Bicyclol photoaffinity probe as well as preparation method and application thereof

Abstract

The invention provides a compound shown in a formula (I) or a salt or a solvate thereof, further provides a kit or a composition containing the compound or the salt or the solvate thereof, a probe containing the compound or the salt or the solvate thereof, and further provides a preparation method and application of the compound or the salt or the solvate thereof. The compound or the salt or the solvate thereof can be used as a bicyclol photoaffinity probe and is applied to target protein identification research of bicyclol. .

Description

Technical Field

[0001] The present application belongs to the field of chemical probes, and specifically relates to a bicyclic alcohol photoaffinity probe and its preparation method and application. Background Art

[0002] Bicyclol, developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences, is my country's first new class I anti-hepatitis drug with independent international intellectual property rights. It is a hexaalkoxybiphenyl compound derived from schisandra chinensis (Schisandra chinensis C), an active ingredient in the traditional Chinese medicine Schisandra chinensis, through structural modification. Studies have shown that bicyclol has a significant protective effect against liver damage caused by various factors, significantly reducing serum transaminase levels and alleviating liver pathological damage. It also exhibits some anti-hepatitis virus activity, significantly improving clinical symptoms in patients with chronic hepatitis B and C. It has a good safety profile and is free of significant toxic side effects. Bicyclol was approved for marketing in 2004 for the treatment of chronic hepatitis and non-viral liver diseases.

[0003] However, as an innovative drug derived from a natural product, bicyclol suffers from unknown targets and mechanisms of action. This lack of target necessitates the development of accurate pharmacological models at the cellular and molecular levels to evaluate the compound's activity, severely impacting the efficiency and accuracy of drug evaluation and screening. Furthermore, the lack of information on drug-target interaction patterns hinders structure-based rational drug design, complicates compound structure optimization and structure-activity relationship studies, and significantly limits the development of new anti-hepatitis drugs (e.g., those with higher selectivity and lower toxicity). Therefore, the lack of a known target has become a major constraint on the research of bicyclol and innovative anti-hepatitis drugs. Summary of the Invention

[0004] The purpose of this application is to provide a bicyclic alcohol photoaffinity probe containing a diazirine group and an alkynyl group, which is used to identify its target protein and solve the problem of unknown targets of bicyclic alcohol's anti-inflammatory and hepatoprotective effects.

[0005] The first aspect of the present application provides a compound represented by formula (I) or a salt or solvate thereof, , where R is or , L is -(CH2)n-, -(CH2)n-NHC(O)-(CH2)m- or -(CH2)nO-(CH2)n-NHC(O)-(CH2)m-, wherein each n or m is independently 1, 2, 3 or 4.

[0006] In certain embodiments, in the compound of formula (I) or a salt or solvate thereof, each n or m is independently 1, 2 or 3.

[0007] In certain embodiments, the compound of Formula (I) or salt or solvate thereof, each n or m is independently 1 or 2.

[0008] In certain embodiments, the compound of Formula (I) or salt or solvate thereof, L is , or .

[0009] In certain embodiments, the compound of Formula (I) or salt or solvate thereof, R is .

[0010] In certain embodiments, the compound of Formula (I) is selected from: .

[0011] A second aspect of the present application provides a method for preparing the compound of the first aspect of the present application or a salt or solvate thereof, comprising: subjecting a compound of Formula I-1 to an amide condensation reaction with a compound of Formula I-2 to obtain a compound of Formula (I), wherein R and L are defined as described in any of the embodiments of the first aspect of the present application, .

[0012] In certain embodiments, the method of the second aspect of the present application, R is or , and L is -(CH2)n- or -(CH2)n-NHC(O)-(CH2)m-, each n and m are defined as described in any of the embodiments of the first aspect of the present application.

[0013] A third aspect of the present application provides another method for preparing the compound of the first aspect of the present application or a salt or solvate thereof, comprising: 1) subjecting a compound of Formula I-1 to an amide condensation reaction with a compound of Formula I-3 and removing the BOC protecting group to obtain a compound of Formula I-4, 2) subjecting the compound of Formula I-4 to an amide condensation reaction with a compound of Formula I-5 to obtain a compound of Formula (I), wherein R is defined as described in any of the embodiments of the first aspect of the present application, and L is -(CH2)n-O-(CH2)n-NHC(O)-(CH2)m-, n and m are defined as described in any of the embodiments of the first aspect of the present application, .

[0014] In certain embodiments, the method of the third aspect of the present application, R is .

[0015] In certain embodiments, the method described in the second or third aspect of the present application further comprises: The bicyclic alcohol or biphenyl diester undergoes a hydrolysis reaction to hydrolyze an ester group into a carboxyl group to obtain a compound represented by formula I-1.

[0016] In certain embodiments, the method for preparing the compound or its salt or solvate comprises: (1) Using bicyclic alcohol as the starting material, the ester group is hydrolyzed to a carboxyl group through a hydrolysis reaction to obtain intermediate 1. (2) Intermediate 1 and 3-aminoethyl-3-(but-3-ynyl)diaziridine (2) undergo amide condensation reaction to obtain compound BC-1; or (1) Using 3-(3-(but-3-yn-1-yl)-3H-naphthyrazine-3-yl)propionic acid (3) as the starting material, N -tert-Butyloxycarbonyl-1,2-ethylenediamine (4) is subjected to amide condensation reaction and removal of the BOC protecting group to obtain intermediate 5. (2) Intermediate 1 and intermediate 5 undergo amide condensation reaction to obtain compound BC-2; or (1) Using 3-(3-(but-3-yn-1-yl)-3H-naphthyridin-3-yl)propionic acid (3) as the starting material, it reacts with tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (6) through an amide condensation reaction and removes the BOC protecting group to obtain intermediate 7. (2) Intermediate 1 and intermediate 7 undergo amide condensation reaction to obtain compound BC-3; or (1) Biphenyl diester is used as the starting material, and one of the ester groups is hydrolyzed into a carboxyl group to obtain intermediate 8. (2) Intermediate 8 reacts with 3-aminoethyl-3-(but-3-ynyl)diaziridine (2) to obtain compound BC-4 through amide condensation reaction; or (1) Biphenyl diester is used as the starting material, and is reacted with tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (6) through an amide condensation reaction and the BOC protecting group is removed to obtain the intermediate 9. (2) Intermediate 9 and 3-(3-(but-3-yn-1-yl)-3H-naphthyridin-3-yl)propionic acid (3) are subjected to amide condensation reaction to obtain compound BC-5. .

[0017] The fourth aspect of the present application provides a kit or composition comprising the compound or its salt or solvate according to any embodiment of the first aspect of the present application.

[0018] In certain embodiments, the kit or composition described in the fourth aspect of the present application further comprises biotin having a terminal azide group or a fluorescent dye having an azide group.

[0019] In certain embodiments, the biotin having a terminal azide group is biotin-azide, and the fluorescent dye having an azide group is TAMRA-azide.

[0020] The fifth aspect of the present application provides a probe comprising the compound or a salt or solvate thereof according to any embodiment of the first aspect of the present application.

[0021] The sixth aspect of the present application provides the use of the compound or its salt or solvate described in any embodiment of the first aspect of the present application, or the kit or composition described in any embodiment of the fourth aspect, or the probe described in any embodiment of the fifth aspect in the identification of bicyclic alcohol target proteins.

[0022] A seventh aspect of the present application provides a method for identifying a bicyclic alcohol target protein, comprising: 1) contacting the compound or salt or solvate thereof according to any embodiment of the first aspect of the present application with a target protein to be identified, 2) Detecting the binding of the compound or salt or solvate thereof according to any embodiment of the first aspect of the present application to the target protein to be identified.

[0023] As used herein, the term "salt" refers to a salt of a compound that is substantially non-toxic to living organisms. Typical salts include those prepared by reacting the compounds disclosed herein with an inorganic or organic acid or an organic or inorganic base. Such salts are known as acid addition salts and base addition salts.

[0024] It will be appreciated by those skilled in the art that most or all of the compounds disclosed in this application are capable of forming salts.

[0025] Acids commonly used to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of suitable salts can include sulfate, pyrosulfate, bisulfate, sulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, acetate, propionate, decanoate, octanoate, acrylate, formate, hydrochloride, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, α-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

[0026] Base addition salts include those derived from inorganic bases such as ammonium hydroxide or alkali metal or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0027] In addition, the methods disclosed in the present application can be implemented using solvates of the compounds or their salts. The solvates may include ethanol solvates, hydrates, and the like.

[0028] Beneficial effects of this application The present application provides a bicyclic alcohol photoaffinity probe represented by formula (I), which contains a diazirine group and an alkynyl group, wherein the diazirine group is the smallest photoaffinity group, and its combination with the alkynyl group serves as a "minimized" molecular chain, which can maintain the probe's in situ target recognition ability.

[0029] The probe can effectively label proteins.

[0030] This probe can be used to conduct ABPP research, thereby revealing the target of bicyclic alcohol, laying the foundation for the study of anti-inflammatory and hepatoprotective mechanisms and the establishment of new anti-hepatitis drug screening models, and providing a theoretical basis and support for the rational design of innovative anti-hepatitis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide further explanation of the present application and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present application, but do not constitute a limitation of the present invention.

[0032] Figure 1 Shows the results of proteomic marker detection after compounds BC-1 and BC-3 were co-incubated with HepG2 cells for 6 hours; Figure 2 Shows the results of proteomic marker detection after compounds BC-4 and BC-5 were co-incubated with HepG2 cells for 6 hours; Figure 3 The Venn diagram analysis results show the proteins significantly enriched by compound BC-4 and the proteins effectively competed by bicyclol. DETAILED DESCRIPTION

[0033] The substantive content of the present application is further described below in conjunction with the specific examples of the present application. It should be understood that the following examples are only used to illustrate the present application and are not intended to limit the scope of protection of the present application. In the following examples, if no specific conditions are specified, the conventional conditions or manufacturer's recommendations are used. The raw materials used, if the manufacturer is not specified, are all conventional products that can be obtained through commercial purchase.

[0034] Although many materials and operating methods used in the following examples are well known in the art, this application is still described in as much detail as possible. It will be clear to those skilled in the art that, unless otherwise specified, the materials and operating methods used in the following examples are well known in the art.

[0035] In the embodiment of this application, 1 H-NMR spectra were determined on a Bruker AV400 (400 Hz) NMR spectrometer (TMS as the internal standard). Mass spectra were determined on a Shimadzu GC / MS-QP2010 mass spectrometer (EI-MS) and an Agilent 100LC-MDS-Trans / SL mass spectrometer (EI-MS), respectively. Column chromatography used 300-400 mesh silica gel (Qingdao Ocean Chemical Plant), and the eluents were petroleum ether-ethyl acetate or dichloromethane-methanol. Thin-layer chromatography (TLC) used GF254 plates (Yantai Jiangyou Silica Gel Development Co., Ltd.). The TLC developing system was petroleum ether-ethyl acetate or dichloromethane-methanol. TLC was visualized using a ZF7 triple-UV analyzer (Henan Gongyi Yuhua Instrument Co., Ltd.).

[0036] Example 1: Synthesis of Intermediate 1

[0037] Bicyclic alcohol (70 mg, 0.18 mmol) was dissolved in MeOH (10 mL), and 5% aqueous KOH solution (5 mL) was added. The mixture was heated at 65°C with stirring for 6 hours. 2 N HCl was added to adjust the pH to 4-5, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated by column chromatography to obtain intermediate 1 as a white solid (65 mg, 96% yield). 1 H NMR (400 MHz, CDCl3) δ 1 H NMR (400 MHz, DMSO) δ 12.38 (s, 1H), 7.26 (s, 1H), 6.77(s, 1H), 6.03 (d, J = 9.5 Hz, 2H), 5.85 (d, J = 10.8 Hz, 2H), 5.01 (t, J = 5.1 Hz,1H), 4.27 – 4.09 (m, 2H), 3.89 (s, 3H), 3.84 (s, 3H). HRMS (ESI) m / zcalculated for C 18 H 17 O9[M+H] + : 377.0794, found: 377.0785. Example 2: Synthesis of probe BC-1

[0038] Intermediate 1 (50 mg, 0.13 mmol) was dissolved in DMF (5 mL), and 3-aminoethyl-3-(but-3-ynyl)bis(aziridine) (2, 18 mg, 0.13 mmol), HATU (52 mg, 0.14 mmol), and DIPEA (67 mg, 0.52 mmol) were added. The mixture was reacted at room temperature for 2 hours. The reaction solution was extracted with ethyl acetate / water (5 mL / 5 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the product was separated by column chromatography to obtain probe BC-1 as a white solid (49 mg, yield 76%). 1 HNMR (400 MHz, DMSO) δ 7.79 (t, J = 5.6 Hz, 1H), 6.84 (s, 1H), 6.70 (s, 1H), 5.95 (d, J= 7.5Hz, 2H), 5.85 (s, 1H), 5.77 (s, 1H), 4.26 – 4.11 (m, 2H), 3.84 (s, 3H), 3.80(s, 3H), 2.91 – 2.71 (m, 3H), 1.92 – 1.86 (m, 2H), 1.50 – 1.44 (m, 2H), 1.15– 1.10 (m, 2H). HRMS (ESI) m / z calculated for C 25 H 26 N3O8[M+H] + : 496.1642,found: 496.1633. Example 3: Synthesis of probe BC-2

[0039] 3-(3-(But-3-yn-1-yl)-3H-diaziridin-3-yl)propanoic acid (3, 8 mg, 0.048 mmol) was dissolved in DMF (1 mL) and added N -tert-Butyloxycarbonyl-1,2-ethylenediamine (4, 8 mg, 0.05 mmol), HATU (19 mg, 0.05 mmol), DIPEA (25 mg, 0.19 mmol) were reacted at room temperature for 1 hour. The reaction solution was extracted with ethyl acetate / water (3 mL / 3 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the concentrate was dissolved in dichloromethane (2 mL). Trifluoroacetic acid (1 mL) was added, and the mixture was reacted at room temperature for 2 hours. The solvent was removed under reduced pressure to obtain crude intermediate 5, which was redissolved in DMF (1 mL). Intermediate 1 (18 mg, 0.048 mmol), HATU (19 mg, 0.05 mmol), and DIPEA (25 mg, 0.19 mmol) were added, and the mixture was reacted at room temperature for 1 hour. The reaction solution was extracted with ethyl acetate / water (3 mL / 3 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated by column chromatography to obtain probe BC-2 as a white solid (9 mg). The total yield for the three steps was 34%. 1 H NMR (400 MHz, CDCl3) δ 7.03 (s, 1H), 6.88 (t, J = 5.3 Hz, 1H), 6.75 (s, 1H), 6.15(t, J = 5.2 Hz, 1H), 6.03 (d, J = 1.3 Hz, 1H), 5.99 (d, J= 1.4 Hz, 1H), 5.92 –5.88 (m, 2H), 4.58 (d, J = 11.5 Hz, 1H), 4.41 (d, J = 11.6 Hz, 1H), 3.96 (s, 6H),3.32 – 3.25 (m, 2H), 3.23 – 3.03 (m, 2H), 2.07 (s, 1H), 2.02 – 1.96 (m, 2H),1.89 – 1.83 (m, 2H), 1.82 – 1.76 (m, 2H), 1.66 – 1.59 (m, 2H). HRMS (ESI) m / zcalculated for C 28 H 31 N4O9[M+H] + : 567.2013, found: 567.2003. Example 4: Synthesis of probe BC-3

[0040] 3-(3-(But-3-yn-1-yl)-3H-bis(aziridin-3-yl)propanoic acid (3, 7 mg, 0.042 mmol) was dissolved in DMF (1 mL), and tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (6, 9 mg, 0.044 mmol), HATU (17 mg, 0.044 mmol), and DIPEA (22 mg, 0.17 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was extracted with ethyl acetate / water (3 mL / 3 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the concentrate was dissolved in dichloromethane (2 mL). Trifluoroacetic acid (1 mL) was added, and the mixture was reacted at room temperature for 2 hours. The solvent was removed under reduced pressure to obtain crude intermediate 7, which was redissolved in DMF (1 mL). Intermediate 1 (16 mg, 0.042 mmol), HATU (17 mg, 0.044 mmol), and DIPEA (22 mg, 0.17 mmol) were added, and the mixture was reacted at room temperature for 1 hour. The reaction solution was extracted with ethyl acetate / water (3 mL / 3 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated by column chromatography to obtain probe BC-3 as a white solid (7 mg). The total yield for three steps was 27%. 1 H NMR (400 MHz, CDCl3) δ 7.08 (s, 1H), 6.80 (brs, 1H), 6.70 (s,1H), 6.26 (brs, 1H), 6.02 (d,J = 17.1 Hz, 2H), 5.92 (d, J = 8.1 Hz, 2H), 4.62(d, J = 11.6 Hz, 1H), 4.41 (d, J = 11.6 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.47 – 3.13 (m, 8H), 2.03 – 1.96 (m, 3H), 1.95 – 1.88 (m, 2H), 1.85 – 1.77(m, 2H), 1.65 – 1.58 (m, 2H). HRMS (ESI) m / z calculated for C 30 H 35 N4O 10 [M+H] + :611.2275, found: 611.2283. Example 5: Synthesis of Intermediate 8

[0041] Dissolve biphenyl diacid (100 mg, 0.24 mmol) in 5% potassium hydroxide solution (5 mL), heat and stir at 100°C for 6 hours, cool to room temperature, and add concentrated hydrochloric acid to acidify to pH = 1. Filter the solid, wash with water until neutral, and dry to obtain 92 mg of biphenyl diacid intermediate in a yield of 99% as a white solid. 1 H NMR (DMSO, 400 MHz): δ ppm 3.88 (s,6H), 5.94 (s, 4H), 7.24 (s, 2H), 10.24 (s, br, 2H); MS (ESI, m / z): 389.2 [MH] - .

[0042] Dissolve biphenyldicarboxylic acid (90 mg, 0.23 mmol) in 5 mL of acetic anhydride, heat and stir at 100°C for 12 hours, remove the solvent under reduced pressure, and recrystallize the residue from toluene to obtain 80 mg of biphenyldicarboxylic anhydride (93% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz): δ ppm 3.97 (s, 6H), 6.05 (s, 2H), 6. 16 (s, 2H), 7. 02 (s, 2H).

[0043] Dissolve biphenyl anhydride (75 mg, 0.20 mmol) in 5 mL of methanol and heat with stirring at 60°C for 9 hours to obtain 77 mg of biphenyl ester acid (intermediate 8) in a yield of 95% as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.43 (s,1H), 7.35 (s, 1H), δ 5.99 – 5.94 (m, 4H), 3.96 (s, 3H), 3.95 (s, 3H), 3.66(s, 3H). HRMS (ESI) m / z calculated for C 19 H 17 O 10 [M+H] + : 405.0743, found:405.0751. Example 6: Synthesis of probe BC-4

[0044] Intermediate 8 (29 mg, 0.073 mmol) and 3-aminoethyl-3-(but-3-ynyl)bis(aziridine) (2, 10 mg, 0.073 mmol) were dissolved in DMF (3 mL). HATU (29 mg, 0.077 mmol) and DIPEA (38 mg, 0.292 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was extracted with ethyl acetate / water (5 mL / 5 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and separated by column chromatography to obtain probe BC-4 as a white solid (35 mg, yield 92%). 1 H NMR (400MHz, CDCl3) δ 7.25 (s, 1H), 7.04 (s, 1H), 6.08 (t, J = 5.4 Hz, 1H), 6.03 (d,J = 1.3 Hz, 1H), 5.98 (d, J = 1.3 Hz, 1H), 5.93 (s, 2H), 3.95 (s, 3H), 3.93(s, 3H), 3.76 (s, 3H), 3.09 – 2.89 (m, 2H), 1.98 – 1.89 (m, 3H), 1.54 – 1.47(m, 3H), 1.39 – 1.30 (m, 1H). HRMS (ESI) m / z calculated for C 26 H 26 N3O9[M+H] +: 524.1591, found: 524.1582. Example 7: Synthesis of Probe BC-5

[0045] Intermediate 8 (45 mg, 0.11 mmol) was dissolved in DMF (3 mL), tert-butyl [2-(2- aminoethoxy)ethyl]carbamate (6, 23 mg, 0.11 mmol), HATU (44 mg, 0.12 mmol), DIPEA (57 mg, 0.44 mmol) were added, and the reaction was allowed to proceed at room temperature for 1 h. The reaction was extracted with ethyl acetate / water (5 mL / 5 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the concentrate was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added, and the reaction was allowed to proceed at room temperature for 2 h. The solvent was removed under reduced pressure to give intermediate 9, which was used directly in the next step without further purification.

[0046] Intermediate 9 (30 mg, 0.05 mmol) and 3-(3-(but-3-yn-1-yl)-3H-diaziridin-3-yl)propanoic acid (3, 8 mg, 0.05 mmol) were dissolved in DMF (1 mL), HATU (20 mg, 0.053 mmol), DIPEA (26 mg, 0.2 mmol) were added, and the reaction was allowed to proceed at room temperature for 1 h. The reaction was extracted with ethyl acetate / water (3 mL / 3 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by column chromatography to give probe BC-5 as a colorless oil (25 mg, 78% yield). 1 H NMR (400 MHz, CDCl3) δ 7.23 (s, 1H), 7.10 (s, 1H), 6.43 (brs, 2H), 6.02 –5.93 (m, 4H), 3.97 (d, J = 3.2 Hz, 6H), 3.78 (s, 3H), 3.57 – 3.31 (m, 4H),3.29 – 3.13 (m, 4H), 2.04 – 1.95 (m, 3H), 1.94 – 1.88 (m, 2H), 1.87 – 1.74(m, 2H), 1.60 (t, J = 7.3 Hz, 2H). HRMS (ESI) m / z calculated for C 31 H 35 N4O 11 [M+H] + : 639.2224, found: 639.2235. Experimental Example 1: Photoaffinity labeling and in-gel fluorescence analysis of proteomes using bicyclol probes Different concentrations of bicyclic alcohol probes (probes BC-1, BC-3, BC-4, and BC-5 prepared in the examples of this application) were incubated with HepG2 cells for 6 hours, the cells were washed with PBS to remove excess probes, the cells were irradiated with ultraviolet light (365 nm) for 15 minutes, lysis buffer was added, and ultrasonic disruption was performed. The cell lysate was centrifuged, the supernatant was taken, and the protein concentration was determined by the BCA method and diluted to 2 mg / mL. 50 μL of each sample was taken, and TAMRA-azide, 1 mM BTTAA / CuSO4, and 2.5 mM sodium ascorbate were added to the sample at a final concentration of 200 μM for click chemistry reaction. The mixed solution was shaken at 25°C for 1 hour, and 13.75 μL loading buffer was added to each sample. The sample was boiled and cooled. 40 μL of each sample was taken for SDS-PAGE gel electrophoresis analysis. The fluorescence signal was detected using a fluorescence scanner ChemiDoc MP, and the protein was detected using Coomassie Brilliant Blue staining. The results are as follows: Figure 1 、 Figure 2 shown.

[0047] The results showed that for probes BC-1 and BC-3, although they could label the proteome within the probe concentration range of 1 μM to 100 μM, they did not show obvious concentration-dependent characteristics, and the DMSO group still had a strong background signal, indicating that this type of probe (containing alcohol hydroxyl groups) is not very efficient in labeling the proteome under in situ conditions of living cells ( Figure 1 ). Probes BC-4 and BC-5 can effectively label proteins, and the labeling intensity is positively correlated with the concentration. Moreover, at the same concentration, the labeling effect of BC-4 is significantly stronger than that of BC-5 ( Figure 2 ).

[0048] Experimental Example 2: Mass spectrometry-based TMT quantitative proteomics using bicyclol probes HepG2 cells were incubated with 25 µM of the bicyclol probe BC-4 for 6 hours (for the vehicle group, an equal volume of DMSO was added; for the competitive group, 250 µM bicyclol was incubated with HepG2 cells for 2 hours before adding BC-4). The cells were washed with PBS to remove excess probe, irradiated with UV light (365 nm) for 15 minutes, added with lysis buffer, and disrupted by sonication. The cell lysate was centrifuged, and the supernatant was collected for protein concentration determination using the BCA assay and diluted to 2 mg / mL. A 100 µL aliquot of each sample was added with biotin-azide (final concentrations of 200 µM), 1 mM BTTAA / CuSO4, and 2.5 mM sodium ascorbate for click chemistry. The mixed solution was shaken at 25°C for 1 hour. Proteins were precipitated with methanol-chloroform, resuspended in 0.05% SDS / PBS, and added to 200 µL of magnetic beads for enrichment at room temperature for 3 hours. The beads were washed three times with 0.05% SDS / PBS, and the supernatant was removed. The beads were resuspended in Urea / TEAB, and DTT and IAA were added sequentially to a final concentration of 10 mM and 20 mM, respectively. The beads were mixed at 35°C for 30 minutes, and the supernatant was removed. The beads were washed three times with 200 mM EPPS (pH 8.5), and protease LysC (0.5 µg / µL) was added and incubated at 37°C with rotation for 16 hours. Trypsin (0.5 µg / µL) was then added and incubated at 37°C with rotation for 6 hours. The digested samples were then added to 200 mM EPPS (pH 8.5) and labeled with TMT. Equal amounts of the corresponding samples were mixed, dried at 45°C, and desalted by HPLC. The resulting peptide samples were then resuspended in 0.1% (v / v) formic acid in water for LC-MS / MS analysis. The mass spectrometry data were analyzed using MaxQuant 1.6.5.0 software.

[0049] The BC-4 significantly enriched proteins (differential proteins between the probe group and the solvent group) and the bicyclol effectively competitive proteins (differential proteins between the probe group and the competitive group) were compared and reproducibly analyzed to draw a Venn diagram, as shown in Figure 2. Figure 3 As shown, there are 57 proteins that overlap (see Table 1 ), which are high-confidence candidate target proteins of bicyclol.

[0050] Table 1 57 high-confidence bicyclol target proteins

[0051] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to those details can be made within the scope of the application as disclosed above and as defined in the following claims. The scope of the application is to be measured only by the appended claims and their equivalents.

Claims

1. A compound represented by formula (I) or a salt or solvate thereof, , where R is or , L is -(CH2)n-, -(CH2)n-NHC(O)-(CH2)m- or -(CH2)nO-(CH2)n-NHC(O)-(CH2)m-, wherein each n or m is independently 1, 2, 3 or 4.

2. The compound according to claim 1, or a salt or solvate thereof, wherein each n or m is independently 1, 2 or 3.

3. The compound according to claim 1 or 2, or a salt or solvate thereof, wherein each n or m is independently 1 or 2.

4. The compound according to claim 3, or a salt or solvate thereof, wherein L is 、 or .

5. The compound according to claim 1 or a salt or solvate thereof, wherein the compound is selected from: 。 6. A method for preparing the compound according to any one of claims 1 to 5, or a salt or solvate thereof, comprising: The compound represented by formula I-1 is subjected to an amide condensation reaction with the compound represented by formula I-2 to obtain the compound represented by (I), wherein R and L are as defined in any one of claims 1 to 4, 。 7. A method for preparing the compound according to any one of claims 1 to 5, or a salt or solvate thereof, comprising: 1) subjecting the compound represented by formula I-1 to an amide condensation reaction with the compound represented by formula I-3 and removing the BOC protecting group to obtain the compound represented by formula I-4, 2) subjecting the compound represented by formula I-4 to an amide condensation reaction with the compound represented by formula I-5 to obtain a compound represented by formula (I), wherein R is defined as any one of claims 1 to 4, L is -(CH2)nO-(CH2)n-NHC(O)-(CH2)m-, and n and m are defined as any one of claims 1 to 4, 。 8. The method of claim 6 or 7, further comprising: The bicyclic alcohol or biphenyl diester undergoes a hydrolysis reaction to hydrolyze an ester group into a carboxyl group to obtain a compound represented by formula I-1.

9. A method for preparing the compound according to claim 5 or a salt or solvate thereof, comprising: (1) Using bicyclic alcohol as the starting material, the ester group is hydrolyzed to a carboxyl group through a hydrolysis reaction to obtain intermediate 1. (2) Intermediate 1 and 3-aminoethyl-3-(but-3-ynyl)diaziridine (2) are subjected to amide condensation reaction to obtain compound BC-1; or (1) Using 3-(3-(but-3-yn-1-yl)-3H-naphthyrazine-3-yl)propionic acid (3) as the starting material, N -tert-Butyloxycarbonyl-1,2-ethylenediamine (4) is subjected to amide condensation reaction and removal of the BOC protecting group to obtain intermediate 5. (2) Intermediate 1 and intermediate 5 undergo amide condensation reaction to obtain compound BC-2; or (1) Using 3-(3-(but-3-yn-1-yl)-3H-naphthyridin-3-yl)propionic acid (3) as the starting material, it reacts with tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (6) through an amide condensation reaction and removes the BOC protecting group to obtain intermediate 7. (2) Intermediate 1 and intermediate 7 undergo amide condensation reaction to obtain compound BC-3; or (1) Biphenyl diester is used as the starting material, and one of the ester groups is hydrolyzed into a carboxyl group to obtain intermediate 8. (2) Intermediate 8 reacts with 3-aminoethyl-3-(but-3-ynyl)diaziridine (2) to obtain compound BC-4 through amide condensation reaction; or (1) Biphenyl diester is used as the starting material, and is reacted with tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (6) through an amide condensation reaction and the BOC protecting group is removed to obtain the intermediate 9. (2) Intermediate 9 and 3-(3-(but-3-yn-1-yl)-3H-naphthyridin-3-yl)propionic acid (3) are subjected to amide condensation reaction to obtain compound BC-5. 。 10. A kit or composition comprising the compound according to any one of claims 1 to 5, or a salt or solvate thereof.

11. The kit or composition of claim 10, further comprising biotin having a terminal azide group or a fluorescent dye having an azide group. 12 . The kit or composition according to claim 11 , wherein the biotin having a terminal azide group is biotin-azide, and the fluorescent dye having an azide group is TAMRA-azide. 13 . A probe comprising the compound according to claim 1 , or a salt or solvate thereof.

14. Use of the compound or salt or solvate thereof according to any one of claims 1 to 5, the kit or composition according to any one of claims 10 to 12, or the probe according to claim 13 in identifying a bicyclol target protein.

15. A method for identifying a bicyclic alcohol target protein, comprising: 1) contacting the compound according to any one of claims 1 to 5, or a salt or solvate thereof, with a target protein to be identified, 2) Detecting the binding of the compound according to any one of claims 1 to 5, or a salt or solvate thereof, to the target protein to be identified.

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