2-(4-hydroxyphenyl) benzothiophene compound taking methylene blue analogue as protein degradation factor and application of 2-(4-hydroxyphenyl) benzothiophene compound

Abstract

The invention discloses a 2-(4-hydroxyphenyl) benzothiophene compound taking a methylene blue analogue as a protein degradation factor and application of the 2-(4-hydroxyphenyl) benzothiophene compound. According to the benzothiophene compound containing the singlet oxygen triggering group tag, a methylene blue analogue is used as a singlet oxygen triggering tag. The compound and the pharmaceutical composition containing the compound can show good reactive oxygen quantum yield under exogenous physical stimulation, have good ERalpha inhibitory activity and degradation activity, can be used as an ERalpha degradation agent, can be used for preparing drugs for treating and / or preventing ERalpha overexpression diseases such as cancers, and have good application prospects. Therefore, the method is widely applied to the medicine field. The benzothiophene compound disclosed by the invention shows excellent biological activity and druggability, and has a great drug development prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a 2-(4-hydroxyphenyl)benzothiophene compound using a methylene blue analog as a protein degradation agent and application thereof. Background Art

[0002] Targeted protein degradation (TPD) has attracted much attention for its unique mode of action by triggering endogenous proteins in a highly selective and effective manner. As a revolutionary drug development strategy, it has great development potential in overcoming drug resistance. At present, protein degradation technology is mainly a biodegradation mechanism. The target protein exists in both normal cells and diseased cancer cells. Due to its lack of selectivity for cancer cells, it will cause the target protein in all cells to degrade and disappear. Its main disadvantages are non-cancer specificity and high toxicity, which brings serious safety risks. Therefore, it is crucial to explore new protein degradation mechanisms, make them controllable, and enable them to be activated in tissues or cells required by cancer cells, in order to solve the non-cancer specificity and high toxicity problems faced by protein degradation technology.

[0003] Estrogen receptor α (ERα) plays an important role in the occurrence and development of breast cancer. Among breast cancer patients, estrogen receptor (ER)-positive breast cancer accounts for 70%, which is an important molecular target for breast cancer treatment. In patients with ER-positive breast cancer who have received long-term treatment, they show resistance to endocrine therapy in clinical practice, which leads to cancer recurrence and metastasis, and the mortality rate of breast cancer has increased year by year. In the clinical treatment of breast cancer, how to overcome the problem of drug resistance has also become a key criterion for the therapeutic effect of this target drug. For the strategy of targeted degradation of ERα target. The ubiquitin-proteasome system is one of the main degradation pathways. The most representative technology is the protein degradation targeting chimera (Proteolysis-Targeting Chimera, PROTAC), which directly binds to the E3 ubiquitin ligase through the ternary complex E3 ligand to degrade the protein by ubiquitin tag. Representative ERα degraders such as ARV-471 have entered Phase III clinical research.

[0004] For PROTAC technology, its structural characteristics lead to its large molecular weight and lack of tissue specificity, resulting in poor oral absorption and membrane permeability. At the same time, PROTAC faces difficulties in forming ternary complexes and poor triplet drugability, which still faces huge challenges in terms of oral drugability and toxic side effects. Therefore, its poor pharmacokinetic properties are the main obstacle to its drugability. Therefore, it is of great significance to explore new degradation strategies, compare the advantages and disadvantages of various degradation strategies, and study the mechanism of action and biological activity of different types of degraders. Singlet oxygen ( 1O2) can cause nonspecific oxidative damage to a variety of biological macromolecules (such as proteins, nucleic acids and lipids). 1 O2 degrades the target protein (POI) to achieve high spatiotemporal resolution, high precision, controllability and reversibility of ERα protein degradation. Compared with PROTAC, it has the advantages of lower molecular weight, better drugability, and no teratogenic risk of thalidomide derivatives. In addition, it only needs to form a binary complex, which is simpler than the formation of PROTAC's ternary complex. At the same time, in terms of structural optimization, it does not need to simultaneously optimize the two ligands that bind the drug molecule to the target protein and the E3 ligase like PROTAC. This feature is also conducive to further drugability research in the later stage, and it is a very promising targeted protein degradation technology. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a 2-(4-hydroxyphenyl)benzothiophene compound using a methylene blue analog as a protein degradation agent and its application. The compound of the present invention selects a raloxifene analog as the basic skeleton in the parent core structure, and introduces a singlet oxygen triggering group into the compound by a side chain introduction method.

[0006] The invention provides a benzothiophene compound containing a singlet oxygen trigger group label, comprising a singlet oxygen trigger group label and a benzothiophene derivative, wherein the singlet oxygen trigger group label is a methylene blue analogue.

[0007] Furthermore, the structure of the benzothiophene compound containing a singlet oxygen trigger group tag is shown in formula (I):

[0008]

[0009] Wherein, n1 is an alkyl chain or alkoxy group composed of C1-C10, and n2 is an alkyl chain or alkoxy group composed of C1-C10.

[0010] Furthermore, the benzothiophene compounds containing singlet oxygen trigger group tags include but are not limited to the following compounds:

[0011] (Z)-3-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(3-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)propanyl)propanamide 15a,

[0012] (Z)-5-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(3-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)propanyl)pentanamide 15b,

[0013] (Z)-7-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)heptylamide 15c,

[0014] (Z)-5-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)pentanamide 15d,

[0015] (Z)-3-((9-(Diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)propanamide 15e.

[0016] The present invention provides a pharmaceutical composition comprising the benzothiophene compound containing a singlet oxygen trigger group tag or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or tautomer thereof.

[0017] The present invention provides a method for preparing a benzothiophene compound containing a singlet oxygen trigger group tag, comprising the following steps:

[0018] S1: preparing a first compound, wherein the first compound is a methylene blue analogue;

[0019] S2: preparing a second compound, wherein the second compound is a benzothiophene derivative;

[0020] S3: subjecting the first compound and the second compound to an amidation reaction to obtain a benzothiophene compound containing a singlet oxygen trigger group tag.

[0021] Furthermore, the specific process of step S1 is as follows:

[0022] S1-1: 1-naphthylamine and methyl 5-bromovalerate were placed in a flask, anhydrous ethanol was added, and the mixture was heated under reflux until the reaction was completed, and the solvent was removed and purified to obtain compound 3;

[0023] S1-2: Dissolve N,N-diethyl-p-phenylenediamine in an aqueous solution of aluminum sulfate, add sodium thiosulfate and zinc chloride in sequence, cool to 0°C, add an aqueous solution of potassium dichromate, react, filter, wash, crystallize, and filter again to obtain compound 5;

[0024] S1-3: Compound 5 and compound 3 are dissolved in a methanol solution, heated, and then silver carbonate is added, refluxed and cooled to room temperature, and the solvent is removed to obtain a dark blue crude product. The dark blue crude product is dissolved in dichloromethane, washed with a saturated sodium carbonate solution, dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the solution is acidified with concentrated hydrochloric acid, and the solution is dried overnight and purified to obtain compound 6;

[0025] S1-4: Add the above compound 6 to a 1,4-dioxane solution, add sodium hydroxide for hydrolysis, extract the organic matter with dichloromethane, remove the solvent, add hydrochloric acid solution for acidification, leave overnight, and purify to obtain a methylene blue analog.

[0026] Furthermore, the specific process of step S2 is as follows:

[0027] S2-1: 4-acetoxybenzoic acid was placed in a round-bottom flask, purged with N2 three times, the mixture was cooled to 0°C, oxalyl chloride was added dropwise, and then DMF was added dropwise, stirred, the solvent was removed and dried to obtain compound 9. Compound 9 was dissolved in DCM and compound 8 was added, cooled to 0°C, three portions of aluminum chloride were added, reacted, quenched, the mixture was extracted, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and purified to obtain compound 10;

[0028] S2-2: Compound 10 was dissolved in a mixed solvent of anhydrous ethanol and water, purged with N2, sodium acetate was added, and the mixture was heated to 100°C for reflux. After the reaction was completed, the mixture was cooled to room temperature, washed, and extracted with ethyl acetate. After drying over anhydrous sodium sulfate, the solvent was removed and purified to obtain compound 11;

[0029] S2-3: heating the mixture of the above compound 11, compound 12 and cesium carbonate until the reaction is completed, cooling to room temperature, washing, extracting, drying over anhydrous sodium sulfate, removing the solvent, and purifying to obtain compound 13;

[0030] S2-4: Dissolve compound 13 in DCM, purge with N2, cool to 0°C, add BBr3 solution, continue the reaction at room temperature, add methanol to terminate the reaction, remove the solvent, dissolve the residue in ethyl acetate, wash the organic phase with saturated sodium carbonate, and extract the mixture with dichloromethane. Combine the organic phases and dry them with anhydrous sodium sulfate. After evaporating the solvent, purify the crude product to obtain benzothiophene derivatives.

[0031] The present invention also provides an application of the benzothiophene compound containing a singlet oxygen trigger group label in the preparation of a drug for targeted degradation of estrogen receptors or an anti-breast cancer drug.

[0032] The present invention also provides an estrogen receptor down-regulator, comprising one or more of the benzothiophene compounds containing a singlet oxygen trigger group tag or a pharmaceutically acceptable salt thereof.

[0033] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0034] (1) The present invention discovered that methylene blue compounds can be used as a singlet oxygen trigger tag, and synthesized a series of estrogen receptor downregulators based on a benzothiophene skeleton and mediated by a singlet oxygen trigger tag. The obtained target compounds all showed a certain ERα binding ability, and by using methylene blue analogs as singlet oxygen trigger tags, not only can the compounds obtain MCF-7 cell inhibitory activity that is superior to the parent compound (benzothiophene compound), but also can have good ERα degradation activity.

[0035] (2) The synthesis of the singlet oxygen trigger tag introduced in the present invention is simple and has a high yield. Compared with PROTAC, the molecular weight of the target molecule obtained is smaller, and it is better in terms of cell membrane permeability and drugability. By introducing methylene blue as a singlet oxygen trigger tag, a successful technical means for selective degradation of target proteins can be achieved. Its modular design strategy makes the rational design of binding pockets more widely applicable, and it is possible to obtain active compounds for non-drugable targets. At the same time, the discovery that methylene blue compounds can be used as a type of singlet oxygen trigger tag can be used as a modular strategy in the future, and also provide ideas and references for the development of new estrogen receptor degraders. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 The synthetic route of the methylene blue analogue proposed in Example 1 of the present invention;

[0038] Figure 2 The synthetic route of benzothiophene derivatives with different length linkers proposed in Example 1 of the present invention is shown in the figure;

[0039] Figure 3 The synthetic route diagram of the final product MRx with a methylene blue analogue label is proposed for Example 1 of the present invention;

[0040] Figure 4The structural formula of (Z)-3-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(3-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)propanyl)propanamide 15a of Example 2 of the present invention;

[0041] Figure 5 The structural formula of (Z)-5-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(3-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)propanyl)pentanamide (15b) of Example 3 of the present invention;

[0042] Figure 6 The structural formula of (Z)-7-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)heptylamide (15c) of Example 4 of the present invention;

[0043] Figure 7 The structural formula of (Z)-5-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)pentanamide (15d) of Example 5 of the present invention;

[0044] Figure 8 The structural formula of (Z)-3-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)propionamide (15e) of Example 6 of the present invention;

[0045] Fig. 9 is the ultraviolet absorption spectrum of compounds 15a-15e in Example 7 of the present invention;

[0046] Fig.10 is the fluorescence emission spectra of compounds 15a-15e in Example 7 of the present invention;

[0047] Fig.11 This is a graph showing the singlet oxygen content generated by light-induced generation of the target compound in Example 8 of the present invention;

[0048] Fig.12Graph showing the ability of the benzothiophene derivatives of the singlet oxygen-triggered label to degrade estrogen receptors in Example 11 of the present invention, wherein A is a protein immunoblot of cells treated with 1.0 μM 15c and light at different incubation times, B is a protein immunoblot of cells treated with different compounds and light, C is a protein immunoblot of cells co-cultured with MG-132 and GSH and 15c, respectively, and D) is the relative content of ERα;

[0049] Fig.13 11 is a graph showing the ability of benzothiophene derivatives with singlet oxygen-triggered tags to degrade estrogen receptors in an example of the present invention, A is a protein immunoblot after cells were treated with different concentrations of 15c, B is a protein immunoblot after cells were treated with light, C is a protein immunoblot after different concentrations of 15c and light treatment, D is a protein immunoblot after 1.0 μM 15c and different light treatment times, E is the relative content of ERα after 1.0 μM 15c and different light treatment times, and F is the relative content of ERα after different concentrations of 15c and light treatment.

[0050] English annotations in the attached figures: Absorbance; Wavelength; FL Intencity; Normalized Absorbance; Irradiation Time; Incubation Time; Vehicle blank control; ERα estrogen receptor α; β-actin internal reference protein; MG132 proteasome inhibitor; GSH glutathione; Grayscale; Concentration. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0052] In the design of the compound of the present invention, raloxifene analogs are selected as the basic skeleton in the parent core structure, and then a singlet oxygen triggering group is introduced into the compound by the method of introducing the side chain. The present invention marks the target protein by the combination of raloxifene analogs and estrogen receptors, and then triggers the generation of singlet oxygen under exogenous physical stimulation (such as light or ultrasound) to cause the degradation of the target protein. In the selection of the singlet oxygen triggering group, methylene blue analogs are selected as degradation tags, and ERα degradation molecules are designed and synthesized. The compound of the present invention can not only maintain the inhibitory activity of the parent compound raloxifene analogs on ERα, but also has excellent degradation activity on ERα, and provides more references for patterned design for estrogen receptor degradation.

[0053] Example 1: Preparation method of benzothiophene compounds containing singlet oxygen trigger tags of the present invention

[0054] The preparation of the benzothiophene compound containing a singlet oxygen triggered tag of the present invention comprises the following steps:

[0055] (1) Synthesis of methylene blue analogs

[0056] The synthetic routes of methylene blue analogs are shown in Figure 1 , specifically including the following steps:

[0057] 1) Synthesis of methylene blue analogue (Compound 3)

[0058] Weigh 1-naphthylamine (4 g, 14.90 mmol) and methyl 5-bromovalerate (methyl 3-bromopropionate, methyl 7-bromoheptanoate, 6.0 g, 15.35 mmol) in a 200 mL round-bottom flask, add 7 mL of anhydrous ethanol, heat and reflux for 15 h, and after TLC detection, concentrate under reduced pressure to remove the solvent, and purify by column chromatography (V 二氯甲烷 :V 石油醚 =1:1) to obtain compound 3 with a yield of 70%.

[0059] 2) Synthesis of methylene blue analogue (Compound 5)

[0060] N,N-diethyl-p-phenylenediamine (2 g, 6.21 mmol) was added to 10 mL of aluminum sulfate (4.62 g, 6.45 mmol) in aqueous solution. Sodium thiosulfate (2.35 g, 13 mmol) and zinc chloride (0.912 g, 6.36 mmol) were added to the mixture in sequence. The reaction mixture was cooled to 0°C in an ice-water bath, and 5 mL of potassium dichromate aqueous solution was slowly added dropwise over 20 min. After the reaction was continued for 2 h, the filter cake was washed with acetone, and the crude product was recrystallized in methanol and filtered again to obtain 0.98 g of dark gray solid compound 5 with a yield of 47%.

[0061] 3) Synthesis of methylene blue analogue (Compound 6)

[0062] Compound 5 (300 mg, 1.0 mmol) and compound 3 (2.3 mmol) were dissolved in 50 mL of methanol solution, placed in a round-bottom flask equipped with a magnetic stirrer and a condenser reflux device and heated to reflux temperature, silver carbonate (700 mg, 2.20 mmol) was slowly added to the reaction mixture, and reflux was continued for 30 min. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to remove the solvent to obtain a dark blue crude product. The crude product was dissolved in 50 mL of dichloromethane, washed with saturated sodium carbonate solution, and the organic phase was dried with anhydrous sodium sulfate. The anhydrous sodium sulfate was removed by filtration, and the solution was acidified with 0.6 mL of concentrated hydrochloric acid. The solution was placed in a fume hood and dried overnight. The obtained dark blue solid was purified by column chromatography (V 二氯甲烷 :V 甲醇 =49:1-9:1) was purified to obtain compound 6.

[0063] 4) Synthesis of methylene blue analogue (Compound 7)

[0064] Compound 6 (1.95 g, 1.0 mmol) was added to 50 mL of 1,4-dioxane solution under stirring, and 1 mol / L sodium hydroxide was added for hydrolysis. After stirring for 4 h, the organic matter was extracted with dichloromethane, and the solvent was removed by concentration under reduced pressure. Then 0.5 mL of 1 mol / L hydrochloric acid solution was added for acidification, and the mixture was placed in a fume hood overnight for 12 h. Compound 7 was further purified by column chromatography.

[0065] (2) Synthesis of benzothiophene derivatives with linkers of different lengths

[0066] The synthetic routes of benzothiophene derivatives with different linker lengths are shown in Figure 2 , specifically including the following steps:

[0067] 1) Synthesis of benzothiophene derivative (Compound 10)

[0068] 4-Acetoxybenzoic acid (0.80 g, 4 mmol) was added to a 50 mL round-bottom flask, purged with N2 three times, the mixture was cooled to 0°C, oxalyl chloride (1 mL, 12 mmol) was slowly added dropwise, and then a few drops of DMF were added dropwise, the mixture was stirred at room temperature for 1 h, the solvent was removed by distillation under reduced pressure and dried to obtain compound 9. Compound 9 was dissolved in 30 mL DCM and compound 8 (0.90 g, 3.2 mmol, 0.8 eq.) was added, then the mixture was cooled to 0°C under N2 atmosphere, three portions of aluminum chloride (0.97 g, 6 mmol) were added in equal amounts in batches within 10 min, and the mixture was slowly heated to room temperature and continued to react for 1 h, then ice water and 1N hydrochloric acid were slowly added to quench the mixture, the mixture was extracted with dichloromethane, the organic phase was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by column chromatography to obtain the desired yellow solid product compound 10 with a yield of 34%.

[0069] 2) Synthesis of benzothiophene derivative (Compound 11)

[0070] Compound 10 (0.80 g, 1.28 mmol, 1.0 eq.) was dissolved in a mixed solvent of anhydrous ethanol: water = 2:1, purged with N2 three times, then sodium acetate (0.75 g, 6.4 mmol, 5.0 eq.) was added, and the mixture was heated to 100°C and refluxed for 16 h. After the reaction, the mixture was cooled to room temperature, washed with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by column chromatography to obtain 0.48 g of yellow oily compound 11 with a yield of 96%.

[0071] 3) Synthesis of benzothiophene derivative (Compound 13)

[0072] A mixture of compound 11, compound 12 (0.50 mL, 2.4 mmol, 2.0 eq.) and cesium carbonate (0.65 g, 1.8 mmol, 1.5 eq.) was heated to reflux for 16 hours. After TLC detection until the reaction was completed, the mixture was cooled to room temperature, washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography to obtain 0.6 g of the desired product compound 13 with a yield of 91%.

[0073] 4) Synthesis of benzothiophene derivative (Compound 14)

[0074] Compound 13 was dissolved in DCM, purged with N2 three times, cooled to 0°C, and BBr3 solution (10.0 mL, 1.0 M in DCM) was slowly added dropwise to the mixture. The mixture was allowed to react for 2 h at room temperature, and then 7 mL of methanol was added to terminate the reaction. After the solvent was removed by concentration under reduced pressure, the residue was dissolved in 70 mL of ethyl acetate, the organic phase was washed with 50 mL of saturated sodium carbonate, and the mixture was extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After evaporating the solvent, the crude product was purified by column chromatography to obtain 0.74 g of compound 14 with a yield of 65%.

[0075] (3) Synthesis of the final product MRx labeled with a methylene blue analog

[0076] After obtaining benzothiophene derivatives 14 with different lengths of side chains, these two compounds were subjected to amidation reaction with methylene blue analogs respectively. Figure 3 , using HATU as a catalyst, DIPEA as a base, and DMF as a solvent, the final product 15 with a methylene blue analog tag containing a linker of different lengths was synthesized. Compared with PROTAC, the raw materials of this type of compound are cheap and easy to obtain, the synthesis is fast, and the operation is relatively simple.

[0077] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0078] Embodiment 2:

[0079] The structural formula of (Z)-3-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(3-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)propanyl)propanamide 15a is as follows Figure 4 As shown, the preparation steps are as follows:

[0080] Compound 7a (60 mg, 0.197 mmol) and compound 14b (60 mg, 0.197 mmol) were dissolved in 15 mL of anhydrous DMF solvent, and DIPEA (52 μL, 0.296 mmol) and TBTU (95 mg, 0.296 mmol) were added. N2 was purged three times and the reaction was carried out at room temperature for 48 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure and the mixture was purified by column chromatography (V 二氯甲烷 :V 甲醇 =12:1) to purify the final product to obtain compound 15a. 1H NMR(400MHz, Methanol-d4)δ9.03–8.94(m,1H),8.54(s,2H),8.29–8.24(m,1H),7.96(d,J=9.5Hz,1H),7.80–7.7 2(m,2H),7.46(d,J=8.8Hz,2H),7.35(d,J=8.8Hz,1H),7.32(dd,J=9.6,2.8Hz,1H),7.18(d,J=2.7Hz,1H),7.05(d ,J=8.7Hz,2H),6.82(dd,J=8.8,2.3Hz,1H),6.65(d,J=8.9Hz,2H),6.52(d,J=8.7Hz,2H),4.01(t,J=5.4Hz,2H), 3.67(t,J=7.2Hz,4H), 3.63(d,J=7.5Hz,3H), 3.54(t,J=5.3Hz,2H), 2.30(t,J=5.4Hz,2H), 1.31(t,J=7.1Hz,6H). 13 C NMR (151MHz, DMSO-d6) δ192.93,172.83,170.40,158.39,156.00,153.09,151.00,140. 61,139.57,139.52,136.94,133.68,133.15,132.29,132.10,131.44,131.24,130.02,1 29.96,129.57,124.78,124.48,124.08,123.98,123.58,117.54,116.15,115.66,114. 63,107.55,105.56,103.40,65.97,45.58,35.82,34.44,29.85,13.16.HRMS(ESI)calcd for C 47 H 42 N4O5S2[M+Na] + ,829.2494;found829.2489.

[0081] Embodiment 3:

[0082] The structural formula of (Z)-5-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(3-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)propanyl)pentanamide (15b) is as follows Figure 5 As shown, the preparation steps are as follows:

[0083] Compound 7b (62 mg, 0.143 mmol) and compound 14b (96 mg, 0.229 mmol) were dissolved in 15 mL of anhydrous DMF solvent, and DIPEA (50 μL, 0.287 mmol) and TBTU (55 mg, 0.172 mmol) were added. N2 was purged three times and the reaction was carried out at room temperature for 48 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure and the reaction mixture was purified by column chromatography (V 二氯甲烷 :V 甲醇 =12:1) to purify the final product to obtain compound 15b. 1 H NMR (400MHz, Methanol-d4) δ8.95(d,J=8.0Hz,1H),8.26(d,J=8.0Hz,1H),7.92(d,J=9.5Hz,1H),7.76(dt,J=21.0,7. 1Hz,2H),7.52(d,J=8.8Hz,2H),7.38(d,J=2.4Hz,1H),7.32–7.26(m,2H),7.15(d,J=2.2Hz,1H),7.13(d,J=2.4Hz,1H) ,7.05(d,J=8.6Hz,2H),6.78(dd,J=8.8,2.2Hz,1H),6.65(d,J=8.9Hz,2H),6.54(d,J=8.6Hz,2H),3.93(t,J=6.1Hz,2 H),3.61(dd,J=13.9,6.7Hz,6H),2.29(t,J=6.5Hz,2H),2.07–1.95(m,4H),1.89(dt,J=16.8,8.3Hz,4H),1.29(s,6H). 13 C NMR(101MHz,Methanol-d4)δ174.37,155.35,153.32,151.36,142.49,140.17,1 37.21,132.96,132.83,131.95,130.89,129.88,129.45,128.03,125.07,124.5 5,124.51,123.30,122.08,117.34,115.04,114.61,113.78,106.45,104.74,10 1.79,65.58,45.41,35.93,30.42,29.38,29.07,22.34,11.64.HRMS(ESI)calcd for C 49 H 47 N4O5S2[M+Na] + ,835.2988;found835.2993.

[0084] Embodiment 4:

[0085] The structural formula of (Z)-7-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)heptylamide (15c) is as follows Figure 6 As shown, the preparation steps are as follows:

[0086] Compound 7c (312.6 mg, 0.677 mmol) and compound 14a (284.1 mg, 0.677 mmol) were dissolved in 15 mL of anhydrous DMF solvent, and DIPEA (236 μL, 1.354 mmol) and TBTU (435 mg, 1.354 mmol) were added. N2 was purged three times and the mixture was reacted at room temperature for 48 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure and the mixture was purified by column chromatography (V 二氯甲烷 :V 甲醇 =12:1) to purify the final product to obtain compound 15c. 1 H NMR (400MHz, Methanol-d4) δ8.96(dd,J=8.2,1.4Hz,1H),8.22(d,J=8.3Hz,1H),7.92(d,J=9.5Hz,1H),7.82(ddd,J=8.3,7.1,1.2Hz,1H), 7.74(ddd,J=8.3,7.1,1.5Hz,1H),7.62–7.57(m,2H),7.30(dd,J=9.5,2.8Hz,1H),7.23(d,J=8.8Hz,1H),7.10(s,1H),7.07(dd,J=5.0,2. 5Hz,2H),7.03–6.99(m,2H),6.81–6.76(m,2H),6.71(dd,J=8.8,2.3Hz,1H),6.54–6.48(m,2H),4.02(t,J=5.2Hz,2H),3.63(q,J=7.1Hz,4 H),3.54–3.47(m,4H),2.20(t,J=7.1Hz,2H),1.77–1.70(m,2H),1.66–1.59(m,2H),1.47–1.41(m,2H),1.34(s,2H),1.30(d,J=7.1Hz,6H). 13CNMR(101MHz,DMSO-d6)δ192.97,172.98,163.16,158.37,155.96,153.44,151.13 ,140.86,139.63,137.15,132.36,132.25,131.59,130.22,130.09,129.91,125.1 0,124.69,124.15,124.05,123.67,117.57,116.16,115.67,114.88,107.57,67.1 3,45.55,44.44,38.44,35.59,28.72,28.68,26.62,25.48,13.13.HRMS(ESI)calcd for C 50 H 49 N4O5S2[M+Na] + ,849.3144; found 849.3149.

[0087] Embodiment 5:

[0088] The structural formula of (Z)-5-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)pentanamide (15d) is as follows Figure 7 As shown, the preparation steps are as follows:

[0089] Compound 7b (40 mg, 0.092 mmol) and compound 14a (66 mg, 0.163 mmol) were dissolved in 15 mL of anhydrous DMF solvent, and DIPEA (52 μL, 0.296 mmol) and TBTU (57 mg, 0.178 mmol) were added. N2 was purged three times and the reaction was carried out at room temperature for 48 h. After the reaction was completed, the solvent was concentrated under reduced pressure and the reaction mixture was purified by column chromatography (V 二氯甲烷 :V 甲醇 =12:1) to purify the final product to obtain compound 15d. 1H NMR (400MHz, DMSO-d6) δ8.52(d,J=7.2Hz,1H),8.33(d,J=6.8Hz,1H),7.64(dd,J=14.9,8 .8Hz,1H),7.57(dd,J=6.4,2.0Hz,4H),7.48–7.42(m,3H),7.16–7.09(m,1H),7.06(dd,J= 6.6,1.5Hz,2H),7.04(t,J=2.6Hz,2H),7.02(dd,J=6.6,1.4Hz,1H),6.92–6.86(m,1H),6. 75–6.70(m,1H),6.69–6.60(m,2H),3.59(s,10H),2.18(s,2H),1.67(s,4H),1.13(s,6H). 13 C NMR(101MHz,Methanol-d4)δ178.90,155.42,152.97,151.08,139.71,136.90,133.55,133.11,132.38,131.55,130.01,124.7 5,123.98,122.17,115.09,114.70,113.89,104.58,101.80,48.26,45.45,43.97,35.44,29.39,22.91,11.81.HRMS(ESI)calcd for C 48 H 45 N4O5S2[M+Na] + ,821.2831; found 821.2836.

[0090] Embodiment 6:

[0091] The structural formula of (Z)-3-((9-(diethylamino)-5H-benzo[a]cyanophenothiazin-5-ylidene)amino)-N-(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)propionamide (15e) is as follows Figure 8 As shown, the preparation steps are as follows:

[0092] Compound 7a (80 mg, 0.197 mmol) and compound 14a (80 mg, 0.197 mmol) were dissolved in 15 mL of anhydrous DMF solvent, and DIPEA (69 μL, 0.395 mmol) and TBTU (127 mg, 0.395 mmol) were added. N2 was purged three times and the reaction was carried out at room temperature for 48 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure and the reaction mixture was purified by column chromatography (V 二氯甲烷 :V 甲醇=12:1) to purify the final product to obtain compound 15e. 1 H NMR(600MHz,DMSO-d6)δ9.87(s,2H),8.49(d,J=7.5Hz,1H),8.24(d,J=7.9Hz,1H),8.19(s,1H), 7.59(d,J=8.1Hz,2H),7.54–7.50(m,1H),7.42(dd,J=17.4,8.6Hz,2H),7.35(s,1H),7.24(d,J=8 .7Hz,1H),7.16(d,J=8.2Hz,2H),7.07(s,1H),6.85(d,J=8.6Hz,1H),6.77(d,J=8.5Hz,2H),6.7 3–6.64(m,4H),3.90(s,2H),3.82(s,2H),3.45–3.40(m,6H),2.52(s,2H),1.12(t,J=6.8Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ192.95,171.91,163.07,158.29,155.91,153.61,148.70,140.73,139.68,132.72,132.63,132.23,130.13,130.10,13 0.07,129.72,129.65,124.75,124.38,124.19,123.73,116.14,115.65,114.70,107.55,105.75,67.15,44.50,38.45,12.96.HRMS(ESI)calcd forC 46 H 41 N4O5S2[M+Na] + ,793.2518; found 793.2522.

[0093] Example 7: Relative affinity determination of compounds

[0094] The affinity of the target compound to ERα was determined by fluorescence polarization. The affinity of the compound was the relative value of the affinity of endogenous E2, and the value of the affinity of E2 to the receptor was set to RBA = 100%. In a 384-well plate, 20 μL of potassium phosphate buffer containing 0.8 μM Eα protein, 150 nM fluorescent ligand and 2.4 μg bovine immunoglobulin was added, and then 20 μL of the target compound solution was added. The compound concentration gradient was 3.16×10 -4 M, 1×10 -4 M, 3.16×10 -5 M, 1×10 -5 M, 3.16×10-6 M, 1×10 -6 M, 3.16×10 -7 M, 1×10 -7 M, 3.16×10 -8 M, 1×10 -8 M, 3.16×10 -9 M. After being placed in a dark place at room temperature for 2 hours, the plate was read on an ELISA reader, and the wavelength at 485 nm was selected as the main wavelength and the wavelength at 528 nm was selected as the reference wavelength. The experimental results were analyzed according to the formula receptor affinity RBA = test substance K i / estradiol K i ×100 to calculate the RBA (Relatively binding affinity) value of each compound, and the results are shown in Table 1. The affinity of the compounds prepared by the present invention to ERα is shown in Table 1, where K d In the value, + represents >1uM, ++ represents 100nM-1uM, +++ represents 10nM-100nM, ++++ represents <10nM, and ND represents not tested. In the RBA value, + represents <1, ++ represents 1-5, +++ represents 5-10, ++++ represents >10, and ND represents not tested.

[0095] Table 1 Relative binding affinities (RBA) of target compounds 15a-15e to ERα and ERβ a

[0096]

[0097] a The relative binding affinity was measured by fluorescence polarization and expressed as IC 50 Estradiol / IC 50 Compound x 100 ± SD (binding affinity of estradiol was set as 100%).

[0098] This example tests the binding affinity of the above five compounds to ERα, and the results are shown in Table 1. Among compounds 15a-15e, the final products obtained all have good binding affinity to ERα, which may be related to the correct positioning of the compounds in the ERα pocket, with the side chain partially exposed outside the pocket. In addition, the obtained compounds 15a-15e can maintain the property of the parent compound benzothiophene as an ER ligand, laying a foundation for the compounds to exert their biological effects.

[0099] Example 8: Fluorescence spectroscopy of compounds

[0100] The ultraviolet absorption spectrum of the target compound is measured by an ultraviolet spectrophotometer, and the maximum absorption wavelength measured by the target compound is used as the excitation wavelength, and the fluorescence emission spectrum of the target compound is measured by a fluorescence spectrophotometer. This example first tests the ultraviolet absorption spectra of the above five compounds in PBS, and the results are as follows: Fig. 9 As shown, compounds 15a-15e have strong absorption in the near-infrared region, with a maximum absorption wavelength of 630 nm. Based on the maximum absorption wavelengths of compounds 15a-15e, their fluorescence emission spectra were tested, and the results are shown in FIG. Fig.10 As shown, under excitation at a wavelength of 630 nm, compounds 15a-15e have a maximum emission wavelength at 720 nm.

[0101] Example 9: Test of the content of reactive oxygen species generated by light-induced generation of compounds in vitro

[0102] The in vitro light-induced singlet oxygen content of the target compound is determined by the photobleaching characteristics of ABDA. ABDA is a singlet oxygen indicator that can react with singlet oxygen to generate corresponding endoperoxides through photobleaching, resulting in a decrease in its absorption wavelength at 400nm. The target compound to be tested is added to a 50μM / L ABDA solution, and the concentration of the target compound is 5μM / L. After irradiation with light of 660nm wavelength for 1min, 2min, 3min, 4min, and 5min, the decrease in the wavelength of 400nm of the sample prepared with the target compound is measured using an ultraviolet spectrophotometer. The sample at 0min of illumination is used as a reference to calculate the content of singlet oxygen produced by light-induced in vitro of the target compound. The ultraviolet absorption spectrum is as follows: Fig.11 The light-induced singlet oxygen content is shown in Table 2.

[0103] Table 2 Singlet oxygen generation of target compounds

[0104]

[0105] This example evaluates the ability of the above five compounds to produce singlet oxygen in vitro by light induction. Compounds 15a-15e labeled with methylene blue analogs all have significant ability to produce singlet oxygen in vitro by light induction at a wavelength of 660 nm for 5 minutes, among which compound 15a has the best effect in generating singlet oxygen and inducing the decrease of ABDA at a wavelength of 400 nm.

[0106] Example 10: Cell viability test

[0107] MCF-7 cells were cultured in DMEM liquid medium containing phenol red and 10% fetal bovine serum. When the cell density reached 80% to 90%, the cells were digested and the cell suspension was plated in a 96-well cell culture plate using DMEM medium without phenol red and 10% FBS. After the cells were completely attached to the plate, the original culture medium was discarded and 100 μL of fresh compound solution prepared in DMEM medium containing 10% FBS was added to each well. The compound concentration gradient was 1×10 -7.5 M, 1×10 -7 M, 1×10 -6.5 M, 1×10 -6 M, 1×10 - 5.5 M, 1×10 -5 M, 1×10 -4.5 M, 1×10 -4 M. After drug treatment, culture for 4 days directly or 6 hours after drug treatment, light was irradiated for 2 minutes and then cultured for 4 days. The culture plate was removed, the culture medium was aspirated, 100 μL of CCK8 working solution was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for 1.5-2 hours. The plate was read on an ELISA reader, and the wavelength of 450 nm was selected as the reference wavelength. The experimental results were analyzed and the IC 50 The results are shown in Table 3. The activities of the compounds prepared by the present invention on MCF-7 cells are shown in Table 3, where + represents >50uM, ++ represents 1uM-50uM, +++ represents 1uM-100nM, ++++ represents <100nM, and ND represents not tested.

[0108] Table 3 Effects of target compounds 15a-15e on the viability of breast cancer cells MCF-7

[0109]

[0110] a IC 50 The data are the mean ± SD of at least three independent experiments.

[0111] This example evaluates the effects of the above four compounds on the viability of MCF-7 cells. Compounds 15a-5e labeled with methylene blue analogs all have a certain effect on the viability of MCF-7 cells, and their activity is better under 2 minutes of illumination than without illumination.

[0112] Example 11: ER degradation activity assay

[0113] In order to prove that the synthesized benzothiophene derivatives with singlet oxygen trigger tags have the ability to degrade estrogen receptors, and to analyze the effect of chain length on protein degradation activity. This example takes the three compounds 15a, 15b, and 15c as examples. MCF-7 was treated with these three compounds (1 μM) under light and dark conditions for 24 hours, and then all proteins were extracted. The effect of the compounds on ERα degradation was analyzed by western blotting. The results are shown in Fig.12 , 13 shown.

[0114] pass Fig.12 As shown in A, after 1.0 μM compound 15c was treated with MCF-7 cells under 660 nm wavelength light conditions for 1 min, ERα was completely degraded at 48 h, and the degradation could continue for 24 h without obvious recovery. Fig.12 B shows that compounds 15a, 15b, and 15c all have degradation activity on ERα after 5 minutes of light treatment. Fig.12 As shown in C and D, ERα will not be degraded under light-only conditions. Compound 15c can significantly degrade ERα under light conditions. After the addition of the proteasome inhibitor MG-132, ERα can still be degraded, but the degradation efficiency is significantly reduced, indicating that the degradation pathway of ERα by compound 15c through ubiquitination is reversed. After the addition of the reactive oxygen scavenger GSH, compound 15c can hardly degrade ERα under light conditions. The above results show that compound 15c specifically degrades ERα through the generation of reactive oxygen species and ubiquitination pathways.

[0115] MCF-7 cells were cultured with different doses of 15c for a certain period of time, and then treated with or without light. Fig.13 As can be seen from A, only 15c treatment of cells has almost no effect on the expression of ERα, and only light treatment of cells has no effect on the expression of ERα. Fig.13 As shown in B. Fig.13 C shows that when 15c is activated by light, the expression of ERα is significantly reduced, and the expression of ERα decreases with the increase of 15c concentration, which proves that 15c has a concentration-dependent effect on the degradation of ERα. At a concentration of 50 μM, the expression level of ERα is reduced to 10%. Fig.13 The results in B exclude the possibility that ERα degradation is induced only by light. Fig.13 The results of D showed that when the concentration of 15c was constant, the degradation efficiency of ERα increased with the increase of illumination time. When the illumination was 5 minutes, the expression level of ERα was only 6%, which confirmed that the degradation of ERα by 15c was also dependent on the illumination time.

[0116] By comparing different compounds containing methylene blue analog tags, it was found that the introduction of singlet oxygen triggered tags can indeed enable the compounds to obtain better ERα degradation activity, verifying the feasibility of methylene blue analog tags as singlet oxygen triggered tags.

[0117] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A benzothiophene compound containing a singlet oxygen trigger group tag, characterized in that: The invention comprises a singlet oxygen trigger group tag and a benzothiophene derivative, wherein the singlet oxygen trigger group tag is a methylene blue analogue.

2. The benzothiophene compound containing a singlet oxygen trigger group tag according to claim 1, characterized in that: The structure of the benzothiophene compound containing a singlet oxygen trigger group tag is shown in formula (I): Wherein, n1 is an alkyl chain or alkoxy group composed of C1-C10, and n2 is an alkyl chain or alkoxy group composed of C1-C10.

3. A pharmaceutical composition, characterized in that A benzothiophene compound containing a singlet oxygen trigger group tag as claimed in claim 1 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or tautomer thereof.

4. A method for preparing a benzothiophene compound containing a singlet oxygen trigger group tag according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1: preparing a first compound, wherein the first compound is a methylene blue analogue; S2: preparing a second compound, wherein the second compound is a benzothiophene derivative; S3: subjecting the first compound and the second compound to an amidation reaction to obtain a benzothiophene compound containing a singlet oxygen trigger group tag.

5. The preparation method according to claim 4, characterized in that: The specific process of step S1 is: S1-1: 1-naphthylamine and methyl 5-bromovalerate were placed in a flask, anhydrous ethanol was added, and the mixture was heated under reflux until the reaction was completed, and the solvent was removed and purified to obtain compound 3; S1-2: Dissolve N,N-diethyl-p-phenylenediamine in an aqueous solution of aluminum sulfate, add sodium thiosulfate and zinc chloride in sequence, cool to 0°C, add an aqueous solution of potassium dichromate, react, filter, wash, crystallize, and filter again to obtain compound 5; S1-3: Compound 5 and compound 3 are dissolved in a methanol solution, heated, and then silver carbonate is added, refluxed and cooled to room temperature, and the solvent is removed to obtain a dark blue crude product. The dark blue crude product is dissolved in dichloromethane, washed with a saturated sodium carbonate solution, dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the solution is acidified with concentrated hydrochloric acid, and the solution is dried overnight and purified to obtain compound 6; S1-4: Add the above compound 6 to a 1,4-dioxane solution, add sodium hydroxide for hydrolysis, extract the organic matter with dichloromethane, remove the solvent, add hydrochloric acid solution for acidification, leave overnight, and purify to obtain a methylene blue analog.

6. The preparation method according to claim 4, characterized in that: The specific process of step S2 is: S2-1: 4-Acetoxybenzoic acid was placed in a round-bottom flask, purged with N2 three times, the mixture was cooled to 0°C, oxalyl chloride was added dropwise, and then DMF was added dropwise, stirred, the solvent was removed and dried to obtain compound 9. Compound 9 was dissolved in DCM and compound 8 was added, cooled to 0°C, three equal portions of aluminum chloride were added in batches, reacted, quenched, the mixture was extracted, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and then purified to obtain compound 10; S2-2: Compound 10 was dissolved in a mixed solvent of anhydrous ethanol and water, purged with N2, sodium acetate was added, and the mixture was heated to 100°C for reflux. After the reaction was completed, the mixture was cooled to room temperature, washed, and extracted with ethyl acetate. After drying over anhydrous sodium sulfate, the solvent was removed and purified to obtain compound 11; S2-3: heating the mixture of the above compound 11, compound 12 and cesium carbonate until the reaction is completed, cooling to room temperature, washing, extracting, drying over anhydrous sodium sulfate, removing the solvent, and purifying to obtain compound 13; S2-4: Dissolve compound 13 in DCM, purge with N2, cool to 0°C, add BBr3 solution, continue the reaction at room temperature, add methanol to terminate the reaction, remove the solvent, dissolve the residue in ethyl acetate, wash the organic phase with saturated sodium carbonate, and extract the mixture with dichloromethane. Combine the organic phases and dry them with anhydrous sodium sulfate. After evaporating the solvent, purify the crude product to obtain benzothiophene derivatives.

7. Use of the benzothiophene compound containing a singlet oxygen trigger group tag according to any one of claims 1 to 2 in the preparation of a drug for targeted degradation of estrogen receptors or an anti-breast cancer drug.

8. An estrogen receptor down-regulator, characterized in that Comprising one or more benzothiophene compounds containing singlet oxygen trigger group tags as claimed in claim 1 or pharmaceutically acceptable salts thereof.

Images