A benzo[b]selenophene STING agonist fluorescent probe molecule and its use

Abstract

The present invention discloses a benzo[b]selenophene STING agonist fluorescent probe molecule, wherein the fluorescent probe molecule is a compound, stereoisomer or pharmaceutically acceptable salt obtained by coupling a benzo[b]selenophene monomer or dimer STING agonist with a photoactivated protective group. These molecules can release the STING agonist by illumination with a specific wavelength, thereby achieving spatiotemporal control of STING activation. The present invention also provides the use of these compounds in the preparation of drugs for diagnosing and treating STING-related diseases, as well as the use of these compounds in the preparation of in vivo fluorescent imaging agents.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine and relates to a benzo[b]selenophene STING agonist fluorescent probe molecule and a use thereof. Background Art

[0002] The emergence of immunotherapy has provided excellent therapeutic effects and new research ideas for curing tumors, and was rated as the most important scientific breakthrough in 2013 by Science magazine (Science 2013, 342, 1417). The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an important innate immune pathway in the body. As a pattern recognition receptor, cGAS can capture and recognize abnormal cytoplasmic DNA signals, catalyze the production of the second messenger cyclic GMP-AMP (cGAMP), thereby activating the endoplasmic reticulum membrane receptor STING, and then initiating the activation of downstream transcription factors, ultimately promoting the expression and secretion of type I interferons and proinflammatory factors, and enhancing innate and acquired immune responses (Drug Discov Today 2020, 25, 230-237). In recent years, the cGAS-STING pathway has been considered an important potential target for tumor immunotherapy (J Hematol Oncol 2019, 12, 35). In the cGAS-STING pathway, the transmembrane receptor STING is considered to be the most critical node for regulating the pathway. In preclinical mouse tumor models, STING agonists showed highly effective anti-tumor activity, which could completely inhibit tumor growth and completely eliminate tumors (Nature. 2018, 564, 439-443; Science. 2020, 369, eaba6098; Science. 2020, 369, 993-999).

[0003] So far, many pharmaceutical companies at home and abroad have laid out the R&D pipeline of STING agonists, including Aduro BioTech's ADU-S100, Merck's MK-1454 and other STING agonists that have entered clinical trials for the treatment of various tumors. However, systemic administration of STING agonists has the risk of causing systemic inflammatory responses, which limits the clinical development of this type of drug (J Clin Med 2020, 9, 3323). Therefore, a new generation of tumor-selective STING agonists is urgently needed.

[0004] The pharmacological activity of the molecule is blocked by photoactivatable protecting groups (PPGs), and then the active molecules and fluorescent groups are released by the non-invasive and spatiotemporal controllability of photochemistry, which can complete the precise treatment of the disease and fluorescence imaging, and realize the integration of diagnosis and treatment. In recent years, this kind of PPGs activated by ultraviolet-visible-near-infrared light has been fully developed (Chem Rev 2020, 120, 13135-13272). Among them, o-nitrobenzyl derivatives activated by ultraviolet-visible light, coumarin derivatives and heptamethine cyanine derivatives activated by near-infrared light are several widely used PPGs.

[0005] In order to overcome the toxic and side effects caused by the current systemic administration of STING agonists and to achieve integrated diagnosis and treatment of diseases, it is of great significance to develop a STING agonist fluorescent probe molecule. Summary of the invention

[0006] Purpose of the invention: The present invention aims to provide a series of fluorescent probe molecules obtained by coupling benzo[b]selenophene STING agonists with PPGs by utilizing the spatiotemporal controllability of light, thereby achieving timed and targeted release and having good drugability and pharmacokinetic properties. These molecules can rapidly release fluorescent groups and effective STING agonists under light of a specific wavelength, achieve selective STING activation, and thus realize integrated diagnosis and treatment of diseases, overcoming the toxic and side effects caused by the current systemic administration of STING agonists.

[0007] The present invention provides a benzo[b]selenophene STING agonist fluorescent probe molecule, wherein the fluorescent probe molecule is a compound, a stereoisomer or a pharmaceutically acceptable salt thereof obtained by coupling a benzo[b]selenophene monomer or a dimer STING agonist with a photoactivated protective group.

[0008] The photoactivated protective group is an o-nitrobenzyl derivative X 1 , Coumarin Derivatives X 2 or heptamethine cyanine derivative X 3 :

[0009]

[0010] in,

[0011] R 1 is selected from hydrogen, methyl or allyl;

[0012] R 2Selected from hydrogen, hydroxy, methyl, tert-butyl, methoxy, OCH2CN, OCH2COOH, OCH2COOCH3 or para-substituted styryl;

[0013] The para substituents include but are not limited to OH, OC1-C3 alkyl, OC1-C3 haloalkyl, CHO and COOH);

[0014] R 3 is selected from hydrogen or methoxy;

[0015] R 4 is selected from hydrogen or methyl;

[0016] R 5 is selected from hydrogen, halogen, methoxy or amino;

[0017] R 6 Selected from hydrogen, hydroxy, methoxy, N(CH2CH3)2, benzyloxy, benzoyloxy or pinacol boronic acid ester;

[0018] R 7 Selected from methyl, ethyl, benzyl, (CH2) m SO3H or (CH2) m COOH;

[0019] R 8 is selected from hydrogen, methoxy, SO3H or COOH;

[0020] R 9 is selected from hydrogen; or R 9 With R 8 Connected to form a benzene ring or a C5-C6 aromatic heterocycle;

[0021] R 10 is selected from hydrogen or methyl;

[0022] R 11 is selected from hydrogen or methyl;

[0023] m is selected from 3, 4 or 5.

[0024] In certain preferred embodiments,

[0025] R 1 is selected from hydrogen or methyl;

[0026] R 2 is selected from hydrogen, hydroxy, methyl or methoxy;

[0027] R 5 is selected from hydrogen, bromine or methoxy;

[0028] R 6 Selected from hydrogen, hydroxy, methoxy or N(CH2CH3)2;

[0029] R 7 Selected from methyl, ethyl, benzyl or (CH2) m SO3H;

[0030] R 8 is selected from hydrogen, methoxy or SO3H;

[0031] m is selected from 3 or 4.

[0032] The fluorescent probe molecule of the present invention preferably has a structure as shown in formula (I):

[0033]

[0034] in,

[0035] X is selected from o-nitrobenzyl derivatives (X 1 ), coumarin derivatives (X 2 ) or heptamethine cyanine derivatives (X 3 ).

[0036] The fluorescent probe molecule of the present invention preferably has a structure as shown in formula (II):

[0037]

[0038] in,

[0039] Y is selected from hydrogen, X 1 , X 2 or X 3 ; and when one side Y is selected from hydrogen, the other side Y is selected from X 1 , X 2 or X 3 ; or both sides Y are selected from X 1 , X 2 or X 3 .

[0040] The benzo[b]selenophene STING agonist fluorescent probe molecules of the present invention are preferably the following compounds:

[0041]

[0042]

[0043] The present invention also provides a method for preparing the above compound:

[0044] When the compound has the structure shown in the general formula (I), and X is selected from X 1 and X 2When the compound BSP (prepared according to patent CN113429387A) is reacted with an o-nitrobenzyl derivative 1 or a coumarin derivative 2 by ester condensation, the compounds of general formulae (Ia) and (Ib) are prepared, respectively. The synthetic route is as follows:

[0045]

[0046] Among them, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined above.

[0047] In some more specific embodiments, R 1 and R 4 is selected from hydrogen or methyl, R 2 and R 3 is selected from hydrogen or methoxy, R 5 Selected from hydrogen, R 6 Selected from hydrogen or N(CH2CH3)2.

[0048] In some more specific embodiments, the condensing agent used in the ester condensation reaction is selected from HATU, HBTU, HOAT, HOBT, DCC, EDC or EDCI, the base used is selected from TEA, DIPEA or DMAP, and the reaction solvent is selected from dichloromethane, tetrahydrofuran or DMF.

[0049] When the compound has the structure shown in the general formula (I), and X is selected from X 3 When the compound BSP is reacted with the compound 3 by ester condensation to obtain the compound 4, the compound 4 is then reacted with the compound 5 to obtain the Zincke salt 6, and the compound 6 is finally reacted with the compound 7 by the Zincke reaction to obtain the compound of the general formula (Ic). The synthetic route is as follows:

[0050]

[0051] Among them, R 7 , R 8 , R 9 , R 10 and R 11 As defined above.

[0052] In some more specific embodiments, R 7 is selected from methyl, ethyl, (CH2)3SO3H or (CH2)4SO3H, R 8 Selected from hydrogen or SO3H, R 9 is selected from hydrogen, or R 8 and R 9 Can be connected to form a benzene ring, R10 is selected from hydrogen or methyl, R 11 is selected from hydrogen or methyl.

[0053] In some more specific embodiments, in the process of preparing compound 4 from compound BSP, the reactant is compound 3, the condensing agent used is selected from HATU, HBTU, HOAT, HOBT, DCC, EDC or EDCI, the base used is selected from TEA, DIPEA or DMAP, and the reaction solvent is selected from dichloromethane, tetrahydrofuran or DMF.

[0054] In some more specific embodiments, in the process of preparing compound 6 from compound 4, the reactant is compound 5, and the reaction solvent is selected from toluene, xylene or acetone.

[0055] In some more specific embodiments, in the process of preparing compound (Ic) of general formula from compound 6, the reactant is compound 7, the reaction reagent is selected from aniline or 4-bromoaniline, the base used is selected from sodium carbonate, potassium carbonate, sodium acetate or potassium acetate, and the reaction solvent is selected from methanol, ethanol, dichloromethane, tetrahydrofuran or DMF.

[0056] When the compound has the structure shown in general formula (II), the synthesis route is as follows:

[0057]

[0058] wherein X is as defined above.

[0059] A specific preparation method of the compound of general formula (II) comprises the following steps:

[0060] (1) Compound 8 reacts with compound 9 (benzyl bromide) under alkaline conditions to obtain compound 10, the reaction reagent is selected from potassium carbonate, sodium carbonate, sodium hydroxide or sodium hydride, and the reaction solvent is selected from DMF or tetrahydrofuran;

[0061] (2) Compound 10 reacts with compound 11 (dimethyl diselenide) under alkaline conditions to obtain compound 12, the reaction reagent is selected from DTT, mercaptoethanol, DBU and potassium carbonate, and the reaction solvent is selected from DMF or tetrahydrofuran;

[0062] (3) Compound 12 and compound 13 (ethyl 2-bromoacetate) were subjected to substitution reaction to obtain compound 14, and the reaction solvent was DMF;

[0063] (4) Compound 14 is cyclized under alkaline conditions to obtain compound 15, the reaction reagent is selected from potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide or lithium hydroxide, and the reaction solvent is selected from DMF or acetonitrile;

[0064] (5) Compound 15 is hydrolyzed and decarboxylated to obtain Compound 16, wherein the reaction reagent is selected from potassium carbonate, sodium hydroxide or strong lithium oxide, and the reaction solvent is selected from water, methanol and tetrahydrofuran;

[0065] (6) Compound 16 and compound 17 (malonic acid monomethyl ester potassium salt) are subjected to condensation reaction to obtain compound 18, the reaction reagent is selected from CDI and MgCl2, and the reaction solvent is selected from DMF or tetrahydrofuran;

[0066] (7) Compound 18 and compound 13 (ethyl 2-bromoacetate) are subjected to substitution reaction under alkaline conditions to obtain compound 19, the reaction reagent is selected from potassium carbonate, sodium methoxide, sodium ethoxide or sodium hydride, and the reaction solvent is selected from DMF or tetrahydrofuran;

[0067] (8) Compound 19 undergoes hydrolysis and decarboxylation simultaneously to obtain compound 20, wherein the reaction reagent is selected from potassium carbonate, sodium hydroxide, lithium hydroxide or hydrochloric acid, and the reaction solvent is selected from acetic acid, water or tetrahydrofuran;

[0068] (9) Compound 20 is protected by ethyl ester to obtain compound 21, the reaction reagent is selected from thionyl chloride or oxalyl chloride, and the reaction solvent is selected from ethanol;

[0069] (10) Compound 21 and compound 22 (1,3-dibromopropane) are subjected to substitution reaction under alkaline conditions to obtain compound 23, the reaction reagent is selected from potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium iodide or crown ether, and the reaction solvent is selected from DMF or tetrahydrofuran;

[0070] (11) Compound 23 and Compound 21 are subjected to substitution reaction under alkaline conditions to obtain Compound 24, the reaction reagent is selected from potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium iodide or crown ether, and the reaction solvent is selected from DMF or tetrahydrofuran;

[0071] (12) Compound 24 is hydrolyzed under alkaline conditions to obtain compound diBSP, the reaction reagent is selected from potassium carbonate, sodium hydroxide or lithium hydroxide, and the reaction solvent is selected from water, methanol and tetrahydrofuran;

[0072] (13) Compound diBSP The compound of formula (II) was prepared by the same synthetic method as the compound of formula (I).

[0073] The present invention also provides the use of the benzo[b]selenophene STING agonist fluorescent probe molecule in the preparation of a drug for activating the cGAS-STING pathway. The drug is a STING agonist and can be used to prevent and / or treat infectious diseases or tumors.

[0074] The present invention also provides the use of the benzo[b]selenophene STING agonist fluorescent probe molecule in the preparation of drugs for preventing and / or treating diseases related to the STING pathway.

[0075] The present invention also provides the use of the benzo[b]selenophene STING agonist fluorescent probe molecule in the preparation of an immune adjuvant. The immune adjuvant can be used to prevent and / or treat infectious diseases or tumors.

[0076] The present invention also provides the use of the benzo[b]selenophene STING agonist fluorescent probe molecule in the preparation of an integrated therapeutic drug, which can be used to diagnose and treat a disease simultaneously.

[0077] In certain preferred embodiments, the disease is a tumor, including but not limited to lung cancer, liver cancer, gastric cancer, breast cancer, melanoma, colorectal cancer, pancreatic cancer, prostate cancer, squamous cell carcinoma, glioma and leukemia.

[0078] The drug of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray or via an implanted reservoir. The drug of the present invention can be administered alone or in combination with other anti-tumor drugs. Oral drugs can be in any oral acceptable dosage form, including but not limited to tablets, capsules, emulsions and suspensions, dispersions and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, adhesives, colorants, fillers, emulsifiers, etc.

[0079] Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.

[0080] Topical medications can be formulated as oils, lotions, creams, etc. Carriers for medications include vegetable or mineral oils, animal fats, and high molecular weight alcohols, etc. Pharmaceutically acceptable carriers are carriers in which the active ingredient is soluble.

[0081] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific drug used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific drug used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0082] The present invention also provides the use of the benzo[b]selenophene STING agonist fluorescent probe molecule in the preparation of an in vivo fluorescent imaging agent.

[0083] Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

[0084] The term "stereoisomers" refers to isomers that result from differences in the way the atoms in a molecule are arranged in space.

[0085] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the general formula (I) and (II) of the present invention and a pharmaceutically acceptable non-toxic base, including an organic base or an inorganic base. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amines include naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as betaine, caffeine, choline, N-ethylpiperidine, N,N'-dibenzylethylenediamine, diethylamine, 2-dimethylaminoethanol, arginine, ethanolamine, ethylenediamine, N-ethylmorpholine, glucosamine, methylglucosamine, 2-diethylaminoethanol, glucosamine, histidine, aminoethanol, hydroxocobalamin, lysine, morpholine, piperazine, piperidine, quaternium, polyamine resins, triethylamine, trimethylamine, tripropylamine, isopropylamine, tromethamine, and the like. Salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, lithium, magnesium, potassium, sodium, zinc and the like.

[0086] 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.

[0087] The term "halogen" or "halo" refers to fluorine, chlorine, bromine or iodine, or fluoro, chloro, bromo or iodo.

[0088] The term "haloalkyl" refers to an alkyl group as defined above wherein one or more hydrogen atoms have been replaced by a halogen.

[0089] The term "aromatic heterocycle" denotes a cyclic group of 5 to 6 ring atoms, wherein one or two of the ring atoms are heteroatoms selected from N, O or S, the remaining ring atoms are C, and the ring has a completely conjugated π electron system.

[0090] Beneficial effects:

[0091] The benzo[b]selenophene STING agonist fluorescent probe molecules prepared by the present invention can remove the photoprotective group after being exposed to light, release the prototype drug, i.e., the corresponding STING agonist, and can achieve timed and fixed-point release, and have good drugability and pharmacokinetic properties. Therefore, the compounds of the present invention can be used to prepare drugs that activate the cGAS-STING pathway, to prepare drugs for preventing and / or treating diseases related to the STING pathway, and to prepare immune adjuvants. In addition, the compounds of the present invention carry fluorescent groups and can be used to prepare fluorescent imaging agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The photorelease results of representative compounds I-7 and II-6 under 808nm and 400nm wavelength light sources, respectively;

[0093] Figure 2 The light-dependent STING agonist activity results of representative compounds I-7 and II-6. DETAILED DESCRIPTION

[0094] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0095] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0096] In the embodiments of the present invention, the ratios of TLC developing agent and column chromatography eluent are all volume ratios.

[0097] Example 1 Preparation of (R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutyric acid-2-nitrobenzyl ester (I-1)

[0098]

[0099] Compound BSP (100 mg, 0.27 mmol), 2-nitrobenzyl alcohol (1a) (41 mg, 0.22 mmol), DCC (N,N'-dicyclohexylcarbodiimide, 84 mg, 0.41 mmol), DMAP (4-dimethylaminopyridine, 1 mg, 0.01 mmol) and dichloromethane (4 mL) were added to a 50 mL reaction bottle and stirred at room temperature for 5 h under light-proof conditions. After TLC (dichloromethane: methanol = 20: 1) detected that the reaction of the raw materials was complete, the reaction solution was concentrated, and the obtained residue was dissolved in ethyl acetate (15 mL), cooled and stirred at -10°C for about 30 min, filtered, and used glacial ethyl acetate (2 mL). The filtrate was concentrated to obtain a crude product, which was purified by silica gel column chromatography (dichloromethane: methanol = 50: 1) to obtain 81 mg of compound I-1 as a light yellow solid with a yield of 59%. 1 H NMR (300MHz, DMSO-d6): δ=8.12–8.06(m,1H),7.74(s,1H),7.63–7.54(m,3H),7.51(s,1H),7.14(s,1 H),5.53–5.41(m,2H),3.82(s,6H),3.16-2.87(m,3H),1.65-1.50(m,2H),0.94(t,J=8.8Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.46,174.51,150.52,147.64,147.23,142.63,137.77,136.48,133.11,133.0 4,131.78,128.59,127.83,123.81,110.45,108.75,64.86,56.43,56.12,44.14,40.03,24.97,11.52ppm. HRMS(ESI + ): Calculated value C 23 H 24 NO7Se + (M+H) + ,506.0713; measured value 506.0719.

[0100] Example 2 Preparation of (2R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoic acid-1-(2-nitrophenyl)ethyl ester (I-2)

[0101]

[0102] Referring to the preparation method described in Example 1, the reaction raw material 1a was replaced with 1-(2-nitrophenyl)ethanol to prepare compound I-2. 1H NMR (300MHz, DMSO-d6): δ=8.05–7.97(m,1H),7.75(s,1H),7.68–7.62(m,1H),7.57–7.50(m,3H),7.14(s,1H),6.06(dq,J1=6. 2Hz, J2=2.1Hz,1H),3.81(d,J=2.9Hz,6H),3.21-3.03(m,2H),2.96-2.85(m,1H),1.64–1.56(m,5H),0.94(t,J=6.4Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.04,174.24,150.52,147.66,147.44,142.63,137.76,136.47,135.92,133.13,1 32.29,128.65,127.06,124.36,110.48,108.74,71.42,56.43,56.11,44.14,40.43,24.96,21.25,11.52ppm. HRMS(ESI + ): Calculated value C 24 H 26 NO7Se + (M+H) + ,520.0869; measured value 520.0871.

[0103] Example 3 Preparation of (R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutyric acid-4,5-dimethoxy-2-nitrobenzyl ester (I-3)

[0104]

[0105] Referring to the preparation method described in Example 1, the reaction raw material 1a was replaced with 4,5-dimethoxy-2-nitrobenzyl alcohol to prepare compound I-3. 1 H NMR (300MHz, DMSO-d6): δ = 7.75 (s, 1H), 7.52 (d, J = 2.7Hz, 2H), 7.27 (d, J = 2.5Hz, 1H), 7.14 (s, 1H), 5.41 (dq, J1 = 11.3Hz, J2 =2.7Hz,2H),3.88(d,J=2.8Hz,6H),3.83(d,J=2.8Hz,6H),3.16-2.87(m,3H),1.64–1.52(m,2H),0.94(t,J=6.3Hz,3H)ppm. 13C NMR (75MHz, DMSO-d6): δ=200.46,174.53,153.09,150.54,148.73,147.64,142.61,141.60,137.75,136.48,133. 11,127.15,113.09,110.46,110.04,108.73,64.75,56.53,56.41,56.12,55.94,44.14,40.03,24.96,11.53ppm. HRMS(ESI + ): Calculated value C 25 H 28 NO9Se + (M+H) + ,566.0924; measured value 566.0918.

[0106] Example 4 Preparation of (R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutyric acid-(2-oxo-2H-benzopyran-4-yl)methyl ester (I-4)

[0107]

[0108] Referring to the preparation method described in Example 1, the reaction raw material 1a was replaced with 4-(hydroxymethyl)-2H-benzopyran-2-one to prepare compound I-4. 1 H NMR (300MHz, DMSO-d6): δ = 7.75 (s, 1H), 7.66-7.56 (m, 2H), 7.50 (s, 1H), 7.44 (dd, J1 = 6.3Hz, J2 = 2.9Hz, 1H), 7.23 (dt, J1 = 6.3Hz, J2 = 2.9 Hz,1H),7.15(s,1H),6.20(s,1H),5.43(s,2H),3.80(d,J=2.9Hz,6H),3.13-2.87(m,3H),1.65–1.52(m,2H),0.94(t,J=6.2Hz,3H)ppm.

[0109] 13C NMR (75MHz, DMSO-d6): δ=200.49,174.60,161.31,153.09,150.53,147.64,146.68,142.60,137.78,136.49,133.10,1 32.93,126.30,125.45,120.91,117.45,114.57,110.46,108.72,66.44,56.41,56.11,44.11,39.64,24.97,11.53ppm.

[0110] HRMS(ESI + ): Calculated value C 26 H 25 7sE + (M+H) + ,529.0760; measured value 529.0765.

[0111] Example 5 Preparation of (R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutyric acid-(7-(diethylamino)-2-oxo-2H-benzopyran-4-yl)methyl ester (I-5)

[0112]

[0113] Referring to the preparation method described in Example 1, the reaction raw material 1a was replaced with 7-(diethylamino)-4-(hydroxymethyl)-2H-benzopyran-2-one to prepare compound I-5. 1 H NMR (300MHz, DMSO-d6): δ=7.74(s,1H),7.52-7.46(m,2H),7.15(s,1H),6.63-6.57(m,2H),6.17(s,1H),5.43(s,2H),3.81(d ,J=2.9Hz,6H),3.50(q,J=6.0Hz,4H),3.16-2.85(m,3H),1.63–1.52(m,2H),1.16(t,J=6.3Hz,6H),0.94(t,J=6.2Hz,3H)ppm. 13C NMR (75MHz, DMSO-d6): δ=200.49,174.61,161.35,154.35,151.97,150.52,147.66,146.56,142.63,137.79,136.48,133.11, 126.90,115.54,114.37,110.47,110.10,108.73,99.65,66.71,56.41,56.10,44.40,44.12,39.66,24.96,12.36,11.53ppm. HRMS(ESI + ): Calculated value C 30 H 34 NO7Se + (M+H) + ,600.1495; measured value 600.1491.

[0114] Example 6 Preparation of (2R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoic acid-1-(7-(diethylamino)-2-oxo-2H-benzopyran-4-yl)ethyl ester (I-6)

[0115]

[0116] Referring to the preparation method described in Example 1, the reaction raw material 1a was replaced with 7-(diethylamino)-4-(1-hydroxyethyl)-2H-benzopyran-2-one to prepare compound I-6. 1 H NMR (300MHz, DMSO-d6): δ = 7.74 (s, 1H), 7.52 (s, 1H), 7.31 (d, J = 8.9Hz, 1H), 7.16 (s, 1H),6.63(d,J=2.7Hz,1H),6.49(dd,J1=6.2Hz,J2=2.7Hz,1H),6.19(s,1H),6.01(q, J=6.0Hz,1H),3.82(d,J=2.9Hz,6H),3.49(q,J=6.0Hz,4H),3.22-2.84(m,3H),1.65– 1.52 (m, 2H), 1.42 (d, J = 6.0Hz, 3H), 1.15 (t, J = 6.0Hz, 6H), 0.94 (t, J = 6.0Hz, 3H) ppm. 13C NMR (75MHz, DMSO-d6): δ=200.08,174.11,161.53,154.30,153.45,152.02,150.52,147.65,142.63,137.78,136.49,133.10,126 .00,114.50,113.41,110.48,108.83,108.70,100.03,75.05,56.41,56.09,44.40,44.10,39.96,25.07,20.66,12.34,11.52ppm. HRMS(ESI + ): Calculated value C 31 H 36 NO7Se + (M+H) + ,614.1652; measured value 614.1645.

[0117] Example 7

[0118]

[0119] Step 1: Preparation of (2R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)2-ethyl-4-oxobutanoic acid-1-(pyridin-4-yl)ethyl ester (4a)

[0120] BSP (500 mg, 1.36 mmol), 4-(1-hydroxyethyl)pyridine (3a, 200 mg, 1.63 mmol), EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 390 mg, 2.03 mmol), DMAP (17 mg, 0.14 mmol) and dichloromethane (14 mL) were added to a 100 mL round-bottom flask, and the resulting reaction mixture was stirred at room temperature for 4 h. After the reaction was complete as detected by TLC (dichloromethane: methanol = 20: 1), 50 mL of water was added to the reaction solution, the organic layer was separated, and the aqueous layer was extracted with dichloromethane (20 mL × 2). The organic layers were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 40: 1) to obtain 490 mg of intermediate 4a as a yellow foamy solid with a yield of 76%. 1H NMR (300MHz, DMSO-d6): δ=8.54-8.48(m,3H),7.71(d,J=2.9Hz,1H),7.50(d,J =1.1Hz,1H),7.38-7.32(m,2H),5.83-5.75(m,1H),3.85(s,3H),3.82(s,3H),3 .50-3.40(m,1H),3.25(dt,J1=17.3Hz,J2=3.8Hz,1H),2.99-2.92(m,1H),1.7 3-1.61(m,2H),1.45(t,J=6.5Hz,3H),0.97(dt,J1=21.1Hz,J2=7.3Hz,3H)ppm. HRMS(ESI + ): Calculated value C 23 H 26 NO5Se + (M+H) + ,476.0971; measured value 476.0980.

[0121] Step 2: Preparation of 4-(1-(((R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)ethyl)-1-(2,4-dinitrophenyl)pyridin-1-ium-4-methylbenzenesulfonate (6a)

[0122] Add intermediate 4a (490 mg, 1.03 mmol), 4-methylbenzenesulfonic acid-2,4-dinitrophenyl ester (5,419 mg, 1.24 mmol) and acetone (10 mL) to a 50 mL round-bottom flask. Stir the resulting reaction mixture at 50 °C for 24 h, during which solids precipitate. After TLC (dichloromethane: methanol = 10: 1) detected that the reaction was complete, stop heating. After the reaction solution was cooled to room temperature, filter it with suction, wash the filter cake with a small amount of acetone, and dry it to obtain 600 mg of intermediate 6a as an off-white solid with a yield of 71%. 1H NMR (300MHz, DMSO-d6): δ = 9.34 (d, J = 6.6Hz, 2H), 9.10 (d, J = 2.5Hz, 1H), 8.95 (dd, J1 = 8.7Hz, J2 = 2.5Hz, 1 H),8.52(s,1H),8.39-8.36(m,3H),7.69(s,1H),7.54(s,1H),7.49(d,J=8.0Hz,2H),7.14(d,J=7.9Hz,2 H),6.20(q,J=6.4Hz,1H),3.85(s,3H),3.83(s,3H),3.54-3.44(m,1H),3.39(dd,J1=17.6Hz,J2=4.3Hz, 1H), 3.04-2.98 (m, 1H), 2.29 (s, 3H), 1.82-1.67 (m, 2H), 1.62 (d, J = 6.7Hz, 3H), 0.96 (t, J = 7.4Hz, 3H) ppm. HRMS(ESI + ): Calculated value C 29 H 28 N3O9Se + (M) + ,642.0985; measured value 642.0982.

[0123] Step 3: Preparation of 2-((1E,3Z,5E)-4-(1-(((R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)ethyl)-7-((E)-1,3,3-trimethylindolin-2-ylidene)hept-1,3,5-trien-1-yl)-1,3,3-trimethyl-3H-indole-1-iodide (I-7)

[0124] Add intermediate 6a (200 mg, 0.25 mmol), 4-bromoaniline (51 mg, 0.30 mmol) and methanol (4 mL) to a 50 mL round-bottom flask, and stir the resulting reaction mixture at room temperature for 30 min. Add 1,2,3,3-tetramethyl-3H-indolium iodide (7a, 222 mg, 0.74 mmol) and sodium acetate (121 mg, 1.45 mmol). The reaction solution was stirred at room temperature for 24 h. After TLC (dichloromethane: methanol = 10: 1) detected that the reaction was complete, the reaction solvent was removed by concentration under reduced pressure, and purified by silica gel column chromatography (dichloromethane: methanol = 30: 1) to obtain 34 mg of compound I-7 as a green solid with a yield of 15%. 1H NMR (300MHz, DMSO-d6): δ = 8.50 (d, J = 13.2Hz, 1H), 8.10 (t, J = 13.3Hz, 2H), 7.69 (d, J = 14.1Hz, 1H), 7.61 ( t,J=8.1Hz,2H),7.50(d,J=10.1Hz,1H),7.45-7.37(m,4H),7.27(t,J=7.3Hz,2H),6.66(t,J=13.8Hz,2H ),6.45(t,J=12.5Hz,2H),6.19-6.13(m,1H),3.81-3.78(m,6H),3.61(s,3H),3.58(s,3H),3.47-3.41(m ,1H),3.31-3.20(m,1H),2.96-2.85(m,1H),1.73-1.53(m,17H),1.02(dt,J1=40.3Hz,J2=7.3Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.06,174.48,164.32,156.54,150.53,147.67,147.12,145.55,142. 63,139.96,138.30,137.78,137.63,136.49,135.56,133.10,132.39,131.10,128.83,128.35,1 27.64,126.95,125.92,123.81,122.93,119.14,118.56,110.44,110.32,108.72,101.77,70.13 ,56.42,56.08,44.12,40.55,40.01,39.90,37.54,32.10,29.17,28.52,25.05,20.35,11.53ppm. HRMS(ESI + ): Calculated value C 47 H 53 N2O5Se + (M) + ,805.3114; measured value 805.3115.

[0125] Example 8 Preparation of 2-((1E,3Z,5E)-4-(2-(((R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)propyl-2-yl)-7-((E)-1,3,3-trimethylindolin-2-ylidene)hept-1,3,5-trien-1-yl)-1,3,3-trimethyl-3H-indole-1-iodide (I-8)

[0126]

[0127] Referring to the preparation method described in Example 7, the reaction raw material 3a was replaced with 2-(pyridin-4-yl)propan-2-ol to prepare compound I-8. 1 H NMR (300MHz, DMSO-d6): δ = 8.51 (d, J = 13.2Hz, 1H), 8.12 (t, J = 13.3Hz, 2H), 7.69 (d, J = 13.9Hz, 1H), 7 .59(t,J=8.1Hz,2H),7.50(d,J=10.1Hz,1H),7.48-7.36(m,4H),7.27(t,J=7.2Hz,2H),6.67(t,J=1 3.9Hz,2H),6.46(t,J=12.1Hz,2H),3.81-3.78(m,6H),3.61(s,3H),3.58(s,3H),3.47-3.41(m,1H) ,3.31-3.20(m,1H),2.96-2.85(m,1H),1.74-1.51(m,20H),1.01(dt,J1=40.3Hz,J2=7.3Hz,3H)ppm.

[0128] 13 C NMR (75MHz, DMSO-d6): δ=200.05,173.21,164.21,156.52,150.52,147.66,147.12,145.55,144. 16,142.63,141.15,139.96,137.78,137.63,136.48,133.12,133.03,132.28,129.31,128.85,1 27.64,127.14,126.96,123.78,122.90,119.23,118.56,110.49,110.32,108.72,102.05,80.57 ,56.41,56.12,43.88,40.58,39.99,39.61,37.54,32.10,29.17,28.52,27.17,24.91,11.52ppm.

[0129] HRMS(ESI + ): Calculated value C 48 H 55 N2O5Se + (M) + ,819.3271; measured value 819.3275.

[0130] Example 9 Preparation of 2-((1E,3Z,5E)-4-(1-(((R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)ethyl)-7-((E)-5-methoxy-1,3,3-trimethylindolin-2-ylidene)hept-1,3,5-trien-1-yl)-5-methoxy-1,3,3-trimethyl-3H-indole-1-iodide (I-9)

[0131]

[0132] Referring to the preparation method described in Example 7, the reaction raw material 7a was replaced with 5-methoxy-1,2,3,3-tetramethyl-3H-indolium iodide to prepare compound I-9. 1 H NMR (300MHz, DMSO-d6): δ = 8.50 (d, J = 13.2Hz, 1H), 8.09 (t, J = 13.2Hz, 2H), 7.70 (d, J = 14.1Hz, 1H), 7.61 ( t,J=8.1Hz,2H),7.51(d,J=10.1Hz,1H),7.43-7.37(m,4H),7.27(t,J=7.6Hz,2H),6.67(t,J=13.9Hz,2H) ,6.45(t,J=12.3Hz,2H),6.19-6.13(m,1H),3.83-3.78(m,12H),3.61(s,3H),3.58(s,3H),3.47-3.41(m ,1H),3.31-3.19(m,1H),2.98-2.85(m,1H),1.73-1.53(m,17H),1.02(dt,J1=39.9Hz,J2=7.6Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.08,174.47,164.09,156.03,155.25,154.77,150.52,147.65,143.9 3,142.63,139.59,138.66,138.31,137.79,137.55,136.48,135.57,133.11,131.10,128.35,125. 94,121.15,119.22,118.71,113.53,111.40,110.63,110.45,108.90,108.72,101.95,70.12,56.4 2,56.11,55.65,44.13,41.00,39.99,39.88,37.57,32.12,29.18,28.55,25.05,20.36,11.53ppm.

[0133] HRMS(ESI + ): Calculated value C 49 H 57 N2O7Se + (M) + ,865.3326; measured value 865.3321.

[0134] Example 10 Preparation of sodium 3-(2-((1E,3Z,5E)-4-(1-(((R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)ethyl)-7-((E)-3,3-dimethyl-1-(3-sulfopropyl)indolin-2-ylidene)hept-1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate (I-10)

[0135]

[0136] Referring to the preparation method described in Example 7, the reaction raw material 7a was replaced with 3-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate to prepare compound I-10. 1 H NMR (300MHz, DMSO-d6): δ = 8.50 (d, J = 13.4Hz, 1H), 8.09 (t, J = 13.4Hz, 2H), 7.70 (d, J = 14.2Hz, 1H), 7.62 (t, J = 8. 1Hz,2H),7.50(d,J=10.1Hz,1H),7.45-7.37(m,4H),7.27(t,J=7.3Hz,2H),6.66(t,J=13.4Hz,2H),6.47(t,J=12 .5Hz,2H),6.18-6.13(m,1H),3.81-3.78(m,6H),3.67-3.55(m,4H),3.47-3.39(m,1H),3.32-3.20(m,1H),3.08- 3.05(m,4H),2.96-2.85(m,1H),2.13-2.07(m,4H),1.75-1.53(m,17H),0.99(dt,J1=40.1Hz,J2=7.2Hz,3H)ppm. 13C NMR (75MHz, DMSO-d6): δ=200.05,174.47,169.22,155.65,150.52,147.65,144.19,144.10,142.64,140 .47,137.81,137.77,137.23,136.49,135.54,133.10,132.00,131.28,128.99,128.05,127.55,125.95 ,124.92,124.06,122.99,119.76,117.74,111.13,110.45,108.74,102.24,70.11,56.43,56.11,51.51 ,50.06,45.18,44.11,43.65,42.60,41.44,39.90,29.05,28.45,25.06,23.41,20.36,18.84,11.50ppm.

[0137] HRMS(ESI - ): Calculated value C 51 H 59 N2O 11 S2Se-(M) - ,1019.2731; measured value 1019.2737.

[0138] Example 11 Preparation of 2-((1E,3Z,5E)-4-(1-(((R)-4-(5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)ethyl)-7-((E)-1-ethyl-3,3-dimethyl-5-sulfonic acid indolyl-2-ylidene)heptyl-1,3,5-trien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium-5-sulfonate sodium salt (I-11)

[0139]

[0140] Referring to the preparation method described in Example 7, the reaction raw material 7a was replaced with 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate to prepare compound I-11. 1H NMR (300MHz, DMSO-d6): δ = 8.52 (d, J = 13.2Hz, 1H), 8.11 (t, J = 13.1Hz, 2H), 7.82-7.78 (m, 1H), 7.69 (d, J=14.1Hz,1H),7.50(d,J=10.3Hz,1H),7.45-7.36(m,3H),7.23(t,J=7.4Hz,2H),6.66(t,J=13.5Hz,2 H),6.45(t,J=12.3Hz,2H),6.19-6.13(m,1H),3.81-3.78(m,6H),3.66-3.57(m,4H),3.47-3.42(m,1H ),3.30-3.18(m,1H),2.96-2.84(m,1H),1.72-1.51(m,23H),1.00(dt,J1=40.0Hz,J2=7.4Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.04,174.47,167.20,155.25,150.53,147.98,147.66,145.56,140.8 5,140.33,137.81,137.79,136.46,136.35,135.56,133.08,131.66,131.24,129.29,128.56,128. 04,125.95,124.82,123.96,120.83,116.95,110.87,110.47,108.72,102.02,70.14,56.42,56.1 1,44.15,42.12,40.66,40.45,39.91,38.73,29.10,28.48,25.06,20.35,13.47,13.11,11.51ppm.

[0141] HRMS(ESI - ): Calculated value C 49 H 55 N2O 11 S2S - (M) - ,991.2418; measured value 998.2413.

[0142] Example 12 Preparation of sodium 4-(2-((1E,3Z,5E,7E)-4-(1-(((R)-4-(5,6-5,6-dimethoxybenzo[b]selenophene-2-yl)-2-ethyl-4-oxobutanoyl)oxy)ethyl)-7-(1,1-dimethyl-3-(4-sulfonic acid butyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)heptyl-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate (I-12)

[0143]

[0144] Referring to the preparation method described in Example 7, the reaction raw material 7a was replaced with 4-(1,1,2-trimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate to prepare compound I-12. 1 H NMR (300MHz, DMSO-d6): δ = 8.67-8.63 (m, 1H), 8.50 (d, J = 13.2Hz, 1H), 8.31-8.25 (m, 2H), 8.07-8.03 (m, 1H), 7.83-7.7 4(m,3H),7.54-7.27(m,8H),7.16(s,1H),7.07(d,J=8.7Hz,2H),6.87(dd,J1=14.7Hz,J2=6.1Hz,1H),6.74-6.70(m,1H ),6.62-6.54(m,1H),6.14(dd,J1=9.2Hz,J2=2.9Hz,1H),5.42-5.34(m,1H),4.44-4.38(m,2H),3.84-3.72(m,8H),3.2 9-3.04(m,4H),2.98-2.79(m,3H),2.05-1.52(m,21H),1.35(d,J=6.1Hz,3H),0.97(dt,J1=40.0Hz,J2=7.0Hz,3H)ppm. 13CNMR (75MHz, DMSO-d6): δ=200.08,174.47,165.42,154.94,150.51,147.66,142.62,141.65,140.52,137.77,137.60,136 .55,136.48,135.57,133.82,133.15,133.09,132.64,131.88,131.28,129.24,128.75,128.04,127.81,127.69,127.22,1 26.83,126.45,126.10,125.94,123.17,122.82,120.42,117.14,110.47,108.77,108.72,101.75,70.14,56.41,56.11,55 .26,52.95,45.44,44.69,44.13,43.67,41.02,39.92,30.92,30.38,25.69,25.06,23.58,23.30,20.35,19.78,11.52ppm.

[0145] HRMS(ESI - ): Calculated value C 61 H 67 N2O 11 S2S - (M) - ,1147.3357; measured value 1147.3352.

[0146] Example 13

[0147]

[0148] Step 1: Preparation of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (10)

[0149] 2-Bromo-5-hydroxy-4-methoxybenzaldehyde (8, 6.9 g, 30.0 mmol), K2CO3 (8.3 g, 60.0 mmol) and DMF (70 mL) were added to a 250 mL reaction bottle, and benzyl bromide (9, 6.1 g, 36.0 mmol) was added. The resulting reaction mixture was heated to 50 ° C and stirred for 2 h. After TLC (petroleum ether: ethyl acetate = 1: 1) detected that the reaction was complete, the heating was stopped, and the reaction solution was cooled to room temperature. The reaction solution was poured into 350 mL of ice water, and the filter cake was collected by suction filtration. The filter cake was washed with water and dried. The dried filter cake was slurried with petroleum ether (200 mL) for 20 min, suction filtered, and the filter cake was washed with petroleum ether and dried to obtain 9.24 g of intermediate 10 as a white solid with a yield of 96%. 1H NMR (300MHz, CDCl3): δ = 10.16 (s, 1H), 7.48 (s, 1H), 7.46–7.30 (m, 5H), 7.07 (s, 1H), 5.16 (s, 2H), 3.95 (s, 3H) ppm. HRMS(ESI + ): Calculated value C 15 H 14 BrO3 + (M+H) + ,321.0121; measured value 321.0122.

[0150] Step 2: Preparation of 5-(benzyloxy)-4-methoxy-2-(methylseleno)benzaldehyde (12)

[0151] Intermediate 10 (6.53 g, 20.3 mmol), DTT (DL-dithiothreitol, 4.18 g, 27.1 mmol) and DMF (70 mL) were added to a 250 mL reaction bottle, and dimethyl diselenide (11, 2.55 g, 13.6 mmol) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene, 5.15 g, 33.9 mmol) were added thereto. After stirring for 27 h at room temperature under nitrogen protection, the reaction solution was poured into 250 mL of water to precipitate. The filter cake was filtered and washed with water to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain intermediate 12 3.95 g, a light yellow solid, with a yield of 87%. 1 H NMR (300MHz, CDCl3): δ = 10.08 (s, 1H), 7.46–7.23 (m, 6H), 6.97 (s, 1H), 5.18 (s, 2H), 3.99 (s, 3H), 2.31 (s, 3H) ppm. HRMS(ESI + ): Calculated value C 16 H 17 O3Se + (M+H) + ,337.0337; measured value 337.0334.

[0152] Step 3: Preparation of ethyl 2-((4-(benzyloxy)-2-formyl-5-methoxyphenyl)seleno)acetate (14)

[0153] Add intermediate 12 (3.95 g, 11.8 mmol) and DMF (30 mL) to a 100 mL reaction bottle, add ethyl 2-bromoacetate (13, 5.91 g, 35.4 mmol), and heat and stir the reaction solution at 150 ° C for 4 h. After TLC (petroleum ether: ethyl acetate = 4: 1) detected that the raw material reaction was complete, the reaction solution was gradually cooled to room temperature. Add water (150 mL) to dilute and extract with ethyl acetate (50 mL) three times. Combine the organic phases, wash with water, wash with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate to obtain the crude intermediate 14 as a brown-yellow solid. The crude product was used directly in the next reaction without additional purification. HRMS (ESI + ): Calculated value C 19 H 21 5S + (M+H) + ,409.0549; Found 409.0548. Step 4: Preparation of ethyl 5-(benzyloxy)-6-methoxybenzo[b]selenophene-2-carboxylate (15)

[0154] The intermediate 14 obtained in the previous step and K2CO3 (4.07 g, 29.5 mmol) were added to a 100 mL reaction bottle, and acetonitrile (40 mL) was added thereto. The reaction solution was stirred under reflux for 4 h. After TLC (petroleum ether: ethyl acetate = 4:1) detected that the raw material reaction was complete, the reaction solution was gradually cooled to room temperature. After the reaction solution was concentrated, water (180 mL) was added to dilute it, and it was extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain intermediate 15 3.48 g, light yellow solid, with a two-step yield of 76%. 1 H NMR (300MHz, DMSO-d6): δ = 8.23 ​​(s, 1H), 7.75 (s, 1H), 7.67 (s, 1H), 7.49-7.32 (m, 5H), 5.19 (s, 2H), 4.32 (q, J = 6.9Hz, 2H), 3.85 (s, 3H), 1.33 (t, J = 7.1Hz, 3H) ppm. HRMS(ESI + ): Calculated value C 19 H 19 oeLh + (M+H) + ,391.0443; measured value 391.0440.

[0155] Step 5: Preparation of 5-(Benzyloxy)-6-methoxybenzo[b]selenophene-2-carboxylic acid (16)

[0156] Intermediate 15 (3.48 g, 8.94 mmol), methanol (35 mL) and tetrahydrofuran (35 mL) were added to a 250 mL reaction bottle, and 2N NaOH aqueous solution (13 mL) was added. The reaction solution was stirred at 60 ° C for 1 h. After TLC (petroleum ether: ethyl acetate = 4: 1) detected that the raw material reaction was complete, the reaction solvent was removed by decompression, and the residue was adjusted to pH 2-3 with 1N HCl. After suction filtration, the filter cake was washed with water and dried to obtain 3.20 g of intermediate 16, an off-white solid, with a yield of 99%. 1 H NMR (300MHz, DMSO-d6): δ = 8.13 (s, 1H), 7.66 (s, 1H), 7.62 (s, 1H), 7.46–7.29 (m, 5H), 5.09 (s, 2H), 3.82 (s, 3H) ppm. HRMS(ESI - ): Calculated value C 17 H 13 oeLh - (MH) - ,360.9985; measured value 360.9991.

[0157] Step 6: Preparation of methyl 3-(5-(benzyloxy)-6-methoxybenzo[b]selenophene-2-yl)-3-oxopropanoate (18) Intermediate 16 (3.20 g, 8.86 mmol), CDI (N,N-carbonyldiimidazole, 4.31 g, 26.6 mmol) and DMF (65 mL) were added to a 250 mL reaction bottle. The resulting reaction solution was stirred at room temperature for 1 h, and MgCl2 (2.53 g, 26.6 mmol) and potassium monomethyl malonate (17, 4.15 g, 26.6 mmol) were added. The reaction was carried out at room temperature for 5 h. After TLC (petroleum ether: ethyl acetate = 2:1) detected that the raw materials had reacted completely, the reaction solution was poured into water (300 mL). The filter cake was washed with water to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain 3.18 g of intermediate 18 as a yellow solid. The yield was 86%. 1 H NMR (300MHz, CDCl3): δ = 8.01 (s, 1H), 7.45–7.27 (m, 7H), 5.19 (s, 2H), 3.96 (s, 3H), 3.97 (s, 2H), 3.73 (s, 3H) ppm. HRMS(ESI + ): Calculated value C 20 H 19 5S + (M+H) + ,419.0392; measured value 419.0393.

[0158] Step 7: Preparation of methyl ethyl 2-(5-(benzyloxy)-6-methoxybenzo[b]selenophene-2-carbonyl)succinate (19)

[0159] Add intermediate 18 (3.18 g, 7.61 mmol), K2CO3 (2.10 g, 15.2 mmol) and DMF (30 mL) to a 100 mL reaction bottle, and add ethyl 2-bromoacetate (13, 1.52 g, 9.13 mmol). The reaction solution was stirred at room temperature for 4 h. After TLC (petroleum ether: ethyl acetate = 2:1) detected that the raw material reaction was complete, water (150 mL) was added to the reaction solution, and extracted three times with ethyl acetate (60 mL). The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude intermediate 19 was obtained. The crude product was directly used in the next step without additional purification. HRMS (ESI + ): Calculated value C 24 H 25 7sE + (M+H) + ,505.0760; measured value 505.0759.

[0160] Step 8: Preparation of 4-(5-hydroxy-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoic acid (20)

[0161] The intermediate 19 obtained in the previous step and acetic acid (15 mL) were added to a 100 mL reaction bottle, and concentrated hydrochloric acid (15 mL) was slowly added. The resulting reaction solution was stirred at 90 ° C for 2 h. After TLC (petroleum ether: ethyl acetate = 2: 1) detected that the raw material reaction was complete, the reaction solution was naturally cooled to room temperature, water (120 mL) was added and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 30: 1) to obtain 1.46 g of intermediate 20 as a light yellow solid, with a two-step yield of 59%. 1 H NMR (300MHz, DMSO-d6): δ = 12.16 (s, 1H), 9.35 (s, 1H), 8.21 (s, 1H), 7.63 (s, 1H) ), 7.36 (s, 1H), 3.85 (s, 3H), 3.26 (t, J = 6.1Hz, 2H), 2.62 (t, J = 6.5Hz, 2H) ppm. HRMS(ESI - ): Calculated value C 13 H 11 5S - (MH) -,326.9777; Found 326.9782. Step 9: Preparation of ethyl 4-(5-hydroxy-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoate (21)

[0162] Add intermediate 20 (1.46 g, 4.47 mmol) and anhydrous ethanol (45 mL) to a 100 mL reaction bottle and place in an ice bath to cool. At 0 ° C, slowly drip SOCl2 (1.59 g, 13.4 mmol), gradually return to room temperature after dripping, and stir under reflux for 1 h. After TLC (dichloromethane: methanol = 20: 1) detected that the raw materials were completely consumed, the reaction was naturally cooled to room temperature. After the reaction solution was concentrated, water (180 mL) was added and extracted three times with ethyl acetate (45 mL). The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4: 1) to obtain intermediate 21 1.51 g, off-white solid, with a yield of 95%. 1 H NMR (300MHz, CDCl3): δ = 8.09 (s, 1H), 7.40 (s, 1H), 7.31 (s, 1H), 4.22 (q, J = 7.1Hz, 2H) ,3.99(s,3H),3.34(t,J=6.7Hz,2H),2.81(t,J=6.9Hz,2H),1.29(t,J=7.1Hz,3H)ppm. HRMS(ESI + ): Calculated value C 15 H 17 5S + (M+H) + ,357.0236; measured value 357.0233.

[0163] Step 10: Preparation of ethyl 4-(5-(3-bromopropyl)-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoate (23)

[0164] Intermediate 21 (753 mg, 2.13 mmol), K2CO3 (587 mg, 4.23 mmol) and DMF (20 mL) were added to a 100 mL reaction bottle, and 1,3-dibromopropane (22, 4.29 g, 21.2 mmol) was added. The reaction solution was stirred at 45 °C for 2 h. After TLC (petroleum ether: ethyl acetate = 4:1) detected that the raw material had reacted completely, water (150 mL) was added for dilution, and ethyl acetate (35 mL) was used for extraction three times. The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain 768 mg of intermediate 23 as a light yellow solid with a yield of 76%. 1H NMR (300MHz, CDCl3): δ = 8.10 (s, 1H), 7.34 (s, 1H), 7.33 (s, 1H), 4.22–4.12 (m, 4H), 3.94 (s, 3H), 3.68 (t, J=6.3Hz,2H),3.34(t,J=6.8Hz,2H),2.79(t,J=6.7Hz,2H),2.45–2.37(m,2H),1.29(t,J=7.1Hz,3H)ppm. HRMS(ESI + ): Calculated value C 18 H 22 BrO5Se + (M+H) + ,476.9810; measured value 476.9814.

[0165] Step 11: Preparation of 4,4'-((propane-1,3-diylbis(oxy))bis(6-methoxybenzo[b]selenophene-5,2-diyl))bis(4-oxobutanoic acid diethyl ester) (24)

[0166] Intermediate 23 (758 mg, 1.61 mmol), intermediate 21 (564 mg, 1.61 mmol), K2CO3 (658 mg, 4.79 mmol), KI (27 mg, 0.15 mmol), 18-crown ether-6 (42 mg, 0.15 mmol) and DMF (18 mL) were added to a 50 mL reaction bottle, and the resulting reaction solution was stirred at 50 ° C for 8 h. After TLC (petroleum ether: ethyl acetate = 2: 1) detected that the raw material reaction was complete, water (100 mL) was added, and ethyl acetate (40 mL) was used for extraction three times. The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain 1040 mg of intermediate 24 as a yellow solid with a yield of 87%. 1 H NMR (300MHz, DMSO-d6): δ = 8.42 (s, 2H), 7.71 (s, 2H), 7.58 (s, 2H), 4.25 (t, J = 5.8Hz, 4H), 4.09 (q, J = 7.1Hz, 4 H), 3.85 (s, 6H), 3.31 (t, J = 6.0Hz, 4H), 2.67 (t, J = 5.9Hz, 4H), 2.30–2.25 (m, 2H), 1.19 (t, J = 7.1Hz, 6H) ppm. HRMS(ESI + ): Calculated value C 33 H 37 O 10 Se2 + (M+H) +,753.0712; measured value 753.0714.

[0167] Step 12: Preparation of 4,4'-((propane-1,3-diylbis(oxy))bis(6-methoxybenzo[b]selenophene-5,2-diyl))bis(4-oxobutanoic acid) (diBSP)

[0168] Intermediate 24 (900 mg, 1.20 mmol), methanol (12 mL) and tetrahydrofuran (12 mL) were added to a 100 mL reaction bottle, and a 2N aqueous NaOH solution (3.6 mL) was added. The reaction solution was stirred at 55°C for 1 h. After TLC (petroleum ether: ethyl acetate = 1:1) detected that the raw material reaction was complete, the reaction solution was concentrated, the pH of the obtained residue was adjusted to 2-3 with 1N HCl, and extracted three times with ethyl acetate (40 mL). The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 30:1) to obtain 571 mg of compound diBSP as a yellow solid with a yield of 69%. 1 H NMR (300MHz, DMSO-d6): δ=12.19(s,2H),8.40(s,2H),7.71(s,2H),7.59(s,2H),4.23(t,J=5 .9Hz,4H),3.85(s,6H),3.27(t,J=5.9Hz,4H),2.61(t,J=6.2Hz,4H),2.31–2.27(m,2H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=194.04,174.20,150.99,147.94,145.13,137.39,135.52,134.61,110.75,108.60,65.63,56.34,33.01,29.08,28.52ppm. HRMS(ESI - ): Calculated value C 29 H 27 O 10 Se2 - (MH) - ,694.9940; measured value 694.9945.

[0169] Example 14 Preparation of 4-(6-methoxy-5-(3-((6-methoxy-2-(4-((2-nitrobenzyl)oxy)-4-oxobutanoyl)benzo[b]selenophene-5-yl)oxy)propoxy)benzo[b]selenophene-2-yl)-4-oxobutanoic acid (II-1)

[0170]

[0171] Compound diBSP (150 mg, 0.22 mmol), 2-nitrobenzyl alcohol (1a) (33 mg, 0.22 mmol), DCC (44 mg, 0.33 mmol), DMAP (1 mg, 0.01 mmol) and dichloromethane (4 mL) were added to a 50 mL reaction bottle and stirred at room temperature for 5 h under light-proof conditions. After TLC (dichloromethane: methanol = 20: 1) detected that the reaction of the raw materials was complete, the reaction solution was concentrated, and the obtained residue was dissolved in ethyl acetate (15 mL), cooled and stirred at -10°C for about 30 min, filtered and washed with glacial ethyl acetate (2 mL), and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 50: 1) to obtain 106 mg of compound II-1 as a light yellow solid with a yield of 59%. 1 H NMR (300MHz, DMSO-d6): δ = 11.62 (s, 1H), 8.11–8.06 (m, 1H), 7.76 (s, 2H), 7.63–7.54 (m, 3H), 7.52 (s, 2H), 7.12 (s, 2H), 5.51(d,J=2.9Hz,2H),4.16(t,J=3.1Hz,4H),3.84(s,6H),3.24-3.07(m,4H),2.69-2.58(m,4H),2.33–2.26(m,2H)ppm. 13 CNMR (75MHz, DMSO-d6): δ = 201.54, 200.90, 174.39, 170.84, 151.03, 147.23, 146.42, 142.36, 137.75, 136.48, 133.63, 133.04,131.46,128.48,127.82,123.51,112.03,108.79,66.73,64.35,56.12,38.27,38.03,30.27,29.41,29.08ppm. HRMS(ESI - ): Calculated value C 36 H 32 NO 12 Se2 - (MH) - ,830.0260; measured value 830.0257.

[0172] Example 15 Preparation of 4,4'-((propane-1,3-diylbis(oxy))bis(6-methoxybenzo[b]selenophene-5,2-diyl))bis(4-oxobutanoic acid)bis(2-nitro)benzyl ester (II-2)

[0173]

[0174] Compound diBSP (150 mg, 0.22 mmol), 2-nitrobenzyl alcohol (1a) (99 mg, 0.65 mmol), DCC (88 mg, 0.66 mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (5 mL) were added to a 50 mL reaction bottle, protected from light, and stirred at room temperature for 6 h. After TLC (dichloromethane: methanol = 20: 1) detected that the reaction of the raw materials was complete, the reaction solution was concentrated, and the residue was dissolved in ethyl acetate (15 mL), cooled and stirred at -10°C for about 30 min, filtered and washed with glacial ethyl acetate (2 mL), and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 100: 1) to obtain 131 mg of compound II-2 as a light yellow solid with a yield of 63%. 1 H NMR (300MHz, DMSO-d6): δ=8.12–8.06(m,2H),7.79(s,2H),7.63–7.54(m,6H),7.52(s,2H),7.12(s,2H),5.50(d,J= 2.8Hz,2H),4.16(t,J=6.0Hz,4H),3.84(s,6H),3.12(t,J=3.3Hz,4H),2.62(t,J=3.3Hz,4H),2.34–2.26(m,2H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.89,170.87,150.01,147.23,146.42,142.36,137.75,136.48,133.62,13 3.04,131.46,128.45,127.82,123.51,112.06,108.79,66.71,64.35,56.12,38.06,30.27,29.09ppm. HRMS(ESI + ): Calculated value C 43 H 39 N2O 14 Se2 + (M+H) + ,967.0726; measured value 967.0724.

[0175] Example 16 Preparation of 4,4'-((propane-1,3-diylbis(oxy))bis(6-methoxybenzo[b]selenophene-5,2-diyl))bis(4-oxobutyrate)bis(4,5-dimethoxy-2-nitro)benzyl ester (II-3)

[0176]

[0177] Referring to the preparation method described in Example 15, the reaction raw material 1a was replaced with 4,5-dimethoxy-2-nitrobenzyl alcohol to prepare compound II-3. 1 H NMR (300MHz, DMSO-d6): δ = 7.78 (s, 2H), 7.52 (d, J = 3.0Hz, 4H), 7.26 (t, J = 2.9Hz, 2H), 7.12 (s, 2H), 5.50 (d, J = 2.9Hz, 4H), 4. 15(t,J=6.1Hz,4H),3.88(d,J=6.0Hz,12H)3.84(s,6H),3.12(t,J=8.7Hz,4H),2.61(t,J=5.9Hz,4H),2.34–2.25(m,2H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.89,170.88,153.07,151.01,148.73,146.42,142.36,141.60,137.73,136.49,1 33.62,127.04,113.10,112.06,110.04,108.79,66.71,64.23,56.52,56.11,55.95,38.06,30.25,29.10ppm. HRMS(ESI + ): Calculated value C 47 H 47 N2O 18 Se2 + (M+H) + ,1087.1149; measured value 1087.1149.

[0178] Example 17 Preparation of 4,4'-((propane-1,3-diylbis(oxy))bis(6-methoxybenzo[b]selenophene-5,2-diyl))bis(4-oxobutyrate)bis(2-oxo-2H-benzopyran-4-yl)methyl ester (II-4)

[0179]

[0180] Referring to the preparation method described in Example 15, the raw material 1a was replaced with 4-(hydroxymethyl)-2H-benzopyran-2-one to obtain compound II-4. 1H NMR (300MHz, DMSO-d6): δ = 7.76 (s, 2H), 7.67–7.56 (m, 4H), 7.50 (s, 2H), 7.43 (dd, J1 = 5.9Hz, J2 = 2.1Hz, 2H), 7.25–7.19 (m, 2H), 7.11 (s, 2H),6.21(s,2H),5.42(s,4H),4.16(t,J=6.0Hz,4H),3.84(s,6H),3.14(t,J=8.1Hz,4H),2.61(t,J=5.7Hz,4H),2.35–2.23(m,2H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.91,170.93,161.34,153.06,146.65,146.43,142.35,137.73,136.46,133.64,13 2.95,126.30,125.47,120.87,117.44,114.85,112.05,108.76,66.71,65.93,56.12,38.07,30.29,28.87ppm. HRMS(ESI + ): Calculated value C 49 H 41 O 14 Se2 + (M+H) + ,1013.0821; measured value 1013.0822.

[0181] Example 18 Preparation of 4-(5-(3-((2-(4-((7-(diethylamino)-2-oxo-2H-benzopyran-4-yl)methoxy)-4-oxobutanoyl)-6-methoxybenzo[b]selenophene-5-yl)oxy)propoxy)-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoic acid (II-5)

[0182]

[0183] Referring to the preparation method described in Example 14, the reaction raw material 1a was replaced with 7-(diethylamino)-4-(hydroxymethyl)-2H-benzopyran-2-one to prepare compound II-5. 1H NMR (300MHz, DMSO-d6): δ=11.63(s,1H),7.76(s,2H),7.52–7.46(m,3H),7.12(s,2H),6.65–6.59(m,2H),6.15(s,1H),5.41(s ,2H),4.15(t,J=5.7Hz,4H),3.83(s,6H),3.50(q,J=6.4Hz,4H),3.15–3.08(m,4H),2.68–2.58(m,4H),2.37–2.28(m,2H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=201.55,200.88,174.38,170.94,161.37,154.35,151.96,150.99,146.53,146.45,142.35,137.76,136.47,133 .64,126.91,115.54,114.64,112.05,110.15,108.81,99.65,66.72,66.13,56.09,44.43,38.26,38.06,30.27,29.42,28.89,12.36ppm. HRMS(ESI - ): Calculated value C 43 H 42 NO 12 Se2 - (MH) - ,924.1043; measured value 924.1044.

[0184] Example 19 Preparation of 4,4'-((propane-1,3-diylbis(oxy))bis(6-methoxybenzo[b]selenophene-5,2-diyl))bis(4-oxobutyrate)bis(7-(diethylamino)-2-oxo-2H-benzopyran-4-yl)methyl ester (II-6)

[0185]

[0186] Referring to the preparation method described in Example 15, the raw material 1a was replaced with 7-(diethylamino)-4-(hydroxymethyl)-2H-benzopyran-2-one to obtain compound II-6. 1H NMR (300MHz, DMSO-d6): δ=7.77(s,2H),7.53–7.46(m,4H),7.14(s,2H),6.63–6.56(m,4H),6.19(s,2H),5.43(s,4H),4.14(t,J=8.9Hz ,4H),3.86(s,6H),3.49(q,J=8.3Hz,8H),3.12(t,J=8.3Hz,4H),2.61(t,J=8.5Hz,4H),2.34–2.26(m,2H),1.12(t,J=6.1Hz,12H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=200.90,170.92,161.36,154.37,151.99,151.03,146.53,146.44,142.36,137.76,136.50, 133.62,126.93,115.54,114.63,110.14,108.76,99.64,66.71,66.18,56.13,44.42,38.05,30.27,28.89,12.36ppm. HRMS(ESI + ): Calculated value C 57 H 59 N2O 14 Se2 + (M+H) + ,1155.2291; measured value 1155.2288.

[0187] Example 20 Preparation of 4-(5-(3-((2-(4-(1-(7-(diethylamino)-2-oxo-2H-benzopyran-4-yl)ethoxy)-4-oxobutanoyl)-6-methoxybenzo[b]selenophene-5-yl)oxy)propoxy)-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoic acid (II-7)

[0188]

[0189] Referring to the preparation method described in Example 14, the reaction raw material 1a was replaced with 7-(diethylamino)-4-(1-hydroxyethyl)-2H-benzopyran-2-one to prepare compound II-7. 1H NMR (300MHz, DMSO-d6): δ = 11.63 (s, 1H), 7.78 (s, 2H), 7.53 (s, 2H), 7.32 (d, J = 8.7Hz, 1H), 7.12(s,2H),6.64(d,J=2.8Hz,1H),6.50(dd,J1=6.1Hz,J2=2.9Hz,1H),6.18(s,1H),6.04( q,J=5.9Hz,1H),4.15(t,J=5.9Hz,4H),3.83(s,6H),3.51(q,J=6.3Hz,4H),3.15–3.08(m,4 H), 2.68–2.58 (m, 4H), 2.37–2.28 (m, 2H), 1.43 (d, J = 8.1Hz, 3H), 1.15 (t, J = 6.5Hz, 6H) ppm. 13 C NMR (75MHz, DMSO-d6): δ=201.58,200.90,174.38,171.63,161.54,153.64,153.45,152.05,151.02,146.44,142.35,137.74,136.47,133 .64,126.01,114.34,113.43,108.84,108.77,100.04,74.36,66.70,56.13,44.43,38.27,38.16,30.27,29.42,29.25,20.65,12.34ppm. HRMS(ESI - ): Calculated value C 44 H 44 NO 12 Se2 - (MH) - ,938.1199; measured value 938.1201.

[0190] Example 21 Preparation of 2-((1E,3Z,5E)-4-(1-((4-(5-(3-((2-(3-carboxypropanoyl)-6-methoxybenzo[b]selenophene-5-yl)oxy)propoxy)-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoyl)oxy)ethyl)-7-((E)-1,3,3-trimethylindolin-2-ylidene)hept-1,3,5-trien-1-yl)-1,3,3-trimethyl-3H-indole-1-iodide (II-8)

[0191]

[0192] Referring to the preparation method described in Example 7, the reaction raw material BSP was replaced with diBSP to prepare compound II-8. 1HNMR (300MHz, DMSO-d6): δ = 11.64 (s, 1H), 7.80 (s, 2H), 7.73–7.46 (m, 7H), 7.42–7.15 (m, 3H), 7.10 (s ,2H),7.07–6.89(m,2H),6.75–6.66(m,2H),6.62–6.52(m,1H),6.24(dd,J1=6.1Hz,J2=2.7Hz,1H),5. 55–5.34(m,1H),4.27(s,3H),4.17(t,J=5.9Hz,4H),3.84(s,6H),3.55(s,3H),3.26–3.01(m,5H),2. 69–2.56(m,4H),2.36–2.23(m,2H),1.71(dd,J1=27.6Hz,J2=15.2Hz,11H),1.28(d,J=6.3Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ = 201.59, 200.92, 174.39, 171.83, 164.32, 156.60, 151.03, 147.15, 146.42, 14 5.54,142.35,139.94,138.83,137.74,137.61,136.46,135.33,133.63,132.26,131.10,128.86,128. 27,127.63,126.94,125.96,123.81,122.92,119.11,118.58,112.06,110.32,108.81,101.76,69.46, 66.72,56.11,40.56,39.98,38.25,38.14,37.55,32.11,30.27,29.45,29.25,29.17,28.53,20.33ppm. HRMS(ESI + ): Calculated value C 60 H 63 N2O 10 Se2 + (M) + ,1131.2808; measured value 1131.2803.

[0193] Example 22 Preparation of 2-((1E,3Z,5E)-4-(1-((4-(5-(3-((2-(3-carboxypropanoyl)-6-methoxybenzo[b]selenophene-5-yl)oxy)propoxy)-6-methoxybenzo[b]selenophene-2-yl)-4-oxobutanoyl)oxy)ethyl)-7-((E)-5-methoxy-1,3,3-trimethylindolin-2-ylidene)hept-1,3,5-trien-1-yl)-5-methoxy-1,3,3-trimethyl-3H-indole-1-iodide (II-9)

[0194]

[0195] Referring to the preparation method described in Example 7, the reaction raw material BSP was replaced by diBSP, and 7a was replaced by 5-methoxy-1,2,3,3-tetramethyl-3H-indolium iodide to prepare compound II-9. 1 H NMR (300MHz, DMSO-d6): δ = 11.65 (s, 1H), 7.79 (s, 2H), 7.52–7.48 (m, 4H), 7.42–7.33 (m, 2H), 7.17–7.11 (m, 3H), 7.01 –6.90(m,3H),6.83(dd,J1=6.2Hz,J2=2.7Hz,1H),6.71(dt,J1=6.4Hz,J2=2.4Hz,1H),6.62–6.53(m,1H),6.23(dd,J 1=6.2Hz, J2=2.7Hz,1H),5.44–5.34(m,1H),4.27(s,3H),4.26–4.11(m,7H),3.86–3.81(m,12H),3.56(s,3H),3.26– 3.01(m,5H),2.71–2.53(m,4H),2.35–2.23(m,2H),1.68(dd,J1=27.4Hz,J2=15.3Hz,11H),1.29(d,J=6.3Hz,3H)ppm. 13CNMR (75MHz, DMSO-d6): δ = 201.56, 200.90, 174.39, 171.83, 164.11, 156.02, 155.26, 154.77, 151.02, 146. 44,143.95,142.35,139.59,138.70,138.31,137.75,137.53,136.48,135.33,133.62,131.13,128.27,125 .97,125.96,121.16,119.21,118.72,113.52,112.08,111.43,110.61,108.91,108.78,101.96,69.43,66. 71,56.11,55.67,40.97,39.99,38.26,38.12,37.57,32.14,30.26,29.45,29.23,29.18,28.52,20.33ppm. HRMS(ESI + ): Calculated value C 62 H 67 N2O 12 Se2 + (M) + ,1191.3019; measured value 1191.3015.

[0196] Example 23 Preparation of sodium 3-(2-((1E,3Z,5E)-4-(1-((4-(5-(3-((2-(3-carboxypropanoyl)-6-methoxybenzo[b]selenophene-5-yl)oxy)propoxy)-6-methoxybenzo[b]selenophene-2-yl)-4-butanoyl)oxy)ethyl)-7-((E)-3,3-dimethyl-1-(3-sulfonylpropyl)indolyl-2-ylidene)heptyl-1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate (II-10)

[0197]

[0198] Referring to the preparation method described in Example 7, the reaction raw material BSP was replaced by diBSP, and 7a was replaced by 3-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate to prepare compound II-10. 1H NMR (300MHz, DMSO-d6): δ=11.60(s,1H),7.77(s,2H),7.73–7.64(m,2H),7.60–7.51(m,4H),7.47–7.35(m,2H),7.28–7.09(m,4 H),7.03(dt,J1=6.3Hz,J2=2.9Hz,1H),6.92–6.82(m,2H),6.74–6.69(m,1H),6.62–6.53(m,1H),6.14(dd,J1=6.3Hz,J2=2.9Hz ,1H),5.44–5.31(m,1H),4.67–4.45(m,2H),4.13(dt,J1=6.1Hz,J2=2.9Hz,1H),4.06–3.93(m,2H),3.84(s,6H),3.45–3.23(m, 2H), 3.22–2.99(m,7H),2.69–2.54(m,4H),2.35–2.04(m,6H),1.66(dd,J1=27.5Hz,J2=15.2Hz,11H),1.31(d,J=6.4Hz,3H)ppm. 13 C NMR (75MHz, DMSO-d6): δ=201.53,200.88,174.39,171.81,169.23,155.66,151.03,146.45,144.18,144.10,1 42.35,140.47,137.83,137.72,137.22,136.46,135.32,133.65,132.03,131.25,129.02,128.07,127.57,12 5.96,124.94,124.03,122.99,119.78,117.76,112.05,111.11,108.75,102.23,69.56,66.72,56.11,51.50, 50.04,45.16,43.66,42.63,41.42,38.27,38.14,30.27,29.42,29.23,29.06,28.48,23.42,20.34,18.85ppm. HRMS(ESI - ): Calculated value C 64 H 69 N2O 16 Se2 - (M) - ,1345.2424; measured value 1345.2419.

[0199] Example 24 Light release experiment of representative compounds

[0200] The compound prepared by the invention contains two types of photoprotective groups, one type is o-nitrobenzyl and coumarin structure which can be removed by ultraviolet-visible light irradiation, and the other type is heptamethine cyanine structure which can be removed by near-infrared light irradiation.

[0201] The present invention selects compound I-7 and compound II-6 as representative compounds to conduct light release experiments. The above representative compounds contain these two types of photoprotective groups and have similar basic skeletons, which can reflect that the compounds prepared by the present invention have the same photolysis properties.

[0202] Compounds I-7 and II-6 were dissolved in DMSO and prepared into a mother solution with a concentration of 10 mM, which was stored at -20°C for future use. The mother solution of the compound was diluted with a mixed solvent of methanol: PBS = 1:1 to a final concentration of 50 μM and then subjected to light treatment. For compound I-7, the wavelength of light was 808 nm; for compound II-6, the wavelength of light was 400 nm. Samples were taken at given times for HPLC analysis and UV absorption spectrum determination. The results are shown in Figure 2. Figure 1 shown.

[0203] Figure 1 A and B respectively represent the change of the UV absorption spectrum of compound I-7 under 808nm light irradiation and the release of the prototype drug BSP. C and D represent the change of the UV absorption spectrum of compound II-6 under 400nm light irradiation and the release of the prototype drug diBSP. Figure 1 It can be seen that both compounds have good photolysis properties and can be quickly and completely converted into the corresponding prototype drugs.

[0204] Therefore, the compound prepared by the present invention has good photolysis properties.

[0205] Example 25 represents the light-dependent STING agonist activity experiment of the compound in THP1-Lucia luciferase reporter gene cells

[0206] The compounds prepared in the present invention all need to release their STING agonist activity through light. Based on the excellent photolysis properties of the compounds, the present invention selects compound I-7 and compound II-6 as representative compounds to conduct light-dependent STING agonist activity experiments.

[0207] THP1-Lucia TM ISG (Invivogen: thpl-isg) cells were diluted with culture medium and 100 μL of cell suspension was inoculated into a 96-well plate so that each well contained 1×10 5cells. Then, 100 μL of the compound to be tested was added to a 96-well plate (the final concentration of the compound was 25 μM, and the final volume of each well was 200 μL), and a light-proof group and an illumination group were set up at the same time, and the dosing wells of the illumination group were exposed to light for a certain period of time. For compound I-7, the illumination wavelength was 808 nm and the illumination time was 5 min; for compound II-6, the illumination wavelength was 400 nm and the illumination time was 1 min. After the illumination, all groups continued to incubate for 24 hours in the dark at 37°C. Then, 10 μL of supernatant was aspirated into a new 96-well white plate, and 50 μL of QUANT-Luc reagent was added. After thorough mixing, the microplate reader was used for measurement immediately. The experiment set up 3 replicate wells. The test results are expressed as excitation folds (fold), which are calculated by (test well-blank well) / (negative well-blank well). The blank wells are cell culture medium, and the negative wells are dimethyl sulfoxide DMSO. The test results are as follows Figure 2 (+ represents the light-exposed group, - represents the light-protected group. The compound diBSP is a benzo[b]selenophene dimer STING agonist. Its STING agonist activity data are shown in Figure 2 B. )

[0208] It can be seen from the figure that compounds I-7 and II-6 cannot activate the STING pathway under light-proof conditions, but can release the prototype drug after corresponding light exposure and restore STING agonist activity. The two corresponding prototype drugs both showed excellent STING agonist activity in the THP1 luciferase reporter gene test and are good STING agonists. Through the light-dependent STING agonist activity of the two representative probe molecules, it can be proved that the compounds prepared by the present invention have similar effects.

[0209] The compounds prepared by the present invention can remove the photoprotective groups after being exposed to light, releasing the prototype drug, i.e., the corresponding STING agonist. Therefore, the compounds of the present invention can be used to prepare drugs for activating the cGAS-STING pathway, to prepare drugs for preventing and / or treating diseases related to the STING pathway, and to prepare immune adjuvants. In addition, the compounds of the present invention carry fluorescent groups and can be used to prepare fluorescent imaging agents.

Claims

1. A benzo[b]selenophene STING agonist fluorescent probe molecule, characterized in that: It has a structure as shown in formula (I) or formula (II): in, X is selected from o-nitrobenzyl derivatives X 1 , Coumarin Derivatives X 2 or heptamethine cyanine derivative X 3 ; Y is selected from hydrogen, X 1 , X 2 or X 3 ; and when one side Y is selected from hydrogen, the other side Y is selected from X 1 , X 2 or X 3 ;or Both sides Y are selected from X 1 , X 2 or X 3 ; o-Nitrobenzyl DerivativesX 1 , Coumarin Derivatives X 2 or heptamethine cyanine derivative X 3 for: in, R 1 is selected from hydrogen, methyl or allyl; R 2 Selected from hydrogen, hydroxy, methyl, tert-butyl, methoxy, OCH2CN, OCH2COOH, OCH2COOCH3 or para-substituted styryl; R 3 is selected from hydrogen or methoxy; R 4 is selected from hydrogen or methyl; R 5 is selected from hydrogen, halogen, methoxy or amino; R 6 R is selected from hydrogen, hydroxy, methoxy, N(CH2CH3)2, benzyloxy, benzoyloxy or pinacol boronic acid ester; 7 Selected from methyl, ethyl, benzyl, (CH2) m SO3H or (CH2) m COOH; R 8 is selected from hydrogen, methoxy, SO3H or COOH; R 9 is selected from hydrogen; or R 9 With R 8 Connected to form a benzene ring or a C5-C6 aromatic heterocycle; R 10 is selected from hydrogen or methyl; R 11 is selected from hydrogen or methyl; m is selected from 3, 4 or 5.

2. The benzo[b]selenophene STING agonist fluorescent probe molecule according to claim 1, characterized in that: R 1 is selected from hydrogen or methyl; R 2 is selected from hydrogen, hydroxy, methyl or methoxy; R 5 is selected from hydrogen, bromine or methoxy; R 6 Selected from hydrogen, hydroxy, methoxy or N(CH2CH3)2; R 7 Selected from methyl, ethyl, benzyl or (CH2) m SO3H; R 8 is selected from hydrogen, methoxy or SO3H; m is selected from 3 or 4.

3. The benzo[b]selenophene STING agonist fluorescent probe molecule according to claim 1, characterized in that Selected from:

4. Use of the benzo[b]selenophene STING agonist fluorescent probe molecule according to any one of claims 1 to 3 in the preparation of a drug for activating the cGAS-STING pathway.

5. Use of the benzo[b]selenophene STING agonist fluorescent probe molecule according to any one of claims 1 to 3 in the preparation of drugs for preventing and / or treating diseases related to the STING pathway.

6. Use of the benzo[b]selenophene STING agonist fluorescent probe molecule according to any one of claims 1 to 3 in the preparation of an immune adjuvant.

7. Use of the benzo[b]selenophene STING agonist fluorescent probe molecule according to any one of claims 1 to 3 in the preparation of an in vivo fluorescent imaging agent.

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