Bicyclol photoaffinity probes, methods of making and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical probes, and particularly relates to a bicyclol photoaffinity probe and a preparation method and application thereof. BACKGROUND

[0002] Bicyclol is the first anti-hepatitis new drug with international independent intellectual property rights in China, which is developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences. It is a compound containing a hexaalkoxy biphenyl structure, which is obtained by structural modification based on the active component of Schisandra chinensis, i.e. schisantherin C. Studies have shown that bicyclol has obvious protective effect on liver injury caused by various factors, can significantly reduce serum transaminase level, and reduce liver pathological damage. At the same time, it has certain anti-hepatitis virus activity, and can well improve the clinical symptoms of patients with chronic hepatitis B and C, and has good safety without obvious toxic and side effects. Bicyclol was approved for marketing in 2004, and is clinically used for the treatment of chronic hepatitis and non-viral liver disease.

[0003] However, as an innovative drug derived from natural products, bicyclol has the problems of unknown action target and unknown action mechanism. The unknown target leads to the inability to establish an accurate pharmacological model at the cellular and molecular levels to evaluate the activity of the compound, which seriously affects the efficiency and accuracy of drug evaluation and screening. At the same time, the lack of information on drug-target interaction mode makes it impossible to carry out structure-based rational drug design, and it becomes difficult to study the structure optimization and structure-activity relationship of the compound, which greatly limits the further development of new anti-hepatitis drugs (such as drugs with higher selectivity and lower toxic side effects). Therefore, the defect of unknown target has become the main factor restricting the research of bicyclol and anti-hepatitis innovative drugs. SUMMARY

[0004] The purpose of the present application is to provide a bicyclol photoaffinity probe containing a bis-aziridine group and an alkyne group, which is used for identifying the target protein thereof, and solves the problem of unknown target of bicyclol anti-inflammatory and liver-protecting effect.

[0005] The first aspect of the present application provides a compound represented by formula (I) or a salt or solvate thereof, wherein R is or ,

[0006] L is -(CH2)n-, -(CH2)n-NHC(O)-(CH2)m- or -(CH2)n-O-(CH2)n-NHC(O)-(CH2)m-, wherein each n or m is independently 1, 2, 3 or 4.

[0007] In some embodiments, in the compound represented by formula (I) or the salt or solvate thereof, each n or m is independently 1, 2 or 3.

[0008] In certain embodiments, the compound of Formula (I) or salt or solvate thereof, each n or m is independently 1 or 2.

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

[0010] In certain embodiments, the compound of Formula (I) or salt or solvate thereof, R is .

[0011] In certain embodiments, the compound of Formula (I) is selected from:

[0012] .

[0013] 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 in any of the embodiments of the first aspect of the present application,

[0014] .

[0015] 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 in any of the embodiments of the first aspect of the present application.

[0016] 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:

[0017] 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,

[0018] 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),

[0019] wherein R is defined as 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 in any of the embodiments of the first aspect of the present application,

[0020] .

[0021] In certain embodiments, in the method of the third aspect of the application, R is .

[0022] In certain embodiments, the method of the second aspect or the third aspect of the application further comprises:

[0023] hydrolyzing bicyclol or difenidin to hydrolyze one ester group to a carboxyl group to obtain a compound shown in formula I-1.

[0024] In certain embodiments, the method for preparing the compound or a salt or solvate thereof comprises:

[0025] (1) taking bicyclol as a starting material, hydrolyzing an ester group to a carboxyl group by a hydrolysis reaction to obtain an intermediate 1,

[0026] (2) subjecting the intermediate 1 to an amide condensation reaction with 3-aminoethyl-3-(but-3-ynyl)diazepine (2) to obtain a compound BC-1;

[0027] or

[0028] (1) taking 3-(3-(but-3-yn-1-yl)-3H-phthalazin-3-yl)propionic acid (3) as a starting material, subjecting the starting material to an amide condensation reaction with tert-butyloxycarbonyl-1,2-ethanediamine (4) and removing a BOC protecting group to obtain an intermediate 5, N

[0029] (2) subjecting the intermediate 1 to an amide condensation reaction with the intermediate 5 to obtain a compound BC-2;

[0030] or

[0031] (1) taking 3-(3-(but-3-yn-1-yl)-3H-phthalazin-3-yl)propionic acid (3) as a starting material, subjecting the starting material to an amide condensation reaction with tert-butyloxycarbonyl-1,2-ethanediamine (4) and removing a BOC protecting group to obtain an intermediate 5,

[0032] (2) subjecting the intermediate 1 to an amide condensation reaction with the intermediate 5 to obtain a compound BC-2;

[0033] or

[0034] (1) taking 3-(3-(but-3-yn-1-yl)-3H-phthalazin-3-yl)propionic acid (3) as a starting material, subjecting the starting material to an amide condensation reaction with tert-butyloxycarbonyl-1,2-ethanediamine (4) and removing a BOC protecting group to obtain an intermediate 5,

[0035] (2) subjecting the intermediate 1 to an amide condensation reaction with the intermediate 5 to obtain a compound BC-2; ​

[0036] or

[0037] (1) Biphenyl disester as starting material, reacted with tert-butyl [2-(2- aminoethoxy) ethyl] carbamate (6) via amide condensation and removal of BOC protecting group to obtain intermediate 9,

[0038] (2) Intermediate 9 reacted with 3-(3-(but-3-yn-1-yl)-3H- xanthen-3-yl)propanoic acid (3) via amide condensation to obtain compound BC-5,

[0039] .

[0040] The fourth aspect of the present application provides a kit or composition comprising the compound or salt or solvate thereof according to any of the embodiments of the first aspect of the present application.

[0041] In certain embodiments, the kit or composition according to the fourth aspect of the present application further comprises biotin with a terminal azido group or a fluorescent dye with an azido group.

[0042] In certain embodiments, the biotin with a terminal azido group is Biotin-azide and the fluorescent dye with an azido group is TAMRA-azide.

[0043] The fifth aspect of the present application provides a probe comprising the compound or salt or solvate thereof according to any of the embodiments of the first aspect of the present application.

[0044] The sixth aspect of the present application provides the use of the compound or salt or solvate thereof according to any of the embodiments of the first aspect of the present application or the kit or composition according to any of the embodiments of the fourth aspect of the present application or the probe according to any of the embodiments of the fifth aspect of the present application in the identification of the bicyclol target protein.

[0045] The seventh aspect of the present application provides a method for identifying the bicyclol target protein, comprising:

[0046] 1) contacting the compound or salt or solvate thereof according to any of the embodiments of the first aspect of the present application with the target protein to be identified,

[0047] 2) detecting the binding of the compound or salt or solvate thereof according to any of the embodiments of the first aspect of the present application to the target protein to be identified.

[0048] The term "salt" used in the present application refers to a salt of a compound which is substantially non-toxic to living organisms. Typical salts include those prepared by reacting a compound disclosed in the present application with an inorganic or organic acid or an organic or inorganic base. Such salts are known as acid addition salts and base addition salts.

[0049] The skilled person will appreciate that most or all of the compounds disclosed in the present application are capable of forming salts.

[0050] Acids commonly employed to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluene sulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid and the like. Examples of suitable salts can include sulfate, pyrosulfate, bisulfate, sulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, acetate, propionate, decanoate, caprylate, 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, a-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- sulfonate, naphthalene-2-sulfonate, mandelate and the like.

[0051] Base addition salts include salts derived from inorganic bases such as ammonium hydroxide or alkali metal or alkaline earth metal hydroxides, carbonates, bicarbonates and the like. Bases used to prepare such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate and the like.

[0052] In addition, the methods disclosed in the present application can be carried out using solvate forms of the compounds or salts thereof. Solvate forms can include ethanol solvates, hydrates and the like.

[0053] Advantages of the present application

[0054] The present application provides a bicyclic alcohol photoaffinity probe represented by formula (I) containing a bis-aziridine group and an alkyne group, wherein the bis-aziridine group is the smallest volume photoaffinity group, and the combination with the alkyne group as "minimized" molecular chain can maintain the in-situ target recognition ability of the probe.

[0055] The probe can effectively label proteins.

[0056] ABPP research can be carried out using the probe, and the action targets of bicyclic alcohol can be revealed, which can lay a foundation for the mechanism research of anti-inflammatory and liver protection and the establishment of a screening model for new anti-hepatitis drugs, and provide a theoretical basis and support for the rational design of innovative anti-hepatitis drugs. BRIEF DESCRIPTION OF DRAWINGS

[0057] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0058] Figure 1 Figure 6 shows the results of detection of proteomic markers after 6 hours of co-incubation of compounds BC-1 and BC-3 with HepG2 cells;

[0059] Figure 2 Figure 7 shows the results of detection of proteomic markers after 6 hours of co-incubation of compounds BC-4 and BC-5 with HepG2 cells;

[0060] Figure 3 Figure 8 shows the results of the analysis of the significant enrichment of proteins by compound BC-4 and the effective competition of proteins by bicyclol. DETAILED DESCRIPTION

[0061] The essential content of the present application is further described below in combination with the specific examples of the present application, and it should be understood that the following examples are only used to illustrate the present application, but do not limit the protection scope of the present application. The specific conditions are not indicated in the following examples, which are carried out according to the conventional conditions or the manufacturer's recommendations. The raw materials used are not indicated by the manufacturer, which are conventional products that can be obtained by purchase in the market.

[0062] Although many of the materials and methods used in the following examples are well known in the art, the present application still describes them as much as possible. It is clear to those skilled in the art that the materials and methods used in the following examples are well known in the art if not specifically mentioned.

[0063] In the examples of the present application, 1 H-NMR nuclear magnetic resonance was measured by a Bruker AV400 type (400 HZ) nuclear magnetic resonance instrument (TMS as an internal standard), and mass spectrometry was measured by a Shimadzu GC / MS-QP2010 type mass spectrometer (EI-MS) and an Agilent 100 LC-MDS-Trans / SL type mass spectrometer (EI-MS), respectively. Column chromatography silica gel is 300-400 mesh silica gel (Qingdao Haizhuan Chemical Plant), and the eluent is a petroleum ether-ethyl acetate system or a dichloromethane-methanol system. Thin layer chromatography (TLC) uses GF254 thin layer chromatography plates (Yantai Jiangyou Silica Gel Development Co., Ltd.); the TLC developing system is a petroleum ether-ethyl acetate system or a dichloromethane-methanol system; and the TLC is irradiated and displayed under a ZF7 type three-purpose ultraviolet analyzer (Henan Gugyi Yuhua Instrument Co., Ltd.).

[0064] Example 1: Synthesis of intermediate 1

[0065]

[0066] Bicyclo alcohol (70 mg, 0.18 mmol) was dissolved in MeOH (10 mL), 5% KOH aqueous solution (5 mL) was added, and the mixture was stirred at 65 °C for 6 h. The pH was adjusted to 4-5 by adding 2 N HCl, and the mixture was extracted with dichloromethane (30 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was separated by column chromatography to give intermediate 1 as a white solid 65 mg in 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 / z calculated for C 18 H 17 O9[M+H] + : 377.0794, found: 377.0785.

[0067] Example 2: Synthesis of probe BC-1

[0068]

[0069] Intermediate 1 (50 mg, 0.13 mmol) was dissolved in DMF (5 mL), 3- aminoethyl-3-(but-3-ynyl)bisaziridine (2, 18 mg, 0.13 mmol), HATU (52 mg, 0.14 mmol), and DIPEA (67 mg, 0.52 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was extracted with ethyl acetate / water (5 mL / 5 mL), and the organic phase was combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was separated by column chromatography to give probe BC-1 as a white solid 49 mg in 76% yield. 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.

[0070] Example 3: Synthesis of probe BC-2

[0071]

[0072] Dissolve 3-(3-(but-3-yn-1-yl)-3H-bisacrididin-3-yl)propionic acid (3, 8 mg, 0.048 mmol) in DMF (1 mL), and add... N -tert-Butoxycarbonyl-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 overall yield of 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.

[0073] Example 4: Synthesis of Probe BC-3

[0074]

[0075] Dissolve 3-(3-(but-3-yn-1-yl)-3H-diazepin-3-yl)propanoic acid (3, 7 mg, 0.042 mmol) in DMF (1 mL), add [2-(2-aminoethoxy)ethyl]carbamic acid tert-butyl ester (6, 9 mg, 0.044 mmol), HATU (17 mg, 0.044 mmol), DIPEA (22 mg, 0.17 mmol), react at room temperature for 1 hour, extract the reaction with ethyl acetate / water (3 mL / 3 mL), combine the organic phases, dry over anhydrous sodium sulfate. Remove the solvent under reduced pressure, dissolve the concentrate in dichloromethane (2 mL), add trifluoroacetic acid (1 mL), react at room temperature for 2 hours, remove the solvent under reduced pressure to obtain crude intermediate 7, redissolve in DMF (1 mL), add intermediate 1 (16 mg, 0.042 mmol), HATU (17 mg, 0.044 mmol), DIPEA (22 mg, 0.17 mmol), react at room temperature for 1 hour, extract the reaction with ethyl acetate / water (3 mL / 3 mL), combine the organic phases, dry over anhydrous sodium sulfate, remove the solvent under reduced pressure, separate by column chromatography to obtain probe BC-3 as a white solid 7 mg, total yield 27% for three steps. 1H 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.

[0076] Example 5: Synthesis of intermediate 8

[0077]

[0078] Diphenyl bistrate (100 mg, 0.24 mmol) was dissolved in 5% potassium hydroxide solution (5 mL), heated and stirred at 100°C for 6 hours, cooled to room temperature, added concentrated hydrochloric acid to pH = 1, the solid was filtered, washed with water to neutral, dried to obtain diphenyl bistrate intermediate 92 mg, yield 99%, 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 [M-H] - .

[0079] Diphenyl bistrate (90 mg, 0.23 mmol) was dissolved in 5 mL of acetic anhydride, heated and stirred at 100°C for 12 hours, the solvent was removed under reduced pressure, the residue was recrystallized with toluene to obtain diphenyl anhydride 80 mg, yield 93%, white solid. 1H NMR (CDCl3, 400 MHz): δ ppm 3.97 (s, 6H), 6.05 (s, 2H), 6. 16 (s, 2H), 7. 02 (s, 2H).

[0080] Biphenyl anhydride (75 mg, 0.20 mmol) was dissolved in 5 mL of methanol and heated and stirred at 60 °C for 9 hours to obtain 77 mg of biphenyl ester acid (intermediate 8) with a yield of 95% and the product was 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.

[0081] Example 6: Synthesis of probe BC-4

[0082]

[0083] Intermediate 8 (29 mg, 0.073 mmol) and 3-aminoethyl-3-(but-3-ynyl)bisacrylidine (2, 10 mg, 0.073 mmol) were dissolved in DMF (3 mL), and 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, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the mixture was separated by column chromatography to obtain probe BC-4 as a white solid, 35 mg, with a yield of 92%. 1H NMR (400MHz, CDC13) 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.

[0084] Example 7: Synthesis of Probe BC-5

[0085]

[0086] 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 hour. 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 hours. The solvent was removed under reduced pressure to obtain intermediate 9, which was used directly in the next step without isolation.

[0087] 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 hour. 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 product was isolated by column chromatography to obtain probe BC-5 as a colorless oil (25 mg, 78% yield). 1H 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.

[0088] Experimental Example 1: Photoaffinity labeling of proteome by bicyclic alcohol probes and in-gel fluorescence analysis

[0089] Different concentrations of bicyclic alcohol probes (probes BC-1, BC-3, BC-4, BC-5 prepared in the application) were incubated with HepG2 cells for 6 hours, and the cells were washed with PBS to remove excess probes. The cells were irradiated with ultraviolet light (365 nm) for 15 minutes, and then lysis buffer was added and the cells were broken by ultrasonic. The cell lysate was centrifuged, and the supernatant was taken. The protein concentration was determined by BCA method, and was diluted to 2 mg / mL. 50 μL of each sample was taken, and 200 μM TAMRA-azide, 1 mM BTTAA / CuSO4, and 2.5 mM sodium ascorbate were added to the sample to carry out click chemistry reaction. The mixed solution was shaken at 25 °C for 1 hour, and then 13.75 μL loading buffer was added. After boiling and cooling, 40 μL of each sample was taken for SDS-PAGE gel electrophoresis analysis. The fluorescence signal was detected by fluorescence scanner ChemiDoc MP, and the protein was detected by Coomassie brilliant blue staining method. The results are shown in Figure 1 、 Figure 2 .

[0090] The results show that for probes BC-1 and BC-3, although the proteome can be labeled in the probe concentration range of 1 μM ~ 100 μM, no obvious concentration-dependent characteristics are shown, and the DMSO group still has a strong background signal, indicating that the labeling efficiency of this type of probe (containing alcohol hydroxyl) on the proteome in the in situ condition of living cells is not high. Figure 1). While probes BC-4 and BC-5 could effectively label proteins, the labeling intensity was positively correlated with the concentration, and the labeling effect of BC-4 was significantly stronger than that of BC-5 at the same concentration. Figure 2 .

[0091] Experimental Example 2: Mass spectrometry-based TMT quantitative proteomics research using bicyclic alcohol probe

[0092] HepG2 cells were incubated with 25 µM bicyclic alcohol probe BC-4 for 6 hours (for the vehicle group, an equal volume of DMSO was added; for the competition group, 250 µM bicyclic alcohol was first incubated with HepG2 cells for 2 hours, and then BC-4 was added), the cells were washed with PBS to remove excess probe, and the cells were irradiated with ultraviolet light (365 nm) for 15 minutes. Lysis buffer was added, and the cells were broken up by ultrasonic. The cell lysate was centrifuged, and the supernatant was taken. The protein concentration was determined by the BCA method, and the sample was diluted to 2 mg / mL. 100 µL of each sample was taken, and Biotin-azide, BTTAA / CuSO4, and sodium ascorbate were added to the sample at final concentrations of 200 µM, 1 mM, and 2.5 mM, respectively, to perform click chemistry reaction. The mixed solution was shaken at 25 °C for 1 hour. The proteins were precipitated with methanol-chloroform, and the protein precipitate was resuspended with 0.05% SDS / PBS. 200 µL of magnetic beads were added, and the mixture was enriched at room temperature for 3 hours. The mixture was washed with 0.05% SDS / PBS for 3 times, and the supernatant was removed. The magnetic beads were resuspended in Urea / TEAB, and DTT and IAA were added to the mixture at final concentrations of 10 mM and 20 mM, respectively. The mixture was mixed at 35 °C for 30 min, and the supernatant was removed. The magnetic beads were washed with 200 mM EPPS (pH=8.5) for 3 times. LysC (0.5 µg / µL) was added, and the mixture was incubated at 37 °C for 16 hours. Trypsin (0.5 µg / µL) was further added, and the mixture was incubated at 37 °C for 6 hours. The enzyme-digested sample was taken, and 200 mM EPPS (pH 8.5) solution was added. TMT was used for labeling. The corresponding samples were mixed in equal amounts, and dried at 45 °C. HPLC was used to remove salt. The obtained peptide sample was resuspended in a solution containing 0.1% (v / v) formic acid in water, and subjected to LC-MS / MS analysis. The data obtained by mass spectrometry were analyzed by MaxQuant 1.6.5.0 software.

[0093] The proteins significantly enriched by BC-4 (the differential proteins of the probe group and the vehicle group) and the proteins effectively competed by bicyclic alcohol (the differential proteins of the probe group and the competition group) were compared and analyzed for repeatability, and a Venn diagram was made, as shown in Figure 3 There were 57 overlapping proteins (see Table 1), which belonged to the bicyclic alcohol candidate target proteins with high confidence.

[0094] Table 1 57 high confidence bicoid targets proteins

[0095]

[0096] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. The scope of the application should be determined, therefore, not with reference to the above description, but instead with reference to the appended claims and equivalents thereof.

Claims

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

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

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

4. The compound of claim 3, or a salt thereof, wherein L is or .

5. The compound of claim 1 or a salt thereof, wherein the compound is selected from the group consisting of: 。 6. A process for preparing a compound according to any one of claims 1 to 5, or a salt thereof, comprising: amide condensation reaction of a compound represented by Formula I-1 with a compound represented by Formula I-2 to obtain a compound represented by Formula (I), wherein R and L are as defined in any one of claims 1 to 5, 。 7. A method for preparing the compound of any one of claims 1 to 5 or a salt thereof, comprising: 1) amide condensation reaction of a compound represented by Formula I-1 with a compound represented by Formula I-3 and removal of BOC protecting group to obtain a compound represented by Formula I-4, 2) amide condensation reaction of a compound represented by Formula I-4 with a compound represented by Formula I-5 to obtain a compound represented by Formula (I), wherein R is as defined in any one of claims 1 to 5, L is -(CH2)n-O-(CH2)n-NHC(O)-(CH2)m-, and n and m are as defined in any one of claims 1 to 5, 。 8. The method of claim 6 or 7, further comprising: hydrolysis reaction of diphenyl double ester to hydrolyze one ester group to carboxyl group to obtain a compound represented by Formula I-1.

9. A method for preparing the compound of claim 5 or a salt thereof, comprising: (1) diphenyl double ester as starting material, hydrolyze one ester group to carboxyl group to obtain intermediate 8, (2) amide condensation reaction of intermediate 8 with 3-amine ethyl-3-(but-3- ynyl) bisaziridine (2) to obtain compound BC-4; or (1) diphenyl double ester as starting material, amide condensation reaction with [2- (2-aminoethoxy) ethyl] carbamic acid tert-butyl ester (6) and removal of BOC protecting group to obtain intermediate 9, (2) amide condensation reaction of intermediate 9 with 3-(3-(but-3-yn-1-yl)-3H- bisaziridine-3-yl) propionic acid (3) to obtain compound BC-5, 。 10. A kit or composition comprising the compound of any one of claims 1 to 5 or a salt thereof.

11. The kit or composition of claim 10, further comprising biotin with terminal azido group or fluorescent dye with azido group.

12. The kit or composition of claim 11, wherein the biotin with terminal azido group is Biotin-azide and the fluorescent dye with azido group is TAMRA-azide.

13. A probe comprising the compound of any one of claims 1 to 5 or a salt thereof.

14. Use of the compound of any one of claims 1 to 5 or a salt thereof or the kit or composition of any one of claims 10 to 12 or the probe of claim 13 in identifying bicyclol target proteins for non-disease diagnosis and treatment purposes.

15. A method for identifying a target protein of bicyclol for non-disease diagnosis and treatment purposes, comprising: 1) contacting the compound or salt thereof according to any one of claims 1-5 with a target protein to be identified, 2) detecting the binding of the compound or salt thereof according to any one of claims 1-5 to the target protein to be identified.

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