Fluoro-substituted benzo [b] selenophenes STING agonist molecule and medical application thereof

Abstract

The invention discloses a fluorine-substituted benzo [b] selenophen STING agonist molecule and medical application thereof, the structural formula of the compound is as shown in formula (I): # imgabs0. The fluorine-substituted benzo [b] selenophen STING agonist molecule disclosed by the invention has more excellent membrane permeability, more obvious antiviral activity and more excellent STING agonist activity, and the physicochemical properties and stability are further improved; the compound can be used as a controllable and effective STING regulating agent and is used for treating virus infectious diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a class of fluorine-substituted benzo[b]selenophene STING agonist molecules and their use in preparing antiviral drugs. Background Art

[0002] Studies have shown that cGAS and STING have been identified as intracellular sensors that activate the interferon pathway in response to viral infection, thereby mediating host defense against a range of DNA and RNA viruses (Cell Host & Microbe. 2016, 19, 150-158). When the body is infected by a virus, host cells produce a coordinated innate immune response, including type I interferons and proinflammatory cytokines, which can eliminate pathogens.

[0003] Currently, multiple animal data support that STING knockout mice are more susceptible to DNA virus infection (Nature. 2009; 461: 788-792). It has been reported that the lack of STING expression and functional innate DNA sensing pathways in human and mouse hepatocytes partially explains the preference of hepatitis B virus for hepatocytes (Hepatology. 2016; 64: 746-759). Importantly, the specific introduction of STING expression in hepatocytes can improve the control of HBV in vivo.

[0004] Although there is still much to be revealed about the functions of cGAS and STING in infected cells, the many different immune evasion strategies adopted by RNA and DNA viruses indicate the importance of this pathway for sensing viral pathogens. Some RNA and DNA viruses use similar mechanisms to inhibit STING-TBK1 interactions and STING function (Cell Host & Microbe. 2016, 19, 150-158). At the same time, multiple studies support that STING also plays a role in immunity against RNA virus infection (Nat. Commun. 2019; 10: 2830), and cGAS-STING activity has been detected in lung samples and skin lesions of patients infected with SARS-CoV-2. The STING-dependent type I IFN signature is mainly mediated by macrophages and adjacent endothelial cells, and mitochondrial DNA is released. Pharmacological inhibition of STING can reduce severe lung inflammation caused by severe acute respiratory syndrome coronavirus 2 and improve disease outcomes in mouse models, and controlling abnormal and prolonged type I interferon responses can reduce tissue damage (Nature. 2022; 603: 145-151). Preclinical data have confirmed that STING agonist adjuvant vaccines can produce strong and long-lasting neutralizing antibodies and T cell responses (Cell Res. 2022; 32: 495-497). In addition to viral infections, STING is also involved in infections of bacteria and protozoan parasites (Trends Parasitol. 2020; 36: 773-784). Therefore, the development of STING agonists to achieve antiviral effects is of positive significance.

[0005] Patent CN 113429387A A benzo[b]selenophene STING regulator, its preparation method and use The disclosed compounds I-14, I-15 and I-16 do not have an advantageous effect on antiviral research, and their in vitro antiviral IC 50 The antiviral activity in vivo is poor, and the introduction of selenium atoms will lead to changes in the electron cloud density, resulting in a decrease in STING agonist activity. The compound structure has problems that need to be improved. Therefore, the present invention designs different structural modifications to optimize the antiviral activity of the compound. Summary of the invention

[0006] The present invention provides a class of fluorine-substituted benzo[b]selenophene STING agonist molecules, which inhibit the release of cytokines such as IFN-I when RNA or DNA viruses invade. This class of molecules can activate the cGAS-STING signaling pathway in the body, thereby achieving the antiviral activity of STING activation.

[0007] One of the objects of the present invention is to provide a compound of general formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof:

[0008]

[0009] in:

[0010] X is selected from C(R 3 ) n 、N(R 3 ) m , O and S;

[0011] R 1 Selected from hydrogen atom, halogen atom, cyano group, OR 4 、N(R 4 ) 2 , C 1 ~C 6 Alkyl, C 1 ~C 6 Halogenated alkyl, OR 4 Substituted C 1 ~C 6 Alkyl and OR 4 Substituted C 3 ~C 6 Cycloalkyl;

[0012] R 2 Selected from hydrogen atoms, hydroxyl groups, double bonds, halogen atoms, amino groups, hydroxymethyl groups, methoxy groups, thiomethyl groups, N(CH 2 CH 3 ) 2 、N(CH 3 ) 2 , benzyloxy, benzoyloxy, C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 3 ~C 6 Cycloalkyl, 3-6 membered heterocyclic group and C 5 ~C 10 Aryl;

[0013] or X and R 2 Can form a 3- to 6-membered ring together with the atoms to which it is attached;

[0014] or R 2 Together with the atoms to which they are attached, they can form a 3- to 6-membered ring;

[0015] R 3 From hydrogen atoms, hydroxyl, amino, hydroxymethyl, methoxy, thiomethyl, N(CH 2 CH 3 ) 2 、N(CH 3 ) 2 , benzyloxy, benzoyloxy, C1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 3 ~C 6 Cycloalkyl, 3-6 membered heterocyclic group and C 5 ~C 10 Aryl;

[0016] R 4 Selected from hydrogen atoms, C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 3 ~C 6 Cycloalkyl, 3-6 membered heterocyclic group and C 5 ~C 10 Aryl;

[0017] n is selected from 0, 1, 2 and 3;

[0018] m is selected from 0 and 1.

[0019] Furthermore, R 1 Selected from hydrogen atom, halogen atom, cyano group, OR 4 、N(R 4 ) 2 , C 1 ~C 3 Alkyl, C 1 ~C 3 Haloalkyl, C 3 ~C 6 Cycloalkyl and OR 4 Substituted C 1 ~C 6 Alkyl group, preferably a hydrogen atom, a fluorine atom, a chlorine atom, a methyl group, an ethyl group, a methoxy group, or an ethoxy group;

[0020] R 2 Selected from hydrogen atoms, hydroxyl groups, double bonds, halogen atoms, C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 3 ~C 6 Cycloalkyl, 3-6 membered heterocyclic group and C 5 ~C 10 Aryl, preferably a hydrogen atom, a hydroxyl group, a double bond, a fluorine atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a methoxy group, an ethoxy group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group;

[0021] R 3 Selected from hydrogen atoms, hydroxyl groups, methoxy groups, N(CH 2 CH3 ) 2 、N(CH 3 ) 2 , benzyloxy, benzoyloxy, C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 3 ~C 6 Cycloalkyl, 3-6 membered heterocyclic group and C 5 ~C 10 Aryl, preferably hydrogen atom, methyl, ethyl, isopropyl;

[0022] R 4 Selected from hydrogen atoms, C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 3 ~C 6 Cycloalkyl, 3-6 membered heterocyclic group and C 5 ~C 10 Aryl;

[0023] n is selected from 0, 1, 2 and 3, preferably 1, 2;

[0024] m is selected from 0 and 1.

[0025] Further, X is selected from CH, CH 2 , NH, O and S, preferably CH, CH 2 and NH.

[0026] Another object of the present invention is to provide the following compounds, stereoisomers or pharmaceutically acceptable salts thereof:

[0027]

[0028]

[0029] In the present invention, the above compounds can be synthesized by the following route:

[0030]

[0031] Among them, R 2 As mentioned above.

[0032] In some more specific embodiments, R 2 It may be a hydrogen atom, a fluorine atom, a methyl group, an ethyl group, a cyclobutyl group, a cyclopropyl group, a double bond, an isopropyl group, a methoxy group, or an ethoxy group.

[0033] In some more specific embodiments, in the process of preparing compound 1 from the raw material 6-bromoresulphaldehyde, the reactant is dimethyl diselenide, the reaction reagents can be DTT, mercaptoethanol, DBU and potassium carbonate, and the reaction solvent can be DMF and tetrahydrofuran.

[0034] In some more specific embodiments, in the process of preparing compound 2 from compound 1, the reactant is ethyl 2-bromoacetate, and the reaction solvent is DMF, N-methylpyrrolidone and DMSO.

[0035] In some more specific embodiments, in the process of preparing compound 3 from compound 2, the reaction reagents can be potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, and the reaction solvents can be DMF and acetonitrile.

[0036] In some more specific embodiments, in the process of preparing compound 4 from compound 3, the reactant can be Selectflour reagent and the solvent can be acetonitrile.

[0037] In some more specific embodiments, in the process of preparing compound 5 from compound 4, the reaction reagents can be potassium carbonate, sodium hydroxide, lithium hydroxide, and the reaction solvent can be 1,4-dioxane, water, methanol and tetrahydrofuran.

[0038] In some more specific embodiments, in the process of preparing compound 6 from compound 5, the reactant is potassium salt of malonic acid monoester, and the reaction reagents can be CDI and MgCl 2 , the reaction solvent can be DMF and tetrahydrofuran.

[0039] In some more specific embodiments, in the process of preparing compound 7 from compound 6, the reactant is a halogenated ester, the reaction reagents can be potassium carbonate, sodium methoxide, sodium ethoxide and sodium hydride, and the reaction solvent can be DMF and tetrahydrofuran.

[0040] In some more specific embodiments, in the process of preparing compound Ia from compound 7, the reaction reagents can be potassium carbonate, sodium hydroxide, lithium hydroxide and hydrochloric acid, and the reaction solvent can be acetic acid, water, methanol and tetrahydrofuran.

[0041] In some more specific embodiments, in the process of preparing compound 8 from compound 5, the reaction reagent can be copper powder, and the reaction solvent can be quinoline.

[0042] In some more specific embodiments, in the process of preparing compound Ⅰb from compound 8, the reactants can be acid anhydrides, carboxylic acids, and acyl halides, the reaction reagents can be Lewis acids such as aluminum chloride, ferric chloride, and boron trifluoride, and the reaction solvent can be dichloromethane or chloroform.

[0043] In some more specific embodiments, in the process of preparing compound 10 from compound 5, the reactant is an amino derivative, the reaction condensation reagent can be HATU, HBTU, HOAT, HOBT, DCC, EDC and EDCI, the base used can be TEA, DIPEA and DMAP, and the reaction solvent can be dichloromethane, tetrahydrofuran and DMF.

[0044] In some more specific embodiments, in the process of preparing compound Ic of general formula from compound 10, the reaction reagents can be potassium carbonate, sodium hydroxide, lithium hydroxide, and the reaction solvent can be 1,4-dioxane, water, methanol and tetrahydrofuran.

[0045] In some more specific embodiments, in the process of preparing compound 17 from compound of formula Ia, the reaction reagent can be dichlorothionyl, oxalyl chloride, and the reaction solvent can be methanol.

[0046] In some more specific embodiments, in the process of preparing compound 18 from compound 17, the reactant can be 1,3-propanedithiol, the reaction reagent can be boron trifluoride ether complex, and the reaction solvent can be dichloromethane or chloroform.

[0047] In some more specific embodiments, in the process of preparing compound 19 from compound 18, the reactant can be diethylaminosulfur trifluoride, and the reaction solvent can be dichloromethane or chloroform.

[0048] In some more specific embodiments, in the process of preparing compound Id from compound 19, the reaction reagents can be lithium hydroxide, potassium hydroxide and sodium hydroxide, and the reaction solvent can be 1,4-dioxane, tetrahydrofuran, methanol and water.

[0049] More specifically, when it is a compound shown as S1-S8, 6-bromoresulphaldehyde is subjected to a substitution reaction under alkaline conditions to obtain selenium-substituted compound 1, compound 1 reacts with ethyl bromoacetate under heating conditions to obtain compound 2, compound 2 undergoes an intramolecular cyclization reaction to obtain compound 3, compound 3 is substituted with a fluorine reagent to obtain compound 4, compound 4 is hydrolyzed under alkaline conditions to obtain compound 5, compound 5 reacts with a Grignard reagent to obtain compound 6, compound 6 is subjected to a substitution reaction under alkaline conditions to obtain a compound of the general formula 7, and the compound of the general formula 7 is hydrolyzed and decarboxylated to obtain a compound of the general formula (Ia).

[0050] When it is a compound shown in S9-S15, compound 5 is subjected to a decarboxylation reaction to obtain compound 8, and compound 8 is subjected to a Friedel-Crafts acylation reaction to obtain a compound of formula (Ib).

[0051] When it is a compound shown in S16-S21, compound 5 and compound 9 are reacted by amide condensation to obtain a compound of formula 10, and the compound of formula 10 is further reacted by ester hydrolysis to obtain a compound of formula (Ic).

[0052] When it is the compound shown in S22 and S23, the compound of general formula Ia is protected by methyl to obtain compound 17, compound 17 reacts with a thiophilic reagent to obtain compound 18, compound 18 is fluorinated to obtain compound 19, and compound 19 is finally subjected to ester hydrolysis reaction to obtain a compound of general formula (Id).

[0053] The third object of the present invention is to provide a pharmaceutical composition, comprising a pharmaceutically effective amount of an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises one or more of the above-mentioned compounds, stereoisomers or pharmaceutically acceptable salts thereof. In the pharmaceutical composition, the excipient comprises a pharmaceutically acceptable carrier, diluent and / or excipient.

[0054] The pharmaceutical composition can be prepared into various types of dosage unit forms according to the therapeutic purpose, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules and injections (solutions or suspensions), etc., preferably tablets, capsules, liquids, suspensions and injections (solutions or suspensions).

[0055] The compounds of the present invention can be administered clinically by oral administration, injection or the like.

[0056] A fourth object of the present invention is to provide the use of the above-mentioned compounds, stereoisomers or pharmaceutically acceptable salts thereof or pharmaceutically acceptable pharmaceutical compositions in the preparation of drugs for activating the cGAS-STING pathway.

[0057] A fifth object of the present invention is to provide the use of the above-mentioned compound, stereoisomer or pharmaceutically acceptable salt thereof or pharmaceutically acceptable pharmaceutical composition in the preparation of a drug, wherein the drug is used to treat diseases related to STING pathway activity.

[0058] Furthermore, the disease associated with STING pathway activity is a viral infection, and the viruses include respiratory syncytial virus (RSV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza virus, SARS virus, new coronavirus (COVID-19), rabies virus, avian influenza virus, porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV), etc.

[0059] Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

[0060] The term "alkyl" refers to a monovalent straight or branched saturated aliphatic hydrocarbon group having a number of carbon atoms within a specified range. The alkyl group may be substituted or unsubstituted. When it is a substituted alkyl group, the substituent is preferably one or more, more preferably 1-3, and most preferably 1 or 2 substituents.

[0061] The term "halogen atom" means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

[0062] The term "haloalkyl" refers to an alkyl group as defined above wherein one or more hydrogen atoms have been replaced by a halogen.

[0063] The term "cycloalkyl" refers to a monocyclic or fused ring ("fused" ring means that each ring in the system shares a pair of adjacent carbon atoms with other rings in the system) group that is all carbon, wherein one or more rings do not have a completely connected π electron system, examples of cycloalkyl (not limited to) are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene. Cycloalkyl groups may be substituted and unsubstituted.

[0064] The term "heterocyclyl" refers to a saturated cyclic group of 3 to 8 ring atoms, one or two of which are selected from N, O or S(O). m (wherein m is an integer from 0 to 2), the remaining ring atoms are C, one or two of which may be optionally replaced by a carbonyl group.

[0065] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 5 to 10 carbon atoms with a completely conjugated π electron system. Non-limiting examples of aryl are phenyl, naphthyl and anthracenyl. Aryl can be substituted or unsubstituted.

[0066] The term "stereoisomers" refers to isomers that result from differences in the way the atoms in a molecule are arranged in space.

[0067] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a pharmaceutically acceptable non-toxic base including an organic base or an inorganic base.

[0068] The present invention includes all possible isomers, as well as salts, solvates and solvated salts of their racemates, enantiomers, diastereomers, tautomers and mixtures.

[0069] Based on the inventor's previous research (CN113429387A), the present invention further optimizes and transforms the related compounds and provides a series of fluorine-substituted benzo[b]selenophene STING agonist molecules. Compared with the benzo[b]selenophene structure designed in the previous research, the fluorine-substituted benzo[b]selenophene molecule of the present invention can utilize the special chemical action of F atoms and selenium elements, and has better membrane permeability, more significant antiviral activity, and more superior STING agonist activity, and the physicochemical properties and stability are further improved, and it can be used as a controllable and effective STING regulator for the treatment of viral infectious diseases. DETAILED DESCRIPTION

[0070] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0072] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0073] The abbreviations involved in the following embodiments are as follows:

[0074] 1 H NMR proton nuclear magnetic resonance spectroscopy

[0075] 13 C NMR 13 C NMR spectroscopy

[0076] CDCl 3 Deuterated chloroform

[0077] CDI N,N-Carbonyldiimidazole

[0078] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

[0079] DCC N,N'-Dicyclohexylcarbodiimide

[0080] DMAP 4-dimethylaminopyridine

[0081] DMF N,N-Dimethylformamide

[0082] DMSO Dimethyl sulfoxide

[0083] DTT DL-dithiothreitol

[0084] EDCI 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0085] EA Ethyl acetate

[0086] HRMS High Resolution Mass Spectrometry

[0087] PE Petroleum Ether

[0088] TLC Thin Layer Chromatography

[0089] Intermediate Preparation 1

[0090]

[0091] Step 1: Preparation of ethyl 3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxylate (4a)

[0092] Compounds 1a to 3a were prepared according to patent CN113429387A. 3.60 g of ethyl 5,6-dimethoxybenzo[b]selenophene-2-carboxylate (3a, 11.5 mmol, 1 eq) was suspended in 36 mL of acetonitrile, and 6.11 g of SelectFlour (17.3 mmol, 1.5 eq) was added and reacted at room temperature for 3 h. After the solvent was removed by rotation, a saturated sodium bicarbonate solution was added to quench; EA was used for extraction (40 mL × 3), the organic phases were combined, washed with water and saturated brine, dried with anhydrous sodium sulfate, and filtered; the filtrate was concentrated and purified by silica gel column chromatography (PE:EA=8:1) to obtain intermediate 4a, a white solid, 0.42 g, with a yield of 11.0%. 1 H NMR (300 MHz, DMSO-d 6 ): δ=7.75(s,1H),7.28(s,1H),4.29(q,J=7.11Hz,2H),3.86(d,J=1.8Hz,6H),1.30(t,J=7.2Hz,3H)ppm.HRMS(ESI + ):cacld for C 13 H 14 FO 4 Se + (M+H) + ,354.9855; found 354.9856.

[0093] Compound 11a can be prepared according to the synthesis method of intermediate 4a described above, and after undergoing a silica gel column chromatography purification step (PE:EA=8:1), the corresponding intermediate 11a can be obtained.

[0094] Step 2: Preparation of 3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxylic acid (5a)

[0095] After suspending 2.30 g of compound 4a (6.95 mmol, 1 eq) in 35 mL of dioxane, 10.5 mL of 2N NaOH (21 mmol, 3 eq) was added and refluxed for 2 h. The mixture was slowly cooled to room temperature, the solvent was removed by vortexing, and the pH was adjusted to acidic (pH = 1-2) using 1N dilute hydrochloric acid under ice bath. After stirring for 30 min, the mixture was filtered and the filter cake was dried to obtain intermediate 5a as an off-white solid, 2.01 g, with a yield of 95.5%. 1 H NMR (300 MHz, DMSO-d 6): δ=13.24(s,1H),7.74(s,1H),7.27(s,1H),3.85(s,6H)ppm.HRMS(ESI - ):cacld for C 11 H 8 FO 4 Se - (MH) - ,302.9577; found 302.9510.

[0096] Compound 12a can be prepared according to the synthesis method of intermediate 5a above, using corresponding starting materials and reactants.

[0097] Step 3: Preparation of methyl 3-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-3-oxopropanoate (6a)

[0098] 110 mg of intermediate 5a (0.36 mmol, 1 eq) was dissolved in 4 mL of DMF, followed by the addition of 176 mg of CDI (1.09 mmol, 3 eq) and the reaction was continued at room temperature for about 1 h; 170 mg of potassium methyl malonate (1.09 mmol, 3 eq) and 103 mg of anhydrous magnesium chloride (1.09 mmol, 3 eq) were added and the reaction was continued at room temperature for 4 h. 1 N dilute hydrochloric acid was added for quenching, and the mixture was filtered and dried to obtain intermediate 6a as a yellow solid (102 mg) with a yield of 83.2%. 1 H NMR (300 MHz, DMSO-d 6 ): δ=7.76(s,1H),7.33(s,1H),4.10(s,2H),3.86(d,J=2.7Hz,6H),3.68(s,3H)ppm.HRMS(ESI + ):cacld forC 14 H 14 FO 4 Se + (M+H) + ,360.9985; found 360.9981.

[0099] Compound 13a can be prepared according to the synthesis method of intermediate 6a described above, using corresponding starting materials and reactants.

[0100] Step 4: Preparation of dimethyl 2-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxylate)succinate (7a)

[0101] 300 mg of intermediate 6a (0.88 mmol, 1 eq) was dissolved in 4 mL of DMF; then 364 mg of potassium carbonate powder (2.64 mmol, 3 eq) and 220 mg of methyl 2-bromopropionate (1.32 mmol, 1.5 eq) were added in sequence, and the mixture was reacted at room temperature overnight. After the reaction was completed, 40 mL of water was added for quenching, and EA was used for extraction (20 mL × 3); the organic phases were combined, washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate; filtered, and the filtrate was concentrated to obtain the crude intermediate 7a, a yellow oily liquid, which was directly used in the next step without further purification. HRMS (ESI + ):cacld for C 17 H 18 FO 5 Se + (M+H) + ,447.0353;found447.0359.

[0102] As shown in Table 1 below, intermediates 7b to 7g can be prepared according to the synthesis method of intermediate 7a above, using corresponding starting materials and reactants.

[0103] Table 1

[0104]

[0105]

[0106] Intermediate Preparation 2

[0107]

[0108] Preparation of 3-fluoro-5,6-dimethoxybenzo[b]selenophene (8a)

[0109] 0.50g of intermediate 5a (1.65mmol, 1eq) and 0.53g of copper powder (8.25mmol, 1eq) were dissolved in 15mL of quinoline and reacted at 170°C for 3h. The mixture was slowly cooled to room temperature, 150mL of 2N hydrochloric acid was added under ice bath conditions, and stirring was maintained for 30min; the mixture was filtered, and the filtrate was extracted with EA (10mL×3), and the organic phases were combined, washed with water and saturated brine, and dried over anhydrous sodium sulfate; the mixture was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (PE:EA=8:1) to obtain intermediate 8a as a white solid, 364mg, with a yield of 85.2%. 1 H NMR (300 MHz, DMSO-d 6): δ=7.68(s,1H),7.47(d,J=5.7Hz,2H),7.17(s,1H),3.82(d,J=2.7Hz,6H)ppm.HRMS(ESI + ):cacld for C 10 H 10 FO 2 Se + (M+H) + ,260.9825; found 260.9829.

[0110] Compound 15a can be prepared according to the synthesis method of intermediate 8a described above, using corresponding starting materials and reactants.

[0111] Intermediate Preparation 3

[0112]

[0113] Preparation of (R)-2-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)butyric acid methyl ester (10a)

[0114] 150 mg of intermediate 5a (0.50 mmol, 1 eq) was dissolved in 5 mL of DMF, followed by the addition of 245 mg of HATU (0.64 mmol, 1.3 eq) and 160 mg of DIPEA (1.24 mmol, 2.5 eq), and the reaction was carried out at room temperature for 30 min; 84 mg of raw material (R)-2-aminobutyric acid methyl ester hydrochloride (0.54 mmol, 1.1 eq) was added, and the reaction was continued for about 2 h. 40 mL of water was added to quench, stirred at room temperature for 30 min, filtered, and the filter cake was slurried with methanol to obtain intermediate 10a, a white solid, 182 mg, with a yield of 90.5%. 1 H NMR (300 MHz, DMSO-d 6 ): δ=8.15(q,J=3.6Hz,1H),7.74(s,1H),7.26(s,1H),4.36(q,J=7.8Hz,1H),3.85(d,J=2 .4Hz,6H),3.67(s,3H),1.94-1.77(m,2H),0.94(t,J=7.5Hz,3H)ppm.HRMS(ESI+):cacld for C 14 H 15 FNO 5 Se + (M+H) + ,404.0407; found 404.0425.

[0115] As shown in Table 2 below, intermediates 10b to 10f can be prepared according to the synthesis method of intermediate 10a above, using corresponding starting materials and reactants.

[0116] Table 2

[0117]

[0118] Intermediate Preparation 4

[0119]

[0120] Step 1: Preparation of methyl 4-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-4-oxobutanoate (17a)

[0121] 100 mg of compound S1 (0.28 mmol, 1 eq) was dissolved in 3 ml of anhydrous methanol, and thionyl chloride (2.78 mmol, 10 eq) was slowly added under ice bath, and the mixture was moved to room temperature for 1 hour. After the reaction was complete, the solvent was spun off, and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution, and then extracted with ethyl acetate until no obvious fluorescence was observed, washed once with water, washed once with brine, dried over anhydrous sodium sulfate, and then spun dry to obtain 95 mg of a light yellow solid product with a yield of 91.6%. HRMS (ESI+): cacld for C 15 H 16 FO 5 Se + (M+H) + ,374.0069; found 373.9997.

[0122] As shown in Table 3 below, intermediate 17b can be prepared according to the synthesis method of intermediate 17a above, using corresponding starting materials and reactants.

[0123] Table 3

[0124]

[0125] Step 2: Preparation of methyl 3-(2-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-1,3-dithian-2-yl)propanoate (18a)

[0126] 95 mg of intermediate 17a (0.255 mmol, 1 eq) was dissolved in 3 ml of dry dichloromethane, and then replaced with nitrogen. 1,3-Propanedithiol (0.51 mmol, 2 eq) and boron trifluoride ether complex (0.255 mmol, 1 eq) were added under ice bath, and stirred for about 2 hours under ice bath conditions, and then moved to room temperature for 48 hours. After the reaction was complete, the reaction was quenched with an appropriate amount of saturated sodium bicarbonate solution, extracted with ethyl acetate, and purified by silica gel column chromatography (PE: EA = 6: 1) to obtain an off-white solid product 18a of about 75 mg, with a yield of 63.6%. HRMS (ESI+): cacld for C 18 H 22 FO 4 S 2 Se + (M+H) + ,464.0031;found464.0002.

[0127] As shown in Table 4 below, intermediate 18b can be prepared according to the synthesis method of intermediate 18a above, using corresponding starting materials and reactants.

[0128] Table 4

[0129]

[0130] Step 3: Preparation of methyl 4,4-difluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]seleno-2-yl)butanoate (19a)

[0131] 75 mg of compound 18a (0.162 mmol, 1 eq) was dissolved in 2 ml of dry dichloromethane, and diethylaminosulfur trifluoride (3.24 mmol, 20 eq) was added, and the mixture was reacted at room temperature for 4.5 hours. After the reaction was complete, the mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, and purified by silica gel column chromatography (PE:EA=4:1) to obtain about 75 mg of off-white solid product 19a, with a yield of 63.6%. 1 HNMR (300 MHz, DMSO-d 6 )δ=7.74(d,J=4.6Hz,1H),7.43(s,1H),3.84(d,J=12.67Hz,6H),3.60(s,3H),2.87-2.2.96(m,2H),2. 72-2.81(m,2H),2.7(t,J=6.9Hz,2H),2.58(t,J=5.6Hz,2H),1.81-1.88(m,2H)ppm.HRMS(ESI+):cacld for C 15 H 16 F 3 O4 Se + (M+H) + ,396.0088;found396.0076.

[0132] As shown in Table 5 below, intermediate 19b can be prepared according to the synthesis method of intermediate 19a above, using the corresponding starting materials and reactants.

[0133] Table 5

[0134]

[0135] Example 1

[0136]

[0137] Preparation of 4-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-4-oxobutanoic acid (S1)

[0138] 125 mg of intermediate 7a (0.29 mmol, 1 eq) was dissolved in 3 mL of glacial acetic acid, and then 1.5 mL of concentrated hydrochloric acid was added dropwise under an ice bath. After the addition was completed, the reaction was refluxed for about 4 h. After the solvent was removed by rotation, 2N NaOH solution was added to adjust the pH to alkaline (pH>11), and EA was used for extraction (10 mL×3), and the organic phase was discarded; the aqueous phase was adjusted to acidic pH (pH=1~2) with 1N dilute hydrochloric acid, and EA was used for extraction again (10 mL×3), and the organic phases were combined, washed with water and saturated brine, and dried with anhydrous sodium sulfate; filtered, the filtrate was concentrated, and purified by silica gel column chromatography (DCM:MeOH=50:1) to obtain 50 mg of the target product as a white solid, with a two-step yield of 42%. 1 H NMR (300 MHz, DMSO-d 6 )δ=12.23(s,1H),7.74(s,1H),7.32(s,1H),3.86(s,6H),3.23(t,J=5.1Hz,2H),2.59(t,J=6.3Hz,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6 )δ=191.61,173.76,151.71,148.91,131.03,130.91,123.93,123.59,122.28,122.17,108.64,104.59,55.97,55.80,35.91,27.74ppm.HRMS (ESI – ):Cacld for C 14 H 12 FO 5 Se –(MH) – ,358.9839; found 358.9842.

[0139] Example 2

[0140]

[0141] Preparation of 2-ethyl-4-(3-fluoro-5,6-dimethoxybenzo[b]seleno-2-yl)-4-oxobutanoic acid (S2)

[0142] Referring to the preparation method described in Example 1, the reaction raw material 7a was replaced with 2-ethyl-3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carbonyl)succinic acid dimethyl ester (7b) to prepare compound S2. 1 H NMR (300 MHz, DMSO-d 6 )δ=12.23(s,1H),7.74(s,1H),7.31(s,1H),3.86(s,6H),3.40-3.25(m,1H),3.14- 3.01(m,1H),2.83-2.71(m,1H),1.69-1.53(m,2H),0.92(t,J=7.5Hz,3H).HRMS(ESI - ):Cacld forC 16 H 16 FO 5 Se - (MH) - ,387.0152; found 387.0137.

[0143] Example 3

[0144]

[0145] Preparation of 4-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-yl)-2-methyl-4-oxobutanoic acid (S3)

[0146] Referring to the preparation method described in Example 1, the reaction raw material 7a was replaced with 2-methyl-3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carbonyl)succinic acid dimethyl ester (7c) to prepare compound S3. 1 H NMR (300 MHz, DMSO-d 6 ): δ=12.22(br,1H),7.74(s,1H),7.31(s,1H),3.86(s,6H),3.34(q,J=9.8Hz,1H),3.05(dd,J 1 =14.2Hz,J 2=3.5Hz,1H),2.96-2.82(m,1H),1.18(d,J=7.1Hz,3H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=191.40,176.67,154.65,151.76,150.94,148.93,131.11,130.99,123.92,123. 60,122.40,122.29,108.65,104.58,55.98,55.80,44.14,34.40,17.07ppm.HRMS(ESI - ):cacld for C 15 H 14 FO 5 Se - (MH) - ,372.9996; found 372.9984.

[0147] Example 4

[0148]

[0149] Preparation of 4-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-2-methoxy-4-oxobutanoic acid (S4)

[0150] According to the preparation method described in Example 1, the reaction raw material 7a was replaced by 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carbonyl)-2,2-dimethylsuccinate (7d) to prepare compound S4. 1 H NMR (300 MHz, DMSO-d 6 )δ=7.83(s,1H),7.34(s,1H),3.75(d,J=12.6Hz,6H),3.19(s,2H),1.26(s,6H)ppm. 13 CNMR (75MHz, DMSO-d 6 ): δ=195.94,180.97,154.34,153.06,151.12,147.01,134.23,132.79,127.89,112.06,108.75,56.01,56.08,52.75,41.47,25.11ppm.HRMS (ESI - ):cacldfor C 16 H 16 FO 5 Se - (MH) -,387.0152; found 387.0149.

[0151] Example 5

[0152]

[0153] Preparation of 1-(2-(3-fluoro-5,6-dimethoxybenzo[b]selenophenyl-2-yl)-2-oxoethyl)cyclobutane-1-carboxylic acid (S5)

[0154] Referring to the preparation method described in Example 1, the reaction raw material 7a was replaced with 1-(1-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-3-methoxy-1,3-dioxopropane-2-yl)cyclobutane-1-carboxylic acid methyl ester (7e) to prepare compound S5. 1 H NMR (300 MHz, DMSO-d 6 ): δ=8.22(s,1H),7.85(s,1H),3.81(d,J=12.9Hz,6H),3.4(s,2H),2.12-2.32(m,2H),2.02-1.91(m,2H),1.69-1.78(m,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=198.95,178.34,155.71,153.76,151.21,148.04,134.69,134.01,128. 09,111.23,110.96,56.30,56.04,49.16,48.07,33.12,16.78ppm.HRMS(ESI - ):cacld for C 17 H 16 FO 5 Se - (MH) - ,399.0152; found 399.0144.

[0155] Example 6

[0156]

[0157] Preparation of 2-fluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-yl)-4-oxobutanoic acid (S6)

[0158] Referring to the preparation method described in Example 1, the reaction raw material 7a was replaced with 2-fluoro-3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carbonyl)succinic acid dimethyl ester (7f) to prepare compound S6. 1H NMR (300 MHz, DMSO-d 6 ): δ=7.84(s,1H),7.45(s,1H),5.19-5.37(m,1H),3.82(d,J=12.6Hz,6H),3.57-3.64(m,1H),3.34-3.42(m,1H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=198.45,172.24,154.93,153.02,151.18,148.11,133.87,132.70,126.01,112.18,108.21,86.38,84.17,56.34,45.04ppm.HRMS (ESI - ):cacld for C 14 H 11 F 2 O 5 Se - (MH) - ,376.9745; found 376.9723.

[0159] Example 7

[0160]

[0161] Preparation of 2,2-difluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-yl)-4-oxobutanoic acid (S7)

[0162] Referring to the preparation method described in Example 1, the reaction raw material 7a was replaced with dimethyl 2,2-difluoro-3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carbonyl)succinate (7 g) to prepare compound S7. 1 H NMR (300 MHz, DMSO-d 6 ): δ=9.92(br,0.5H),8.03(s,1H),7.64(s,1H),4.08(t,J=12.6Hz,2H),3.81(d,J=12.9Hz,6H)ppm. 13 CNMR (75MHz, DMSO-d 6 ): δ=196.13,167.25,155.12,153.78,152.27,147.31,134.22,132.76,126.31,115.12,112.96,111.65,108.01,56.74,47.25ppm.HRMS (ESI - ):cacld for C14 H 10 F 3 O 5 Se - (MH) - ,394.9651; found 394.9648.

[0163] Example 8

[0164]

[0165] Preparation of 2-(E)-4-(3-fluoro-5,6-dimethoxybenzo[b]selenophenyl-2-yl)-4-oxobut-2-enoic acid (S8)

[0166] 151 mg (1.54 mmol, 4 eq) of maleic anhydride was dissolved in 4 mL of anhydrous DCM, and then cooled in an ice bath; 351 mg (2.32 mmol, 6 eq) of anhydrous aluminum chloride was added, and the reaction was continued in an ice bath for 30 min; then, 100 mg of intermediate 8a (0.385 mmol, 1 eq) was dissolved in 1 mL of anhydrous DCM, and then slowly added dropwise, and after the dropwise addition was completed, the mixture was moved to room temperature for about 3 h. 30 mL of 1N dilute hydrochloric acid was added to quench, and DCM was used for extraction (10 mL×3), and the organic phases were combined, washed with water and saturated brine, and then dried with anhydrous sodium sulfate; filtered, the filtrate was concentrated, and purified by silica gel column chromatography (DCM: MeOH = 50: 1) to obtain compound S8 as a light yellow solid, 42 mg, with a yield of 30.5%. 1 H NMR (300 MHz, DMSO-d 6 ): δ=8.12(s,1H),7.83(d,J=15.9Hz,1H),7.69(s,1H),6.74(d,J=15.9Hz,1H),3.88(d,J=13.1Hz,6H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=187.50,169.03,153.13,152.78,151.03,147.55,146.07,134.21133.96,131.45,130.06,112.31,111.65,55.71ppm.HRMS (ESI - ):cacld for C 14 H 10 FO 5 Se - (MH) - ,356.96836 found 356.9670.

[0167] Example 9

[0168]

[0169] Preparation of 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxyl)cyclobutane-1-carboxylic acid (S9)

[0170] Referring to the preparation method described in Example 8, the reaction raw material maleic anhydride was replaced with 3-oxabicyclo[3.2.0]heptane-2,4-dione to prepare compound S9. 1 H NMR (300 MHz, DMSO-d 6 ): δ=12.06(s,1H),7.72(s,1H),7.28(s,1H),4.17(q,J=7.4Hz,1H),3.85 (s,6H),3.57(q,J=5.3Hz,1H),2.42-2.34(m,1H),2.27-2.11(m,3H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=191.97,174.18,153.91,151.57,150.21,148.86,131.10,130.98,123.95,123.61, 122.36,122.25,108.64,104.55,55.96,55.76,45.58,41.31,21.40,20.55ppm.HRMS(ESI - ):cacld for C 16 H 14 FO 5 Se - (MH) - ,384.9996; found 384.9990.

[0171] Example 10

[0172]

[0173] Preparation of 4-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carbonyl)cyclopropane-1-carboxylic acid (S10)

[0174] Referring to the preparation method described in Example 8, maleic anhydride was replaced with 3-oxabicyclo[3.1.0]hexane-2,4-dione to prepare compound S10. 1 H NMR (300 MHz, DMSO-d 6): δ=12.22(s,1H),7.74(s,1H),7.33(s,1H),3.86(d,J=1.5Hz,6H),2.92(q,J= 7.8Hz,1H),2.26(q,J=7.1Hz,1H),1.53(q,J=5.8Hz,1H),1.35-1.29(m,1H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=187.49,171.44,154.83,151.79,151.10,148.98,131.38,131.06,124.00,123.67, 122.95,122.86,108.65,104.65,56.03,55.84,28.46,28.41,22.65,11.59ppm.HRMS(ESI - ):cacld for C 15 H 12 FO 5 Se - (MH) - ,370.9839;found370.9848.

[0175] Embodiment 11

[0176]

[0177] Preparation of 1-(2-(3-fluoro-5,6-dimethoxybenzo[b]selenophenyl-2-yl)-2-oxoethyl)cyclopropane-1-carboxylic acid (S11)

[0178] Referring to the preparation method described in Example 8, maleic anhydride was replaced with 5-oxaspiro[2.4]heptane-4,6-dione to prepare compound S11. 1 H NMR (300 MHz, DMSO-d 6 ): δ=7.83(s,1H),7.52(s,1H),3.88(d,J=13.5Hz,6H),3.29(s,2H),2.22-2.37(m,2H),2.08-2.12(m,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=197.34,180.03,156.21,154.97,151.14,147.67,135.03,133.99,128.16,112.37,108.01,57.87,47.33,23.11,16.04ppm.HRMS (ESI -):cacld for C 16 H 14 FO 5 Se - (MH) - ,384.9996;found384.9989.

[0179] Example 12

[0180]

[0181] Preparation of 4-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-2-methoxy-4-oxobutanoic acid (S12)

[0182] Referring to the preparation method described in Example 8, maleic anhydride was replaced with 3-methoxydihydrofuran-2,5-dione to prepare compound S12. 1 H NMR (300 MHz, DMSO-d 6 ): δ=9.44(br,0.7H),7.93(d,J=5.2Hz,1H),7.65(s,1H),4.22-4.41(m,1H),3.75(d,J=12.7Hz,6H),3.23-3.61(m,1H),3.47-3.34(m,4H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=194.56,175.34,153.83,152.15,152.18,147.33,135.021,132.99,126.78,112.14,108.31,76.78,58.30,56.87,43.654ppm.HRMS (ESI - ):cacld for C 15 H 14 FO 6 Se - (MH) - ,388.9945; found 388.9938.

[0183] Example 13

[0184]

[0185] Preparation of 4-(3-fluoro-5,6-dimethoxybenzo[b]selen-2-yl)-2-hydroxy-4-oxobutanoic acid (S13)

[0186] Referring to the preparation method described in Example 8, maleic anhydride was replaced by 2-O-acetylmalonic anhydride to prepare compound S13.1 H NMR (300 MHz, DMSO-d 6 ): δ=8.87(s,1H),8.03(s,1H),7.71(s,1H),5.33(d,J=6.2Hz,1H),4.34-4.27(m ,1H),3.86(d,J=13.4Hz,6H),3.57(t,J=12.5Hz,1H),3.31(t,J=11.5Hz,1H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=193.17,175.98,154.83,152.75,151.58,147.14,134.19,132.85,127.04,110.88,108.01,68.83,56.30,46.47ppm.HRMS (ESI - ):cacld for C 14 H 12 FO 6 Se - (MH) - ,374.9789; found 374.9781.

[0187] Embodiment 14

[0188]

[0189] 5-(3-Fluoro-5,6-dimethoxybenzo[b]selenophene-2-yl)-2-methylene-4-oxobutanoic acid (S14)

[0190] Referring to the preparation method described in Example 8, maleic anhydride was replaced with 3-methylenedihydrofuran-2,5-dione to prepare compound S11. 1 H NMR (300 MHz, DMSO-d 6 ): δ=12.58(s,1H),7.76(s,1H),7.33(s,1H),6.23(s,1H),5.78(s,1H),4.01(s,2H),3.87(s,6H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=190.04,167.43,154.77,151.81,151.05,148.97,135.18,131.23,131.11,128.3 6,123.88,123.55,122.10,122.00,108.68,104.63,56.00,55.82,43.89ppm.HRMS(ESI- ):cacld forC 15 H 12 FO 5 Se - (MH) - ,370.9839; found 370.9843.

[0191] Embodiment 15

[0192]

[0193] Preparation of 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxylic acid)cyclopentane-1-carboxylic acid (S15)

[0194] Referring to the preparation method described in Example 8, maleic anhydride was replaced by 1,2-cyclopentanedicarboxylic anhydride to prepare compound S15. 1 H NMR (300 MHz, DMSO-d 6 ): δ=7.87(s,1H),7.46(s,1H),4.17-46(m,1H),3.75(d,J=15.0Hz,6H),2.76-2.88(m,1H),1.93-2.12(m,1H),1.29-1.74(m,5H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=200.02,178.04,156.56,153.48,151.58,147.32,132.21,125.93,112.13,109.00,57.26,55.19,47.15,30.19,24.72ppm.HRMS(ESI - ):cacld for C 17 H 16 FO 5 Se - (MH) - ,399.0152; found 399.0147.

[0195] Example 16

[0196]

[0197] Preparation of (3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxyl)glycine (S16)

[0198] 161 mg of intermediate 10a (0.43 mmol, 1 eq) was added to a 50 mL round-bottom flask, 5 mL of dioxane was added to dissolve, 0.65 mL of 2N NaOH solution was added, and the mixture was reacted at 55°C for about 2 h. The mixture was slowly cooled to room temperature, the solvent was removed by vortexing, and dilute hydrochloric acid was added to adjust the pH to acidic (pH = 1-2), and the mixture was filtered with suction. The filter cake was slurried with methanol to obtain compound S16 as a yellow-white solid, 134 mg, with a yield of 90.4%. 1 H NMR (300 MHz, DMSO-d 6 ): δ=12.66(s,1H),8.17(q,J=6.0Hz,1H),7.73(s,1H),7.26(s,1H),3.94(d,J=5.7Hz,2H),3.85(d,J=3.7Hz,6H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=171.15,160.85,151.24,150.68,148.77,147.64,129.39,129.27,123.8 5,123.51,116.95,116.85,108.61,104.03,55.88,55.78,41.57ppm.HRMS(ESI - ):cacld forC 13 H 11 FNO 5 Se - (MH) - ,359.9792; found 359.9800.

[0199] Embodiment 17

[0200]

[0201] Preparation of 2-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)butyric acid (S17)

[0202] Referring to the preparation method described in Example 16, (R)-2-aminobutyric acid methyl ester was replaced with 2-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)butyric acid methyl ester to prepare compound S17. 1 H NMR (300 MHz, DMSO-d 6): δ=9.31(d,J=8.7Hz,1H),8.05(s,1H),7.82(s,1H),4.18-4.29(m,1H),3.78(d,J =12.9Hz,6H),1.76-2.08(m,1H),1.32-1.72(m,1H),1.09(t,J=6.2,7.5Hz,3H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=175.41,166.03,161.22,158.14,152.45,147.26,133.28,124.43,111.72,108.19,56.17,54.20,25.52,9.71ppm.HRMS (ESI - ):cacld for C 15 H 15 FNO 5 Se - (MH) - ,388.0105; found 388.0199.

[0203] Embodiment 18

[0204]

[0205] Preparation of 1-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)cyclopropane-1-carboxylic acid (S18)

[0206] Referring to the preparation method described in Example 16, (R)-2-aminobutyric acid methyl ester was replaced with 1-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)cyclopropane-1-carboxylic acid methyl ester to prepare compound S18. 1 H NMR (300 MHz, DMSO-d 6 ): δ=12.48(s,1H),8.47(s,1H),7.72(s,1H),7.23(s,1H),3.84(d,J=1.4Hz,6H),1.41(s,2H),1.17(s,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=173.80,161.64,151.24,150.62,148.73,147.64,129.26,129.15,123.88,1 23.55,117.19,117.08,108.59,103.95,55.86,55.77,33.46,17.00ppm.HRMS (ESI -):cacld for C 15 H 13 FNO 5 Se - (MH) - ,385.9948; found 385.9941.

[0207] Embodiment 19

[0208]

[0209] Preparation of 1-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)cyclobutane-1-carboxylic acid (S19)

[0210] Referring to the preparation method described in Example 16, (R)-2-aminobutyric acid methyl ester was replaced with 1-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)cyclobutane-1-carboxylic acid methyl ester to prepare compound S19. 1 H NMR (300 MHz, Acetone-d 6 ): δ=7.70(d,J=5.8Hz,1H),7.63(s,1H),7.26(s,1H),3.91(d,J=2.1Hz,6H),2.73-2.56(m,4H),2.13-2.05(m,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=174.63,160.30,151.29,150.63,148.78,147.69,129.10,123.90,123. 56,117.01,108.63,103.94,58.18,55.90,55.80,31.07,15.21ppm.HRMS(ESI - ):cacld for C 16 H 15 FNO 5 Se - (MH) - ,400.0105;found 400.0100.

[0211] Embodiment 20

[0212]

[0213] Preparation of 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)oxetane-3-carboxylic acid (S20)

[0214] Referring to the preparation method described in Example 16, (R)-2-aminobutyric acid methyl ester was replaced with 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)oxetane-3-carboxylic acid methyl ester to prepare compound S20. 1 H NMR (300 MHz, DMSO-d 6 ): δ=13.15(s,1H),8.95(s,1H),7.75(s,1H),7.27(s,1H),4.85(d,J=6.6Hz,2H),4.71(d,J=6.6Hz,2H),3.86(d,J=4.4Hz,6H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=172.14,160.83,151.84,150.81,148.83,148.22,129.56,129.45,123.77,1 23.45,116.09,116.01,108.63,104.03,77.13,58.19,55.90,55.82ppm.HRMS(ESI - ):cacld forC 15 H 13 FNO 6 Se - (MH) - ,401.9898; found 401.9951.

[0215] Embodiment 21

[0216]

[0217] Preparation of 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)propionic acid (S21)

[0218] Referring to the preparation method described in Example 16, (R)-2-aminobutyric acid methyl ester was replaced with 3-(3-fluoro-5,6-dimethoxybenzo[b]selenophene-2-carboxamido)propionic acid methyl ester to prepare compound S21. 1 H NMR (300MHz, CD 3 OD): δ=7.54(s,1H),7.36(s,1H),7.19(s,1H),3.96(d,J=2.9Hz,6H),3.71(t,J=5.6Hz,2H),2.67(t,J=6.0Hz,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6): δ=173.12,160.58,160.48,150.82,150.57,148.74,147.23,129.18,129.07,123.91,1 23.57,117.49,117.37,108.61,103.98,55.88,55.78,35.67,35.55,33.80ppm.HRMS(ESI - ):cacld for C 14 H 13 FNO 5 Se - (MH) - ,373.9948; found 373.9374.

[0219] Embodiment 22

[0220]

[0221] Preparation of 4,4-difluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]seleno-2-yl)butyric acid (S22)

[0222] 120 mg of intermediate 19a (0.30 mmol, 1 eq) was added to a 50 mL round-bottom flask, 5 mL of dioxane was added to dissolve, 0.5 mL of 2N NaOH solution was added, and the mixture was reacted at 55°C for about 2 h. The mixture was slowly cooled to room temperature, the solvent was removed by vortexing, and dilute hydrochloric acid was added to adjust the pH to acidic (pH = 1-2), and the mixture was filtered with suction. The filter cake was slurried with methanol to obtain compound S22 as a yellow-white solid, 98 mg, with a yield of 84.8%. 1 H NMR (300 MHz, DMSO-d 6 ): δ=8.32(s,1H),7.61(s,1H),3.75(d,J=12.7Hz,6H),2.62-2.75(m,2H),2.39-2.48(m,2H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=177.28,152.45,147.12,144.52,134.78,131.26,131.33,126.08,125.7 7,122.28,120.20,118.34,113.36,56.50,33.88,33.57,28.19ppm.HRMS(ESI - ):cacld for C 14 H 12 F 3 O 4 Se - (MH)- ,380.9095; found 380.9087.

[0223] Embodiment 23

[0224]

[0225] Preparation of 2-ethyl-4,4-difluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]seleno-2-yl)butanoic acid (S23)

[0226] Referring to the preparation method described in Example 22, 4,4-difluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]seleno-2-yl)butanoic acid methyl ester (19a) was replaced with 2-ethyl-4,4-difluoro-4-(3-fluoro-5,6-dimethoxybenzo[b]seleno-2-yl)butanoic acid methyl ester (19b) to prepare compound S21. 1 H NMR (300MHz, CD 3 OD): δ=7.78(s,1H),7.46(s,1H),3.77(d,J=12.5Hz,6H),2.70-2.86(m,1H),2.50-2.67(m,1 H),2.34-2.39(m,1H),1.68-1.76(m,1H),1.47-1.52(m,1H),1.12(t,J=6.8,4.3Hz,3H)ppm. 13 C NMR (75 MHz, DMSO-d 6 ): δ=180.11,153.23,146.57,144.36,134.21,131.22,124.75,121.09,119.26,117.36,112.46,56.23,42.15,40.15,25.73,11.56ppm.HRMS (ESI - ):cacld for C 16 H 16 F 3 O 4 Se - (MH) - ,409.0171; found 409.0168.

[0227] Embodiment 24

[0228] Anti-RSV infection activity test of representative compounds

[0229] Calu3 cells were cultured at 2×10 5Cells / well were inoculated in a 24-well plate and cultured overnight in complete medium (10% FBS). Subsequently, 300 μL of RSV virus was added to each well to infect the cells, and the infection was carried out at 37°C for 6 h. After the infection, the medium was replaced with low serum medium (1% FBS) containing the corresponding concentration of drugs; at the same time, the chamber was placed in the well, and 100 μL of THP1 cell suspension was added to each chamber at a density of 2×10 4 cells / well. Low serum blank medium was used as negative control at the same time, and the treatment method was the same as that of the drug administration group. 48 hours after administration, the total RNA in Calu3 cells was extracted using the FreeZol Reagent kit, and the relative content of RSV RNA was detected by qPCR. The primer sequences used are shown in the following table.

[0230] Table 6 Primer sequences used for total RNA extraction

[0231]

[0232] In order to verify the anti-viral infection ability of STING agonists, RSV virus was used to infect Calu3 cells, and the activity was verified by constructing a model of co-incubation of THP1 and Calu3 cells.

[0233] The experimental results show that the series of compounds of the present invention can significantly inhibit the expression of RSV mRNA in cells, and the mRNA inhibition level of individual representative structures is equivalent to that of AK0529. 50 The representative structure IC in the present invention is found 50 The value is about one-fold lower than that of AK0529, which also proves that the infection of RSV virus can be inhibited by STING agonist. And the activity is higher than the representative compounds I-3, I-10, I-15, I-24 in the previous research patent (CN 113429387A) and C1 in the patent (CN116332903 A), which all indicate that the compounds of the present invention have significant activity in resisting RSV infection.

[0234] Table 7 Representative compounds for in vitro RSV inhibition and related RNA level detection

[0235]

[0236]

[0237] Note: The data in the table are expressed as mean±SD, n=3.

[0238] Embodiment 25

[0239] Representative compound STING agonist activity assay

[0240] EC values ​​of compounds S3, S9, S10, and S14 in THP1-ISRE Luc cells 50 The experimental results showed that, except for compound S10, S3, S9 and S14 all had significant STING agonist activity, EC 50 The activity of the compounds is higher than that of the representative compounds I-14, I-15 and I-16 in the previous patent (CN 113429387A).

[0241] Table 9 represents the STING agonist activity fold EC of the compounds 50 value

[0242]

[0243]

[0244] Table 10 Representative compounds in luciferase reporter gene assay agonist activity

[0245]

[0246] Note: [a] The excitation fold of the compound (20 μM) and the control group in ISG-THP1 cells; * indicates the strength of the excitation fold; data are expressed as mean, n=3.

[0247] Embodiment 26

[0248] PK properties of representative compounds of the present invention

[0249] The PK properties of representative compounds were tested accordingly, and the results showed that compound S9 had a high in vivo exposure and a half-life (t 1 / 2 ) is 2.72h; the oral bioavailability is as high as 99.04%, which is conducive to the gastrointestinal absorption of the compound after oral administration. Compound S9 has good pharmacokinetic properties and has further development value.

[0250] Table 8 PK data of representative compounds

[0251]

[0252] Embodiment 27

[0253] In vivo evaluation of representative compounds against RSV infection

[0254] For the representative compound S9, three dose gradients of 1.5mpk, 2.5mpk, and 5mpk were used, and the positive controls were ribavirin and the representative compound I-15 (5mpk) in the previous patent (CN 113429387 A). The drug was administered orally, and the weight change was recorded every day. The lung tissue was taken for qPCR detection of viral RNA on the day after the last administration, and the plasma and lung IFNB were detected by Elisa. The experimental data showed that compound S9 had a dose-dependent resistance to RSV virus, and was significantly superior to I-15 and ribavirin at a dose of 5mpk.

[0255] Table 11 Representative compounds for in vivo evaluation against RSV infection

[0256]

[0257] Note: The data in the table are expressed as mean±SD, n=6; ns means no effect; statistically significant differences were calculated by one-way ANOVA test (**P<0.01, ***P<0.001, ****P<0.0001).

[0258] The present invention is based on the representative compounds I-14, I-15 and I-16 in the inventor's previous patent (CN 113429387 A). The structures do not have an advantageous effect on antiviral research. The in vitro antiviral IC 50 The antiviral activity in vivo is poor, and the introduction of selenium atoms will lead to changes in the electron cloud density, resulting in a decrease in STING agonist activity. There are problems in the structure that need to be improved, so it is considered to try different structural modifications to optimize the antiviral activity of the compound. Before designing the F-substituted compound, the inventor tried to make relevant substitutions of different halogen atoms such as Br atoms, but they all lost the STING agonist activity. The introduction of F atoms can innovatively start from the electron cloud density, so that the compound structure that did not have STING agonist activity or good antiviral activity in previous studies can restore STING agonist activity or improve its antiviral ability. At the same time, the original physical and chemical properties of the compound are improved, the stability and membrane permeability are improved; the effect and use that are different from tumor treatment are achieved, and the indications of STING agonists are innovatively expanded.

Claims

1. A compound, stereoisomer or pharmaceutically acceptable salt thereof represented by general formula (I): in: X is selected from C(R 3 ) n 、N(R 3 ) m , O and S; R 1 Selected from hydrogen atom, halogen atom, cyano group, OR 4 、N(R 4 )2, C1~C6 alkyl, C1~C6 haloalkyl, OR 4 Substituted C1-C6 alkyl and 4 Substituted C3-C6 cycloalkyl; R 2 A group selected from the group consisting of a hydrogen atom, a hydroxyl group, a double bond, a halogen atom, an amino group, a hydroxymethyl group, a methoxy group, a thiomethyl group, N(CH2CH3)2, N(CH3)2, a benzyloxy group, a benzoyloxy group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a 3-6 membered heterocyclic group, and a C5-C 10 Aryl; or X and R 2 Can form a 3- to 6-membered ring together with the atoms to which it is attached; or R 2 Can form a 3- to 6-membered ring together with the atoms to which it is attached; R 3 A hydrogen atom, a hydroxyl group, an amino group, a hydroxymethyl group, a methoxy group, a thiomethyl group, N(CH2CH3)2, N(CH3)2, a benzyloxy group, a benzoyloxy group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a 3- to 6-membered heterocyclic group, and a C5-C 10 Aryl; R 4 is selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic group and C5-C 10 Aryl; n is selected from 0, 1, 2 and 3; m is selected from 0 and 1.

2. The compound, stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 1 Selected from hydrogen atom, halogen atom, cyano group, OR 4 、N(R 4 )2, C1~C3 alkyl, C1~C3 haloalkyl, C3~C6 cycloalkyl and 4 Substituted C1-C6 alkyl, preferably a hydrogen atom, a fluorine atom, a chlorine atom, a methyl group, an ethyl group, a methoxy group, or an ethoxy group; R 2 is selected from the group consisting of a hydrogen atom, a hydroxyl group, a double bond, a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a 3-6 membered heterocyclic group, and a C5-C 10 Aryl, preferably a hydrogen atom, a hydroxyl group, a double bond, a fluorine atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a methoxy group, an ethoxy group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group; R 3 A group selected from hydrogen, hydroxy, methoxy, N(CH2CH3)2, N(CH3)2, benzyloxy, benzoyloxy, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic group and C5-C 10 Aryl, preferably hydrogen atom, methyl, ethyl, isopropyl; R 4 is selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic group and C5-C 10 Aryl; n is selected from 0, 1, 2 and 3, preferably 1, 2; m is selected from 0 and 1.

3. The compound, stereoisomer or pharmaceutically acceptable salt thereof according to 1, characterized in that: X is selected from the group consisting of CH, CH2, NH, O and S, preferably CH, CH2 and NH.

4. The compound, stereoisomer or pharmaceutically acceptable salt thereof as shown below:

5. A pharmaceutical composition, characterized in that The invention comprises a pharmaceutically effective amount of an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is one or more of the compounds, stereoisomers or pharmaceutically acceptable salts thereof according to any one of claims 1 to 4.

6. Use of the compound, stereoisomer or pharmaceutically acceptable salt thereof or pharmaceutically acceptable composition according to any one of claims 1 to 4 in the preparation of drugs for activating the cGAS-STING pathway.

7. Use of a compound, stereoisomer or pharmaceutically acceptable salt thereof or pharmaceutically acceptable pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating a disease associated with STING pathway activity.

8. The method for use according to claim 7, characterized in that: The disease associated with STING pathway activity is viral infection.

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