (Invitrogen) complex for 24h. The preparation method of the transfection complex comprises the following steps: mu.l of the solution of the dsDNA was added to 10. Mu.l of Opti-MEM (Gibco) at 0.2. Mu.g, and 10. Mu.l of Opti-MEM was added to 0.2. Mu.l of Lipofectamine2000, and after mixing, 20. Mu.l of the complex was added to a 96-well plate; using QUANTI-Luc ^TM The reagent (InvivoGen) detects luciferase activity. Adding 20 μ L cell culture supernatant into 96-well opaque white plate, and adding 50 μ L QUANTI-Luc ^TM Reagents, values were read on a multifunctional microplate reader (BioTek, winooski, VT). Relative luciferase activity calculation method: lipofectamine 2000-affected cells as negative control, lipofectamine2000: dsDNA complex-affected cells as positive control, relative luciferase activity = (RLU sample-RLU negative control)/(RLU positive control-RLU negative control), RLU represents original luciferase reading, and IC was obtained by fitting compound concentration and relative luciferase activity using Graphpad prism8.0 software ₅₀ 。

This experiment also investigated the growth proliferation inhibitory activity of the test compounds on cells and obtained CC ₅₀ Values, specific experimental manipulations were as follows:

RAW-Lucia ISG (InvivoGen) cells in logarithmic growth phase were cultured at 5X 10 ⁴ One/well was seeded in 96-well plates, different concentrations of compounds were added and pre-incubated in an incubator at 37 ℃ for 24h. DMSO groups served as control wells for compounds. After 24h of incubation, 10. Mu.L of CCK8 solution was added to each well, the plate was incubated in an incubator for 2 hours, and the absorbance at 450nm was measured with a microplate reader. The absorbance of the experimental wells is As (containing cells, culture medium, CCK-8 solution and compound), the absorbance of the control wells is Ac (containing cells, culture medium, CCK-8 solution and DMSO), the absorbance of the blank wells is Ab (containing culture medium, CCK-8 solution), and the cell survival rate = [ (As-Ab)/(Ac-Ab)]X100%. CC was determined by fitting compound concentration to cell viability using Graphpad prism8.0 software ₅₀ The value is obtained.

The activity results for some of the exemplified compounds are shown in table 1 below:

TABLE 1

The results show that: all detection compounds have good inhibitory activity on dsDNA-induced cGAS signal pathway activation in RAW-Lucia ISG cells; wherein, the mouse-derived cGAS activity of most compounds is superior to that of the positive compound G140, and the compounds have no obvious inhibitory activity on the growth and proliferation of cells.

2. Detection of human cGAS inhibitory Activity of some exemplary Compounds in THP1-Dual cells

The THP1-Dual cell is derived from a human THP-1 monocyte cell line and can stably express a reporter gene (interferon stimulation response element-luciferase) plasmid. Under the stimulation of exogenous signals such as dsDNA/cGAMP, interferon Regulatory Factor (IRF) signal pathways can be activated, and the expression of downstream reporter gene luciferase is induced. Luciferase catalyzes a luciferin (luciferin) substrate to be oxidized to generate oxylucerin, bioluminescence is emitted in the oxidation process, the fluorescence value can be measured through a multifunctional microplate reader, and the value can indirectly reflect the activity of human-derived cGAS. In the stage of primary screening of the compound, dsDNA is adopted to induce IRF signal pathway activation, and a cGAS inhibitor with good activity is screened out. Subsequent stimulation of cGAS downstream STING-mediated IRF signaling pathway activation with cGAMP, without luciferase inhibitory activity, suggests that the exemplified compounds specifically act on cGAS, but not STING and its downstream signaling pathways. Finally, we also examined the growth proliferation inhibitory activity of the compounds on cells, i.e., cytotoxicity. The specific experimental operations were as follows:

a. detecting the Effect of Compounds on dsDNA stimulated cGAS Signaling pathway luciferase Activity in THP1-Dual cells

THP1-Dual (Invivogen) cells in logarithmic growth phase were counted at 5X 10 ⁴ One well was seeded in 96-well plates, different concentrations of compound were added and preincubated for 1h in 37 ℃ incubator. The DMSO group served as a negative control for the compounds. Cells were transfected with 2. Mu.g/ml dsDNA (Sigma) and Lipofectamine2000 (Invitrogen) complex for 24h. The preparation method of the transfection complex comprises the following steps: mu.l of Opti-MEM (Gibco) was added to 0.4. Mu.g of dsDNA, and 10. Mu.l of Opti-MEM was added to 0.4. Mu.l of Lipofectamine2000, and after mixing, 20. Mu.l of the complex was added to a 96-well plate; using QUANTI-Luc ^TM Luciferase activity was detected by reagent (InvivoGen). Adding 20 μ l cell culture supernatant into 96-well opaque white plate, and adding 50 μ l QUANTI-Luc ^TM Reagents, values were read on a multifunctional microplate reader (BioTek, winooski, VT).

Relative luciferase activity calculation method: lipofectamine 2000-affected cells were used as negative controls, lipofectamine 2000.

b. Testing the Effect of Compounds on cGAMP-induced cGAS signaling pathway luciferase Activity in THP1-Dual cells

THP1-Dual cells 5 x 10 ⁴ One/well is planted in a 96-well plate, compounds with better luciferase inhibitory activity with different concentrations are added, and the mixture is pre-incubated for 1h in an incubator at 37 ℃. The DMSO group served as a negative control for the compounds. Cells were transfected with 2.5. Mu.g/mL cGAMP (Invivogen) complex with Lipofectamine2000 (Invitrogen) for 24h. Luciferase activity was detected (method as in a).

c. Detecting the growth proliferation inhibitory Activity of Compounds on test cells

THP1-Dual (InvivoGen) cells in logarithmic growth phase were counted at 5X 10 ⁴ One well was seeded in 96-well plates, different concentrations of compound were added and preincubated in an incubator at 37 ℃ for 24h. DMSO groups served as control wells for compounds. After 24h of incubation, 10. Mu.L of CCK8 solution was added to each well, the plates were incubated in an incubator for 2 hours, and the absorbance at 450nm was measured with a microplate reader. The absorbance of the experimental wells is As (containing cells, culture medium, CCK-8 solution and compound), the absorbance of the control wells is Ac (containing cells, culture medium, CCK-8 solution and DMSO), the absorbance of the blank wells is Ab (containing culture medium, CCK-8 solution), and the cell survival rate = [ (As-Ab)/(Ac-Ab)]X 100%. The CC50 values were fitted to compound concentrations and cell viability by Graphpad prism8.0 software.

The luciferase activity of the cGAS-STING signaling pathway stimulated by the compounds on dsDNA or cGAMP, as well as the results of cytotoxicity assays, are shown in table 2 below:

TABLE 2

The results show that: the exemplified compounds all stimulated the cGAS signaling pathway luciferase activity better towards THP1-Dual cell dsDNA, with the best S7 and S17 activities and no inhibition of cGAMP-induced luciferase activity, indicating a specific effect on cGAS, but not STING and its downstream signaling pathway.

3. Water solubility of some exemplary Compounds

1.00mg of compound was accurately weighed using a one-ten-thousandth electronic balance into a 2mL volumetric flask, and the volume was fixed using methanol as a solvent. Then, the solutions were diluted to 250, 125, 62.5 and 31.25. Mu.g/mL standard solutions in this order. A standard curve of concentration-peak area was prepared by using an HPLC analytical column and including 5 gradients. Then the saturated compound solution to be measured (or diluted in advance, and the peak area of the response is preferably in the standard curve) is used for detecting the peak area of the compound by HPLC, and the solubility is calculated by utilizing the fitted standard curve.

The solubility of compounds G140, S7 (HCl salt) and S17 (HCl salt) in water is shown in fig. 1. The results show that: the positive compound G140 was completely insoluble in water, whereas the hydrochloride salts of the compounds S7 and S17 of the present invention had good water solubility, 3.79 and 27.65mg/mL, respectively; the hydrochlorides of the remaining compounds all had a water solubility of greater than 3.0mg/mL.

4. Immunoblotting (Western Blot) to examine the effect of representative compounds on dsDNA-induced activation of the cGAS signaling pathway in THP1-Dual cells

Detection was performed using a conventional Western Blot. THP1-Dual (Invivogen) cells in logarithmic growth phase 2.5 x 10 ⁵ One well was inoculated in a 24-well plate, compounds of different concentrations were added, and preincubation was carried out for 1h at 37 ℃ in an incubator. The DMSO group served as a negative control for the compounds.

Cells were transfected with 2. Mu.g/ml dsDNA and Lipofectamine2000 complexes for 2h stimulation. The preparation method of the transfection compound comprises the following steps: mu.g dsDNA was added to 50. Mu.L of Opti-MEM and 1. Mu.L of Lipofectamine2000 was added to 50. Mu.L of LOpti-MEM, and after mixing, 100. Mu.L of the complex was added to a 24-well plate, and the cell harvest was lysed with a lysate. Taking a proper amount of sample to carry out SDS-PAGE electrophoresis, transferring the protein to a nitrocellulose membrane by using a semi-dry electrotransfer system after the electrophoresis is finished, and placing the nitrocellulose membrane in a confining liquid (SuperBlock) ^TM T20 (TBS) Blocking Buffer) was blocked for 1h at room temperature, and then the membranes were placed in primary antibody solutions (1 ^TM T20 (TBS) Blocking Buffer) was incubated overnight at 4 ℃. Washed 5 times with TBS containing 0.1-vol Tween 20 for 5min each. The membrane was placed in a secondary antibody solution (horseradish peroxidase-labeled goat anti-rabbit IgG,1 20000 diluted in TBS containing 0.1% Tween 20) for a reaction at room temperature for 1h. After washing the membrane 5 times as above, it was developed with ECL plus reagent and photographed with Image Quant LAS 4000.

The western blotting was used to detect the effect of compounds S7 and S17 on dsDNA-stimulated phosphorylation of the cGAS signaling pathway in THP1-Dual cells, and the results are shown in FIGS. 2 to 3. The results show that: the compounds S7 and S17 have good inhibition effect on a cGAS signal pathway in THP1-Dual cells, and can regulate the phosphorylation levels of TBK1, IRF3 and STING in a dose-dependent manner.

5. Anti-inflammatory activity study of compound on LPS-induced acute inflammation mouse animal model

Experiments were performed with 20g female BALB/c mice, grouped by body weight. An amount of compound powder was weighed out and dissolved in DMSO, followed by corn oil addition (10% DMSO +90% Corn oil), and test compound was injected intraperitoneally 1h before LPS (Sigma) (dose: 30 mg/kg). After 1h of administration, 5mg/kg LPS is injected into the abdominal cavity, blood is collected in the orbit at 1.5h, serum is taken, and the levels of inflammatory cytokines TNF-alpha, IL-6 and IL-1 in the serum are detected by adopting an ELISA method. ( The normal group was neither induced with LPS nor compound added; blank group was induced with LPS only, without compound; dexamethasone, ru.521, G140, S7 and S17 are administered 1h before LPS induction, and compound is added )

FIG. 4 is a graph showing the results of the inhibition of TNF-. Alpha.release activity of tumor necrosis factor in LPS-induced acute inflammatory mouse animal models by compounds S7 and S17 according to the example of the present invention; FIG. 5 is a graph showing the results of inhibition of the release activity of inflammatory factor IL-6 by compounds S7, S17 according to the example of the present invention in a mouse model of LPS-induced acute inflammation; FIG. 6 is a graph showing the results of experiments in which compounds S7 and S17 according to the example of the present invention inhibit the release activity of inflammatory factor IL-12 in a mouse animal model of LPS-induced acute inflammation.

FIGS. 4-6 show that: the compounds S7 and S17 can obviously inhibit the release of inflammatory factors in an LPS-induced acute inflammatory mouse model; wherein, the effect of inhibiting the release of TNF-alpha and IL-2 is better than positive compounds G140 and Ru.521, and the effect is equivalent to that of a classical anti-inflammatory drug dexamethasone clinically used.

6. Conclusion

The invention provides a cGAS small molecule inhibitor with greatly improved water solubility, and finds that the cGAS small molecule inhibitor has better inhibition effect on stimulating a cGAS signal path through dsDNA in THP1-Dual cells and RAW-Lucia ISG cells. Excessive activation of the cGAS pathway is involved in inflammatory and autoimmune diseases, such as sepsis, acute pancreatitis, rheumatoid arthritis, systemic lupus erythematosus and Aicardi-Goutieres syndrome, and thus, the compound represented by formula I provided by the invention, or its isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof can provide a promising therapeutic strategy for preventing inflammatory and autoimmune diseases.

Claims (10)

1. A compound of formula I, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

R ₁ is hydrogen, C1-C3 alkyl, or C1-C3 alkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

R ₂ and R ₃ Each independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C6 aryl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C6 aryl, C5-C6 heteroaryl substituted by halogen, amino, cyano, hydroxy, alkoxy or-COORd;

or, R ₂ And R ₃ Are connected to form a three-to six-membered N-containing or O-containing heterocyclic group;

or, R ₂ And R ₃ One and R ₈ Are linked to form a three-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen, saturated or unsaturated five-to six-membered heterocyclic group, or saturated or unsaturated five-to six-membered heterocyclic group substituted by halogen, amino, cyano, C1-C3 alkyl or C1-C3 haloalkyl;

R ₅ 、R ₆ and R ₇ Each independently hydrogen, halogen, cyano, hydroxy, alkoxy, C1-C3 alkyl, -CH = CH ₂ C ≡ CH, C3-C4 cycloalkyl, or C1-C3 alkyl, C3-C4 cycloalkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

R ₈ is hydrogen, C1-C3 alkyl, or C1-C3 alkyl substituted by halogen, amino, cyano, hydroxy or alkoxy;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a three-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, cyano, hydroxy, alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with a group selected from the group consisting of halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

2. The compound according to claim 1, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof,

R ₁ is hydrogen or C1-C3 alkyl;

R ₂ and R ₃ Each independently hydrogen, C1-C3 alkyl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C3 alkyl, C5-C6 heteroaryl substituted by halogen, amino, hydroxy, alkoxy or-COORd;

or, R ₂ And R ₃ Linked to form a three-to six-membered heterocyclic group containing 1-2 heteroatoms selected from: o, N;

or, R ₂ And R ₃ One and R ₈ Are linked to form a five-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen, unsaturated five-to six-membered heterocyclic group, or unsaturated five-to six-membered heterocyclic group substituted by halogen, amino or C1-C3 alkyl;

R ₅ 、R ₆ and R ₇ Each independently hydrogen or halogen;

R ₈ is hydrogen or C1-C3 alkyl;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a five-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, hydroxy, alkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

3. The compound according to claim 1, or an isomer, prodrug, solvate, hydrate or pharmaceutically acceptable salt thereof,

R ₁ is hydrogen;

R ₂ and R ₃ Each independently hydrogen, C1-C3 alkyl, C5-C6 heteroaryl, -C (= O) Ra, -P (= O) RbRc, or C1-C3 alkyl, C5-C6 heteroaryl substituted with halogen, hydroxy or-COORd;

or, R ₂ And R ₃ Linked to form a three-to six-membered heterocyclic group containing 1-2 heteroatoms selected from: o, N;

or, R ₂ And R ₃ One and R ₈ Are linked to form a five-to six-membered N-containing heterocyclic group;

R ₄ is hydrogen, halogen or

R ₅ 、R ₆ And R ₇ Each independently hydrogen or halogen;

R ₈ is hydrogen;

or, R ₈ And R ₂ And R ₃ One of which is linked to form a five-to six-membered N-containing heterocyclic group;

ra, rb, rc, and Rd are each independently hydrogen, halogen, amino, hydroxy, alkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl, or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 cycloalkenyl, C6 aryl, C5-C6 heteroaryl substituted with halogen, amino, cyano, hydroxy, alkoxy, haloalkyl, aryl, or heteroaryl.

4. The compound of claim 1, or an isomer, prodrug, solvate, hydrate thereof, or a pharmaceutically acceptable salt thereof, selected from the group consisting of compounds numbered S1 through S30, or an isomer, prodrug, solvate, hydrate thereof, or a pharmaceutically acceptable salt thereof:

5. use of a compound of any one of claims 1 to 4, an isomer, prodrug, solvate, hydrate thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use as a cGAS inhibitor.

6. Use according to claim 5, characterized in that the cGAS inhibitor is used for the treatment and/or prevention of clinical diseases and for laboratory diagnosis and/or detection.

7. Use of a compound of any one of claims 1 to 4, or an isomer, prodrug, solvate, hydrate thereof or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for anti-inflammatory, anti-infective, or preventing and/or treating an autoimmune disease.

8. The use according to claim 7, wherein the anti-inflammatory, anti-infective, prophylactic and/or therapeutic treatment of autoimmune diseases comprises anti-viral, prophylactic and/or therapeutic treatment of bacterial infection autoimmune diseases, prophylactic and/or therapeutic treatment of cardiovascular diseases, prophylactic and/or therapeutic treatment of neurodegenerative diseases, prophylactic and/or therapeutic treatment of inflammatory bowel diseases, prophylactic and/or therapeutic treatment of diabetes, prophylactic and/or therapeutic treatment of arthritis, anti-tumor immunity.

9. A pharmaceutical composition for use as a cGAS inhibitor comprising:

(i) One or more therapeutically and/or prophylactically effective amounts of a compound of any one of claims 1-4, or an isomer, prodrug, solvate, hydrate thereof, or a pharmaceutically acceptable salt thereof; and

(ii) A pharmaceutically acceptable carrier.

10. A pharmaceutical composition for anti-inflammatory, anti-infective, preventing and/or treating autoimmune diseases, comprising:

(i) One or more therapeutically and/or prophylactically effective amounts of a compound of any one of claims 1 to 4, or an isomer, prodrug, solvate, hydrate thereof, or a pharmaceutically acceptable salt thereof; and

(ii) A pharmaceutically acceptable carrier.

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