A photoaffinity probe targeting cyclin-dependent kinase 9 and its preparation method and application

Abstract

The present application relates to the technical field of photoaffinity probes, and in particular, to a photoaffinity probe targeting cyclin-dependent kinase 9, and its preparation method and application, which can screen CDK9 inhibitors and identify CDK9 target proteins. The preparation method of the photoaffinity probe comprises: reacting (1S, 3R)-3-(tert-butyloxycarbonylamino)cyclohexanecarboxylic acid, N,N-dimethylformamide, N,N'-carbonyldiimidazole and ammonium acetate to obtain compound I; reacting 4-fluoro-2-methoxyphenylborane, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) to obtain compound II; reacting compound I and compound II to obtain compound III; reacting compound III to obtain compound IV; reacting compound IV and 2-(3-butynylaziridine-3-yl)acetic acid to obtain a photoaffinity probe targeting cyclin-dependent kinase 9.

Description

Technical Field

[0001] The present application relates to the technical field of photoaffinity probes, and in particular to a photoaffinity probe targeting cyclin-dependent kinase 9, and a preparation method and application thereof. Background Art

[0002] Cyclin-dependent kinases (CDKs) are a class of serine / threonine protein kinases that are crucial factors in cell cycle regulation, acting synergistically with cyclins (Genome Biology 2014, 15, 122-131). The human genome encodes over 20 CDKs and over 30 cyclins. Among them, CDK9 (cyclin-dependent kinase 9) and its related cyclin T are core molecules involved in transcriptional regulation and RNA processing. CDK9 is the catalytic subunit of the positive transcription elongation factor P-TEFb. When negative transcription elongation factors (NELF and N-TEFs) negatively regulate cellular transcription, positive P-TEFb participates in the transcription elongation inhibition system of NELF and N-TEFs, disengaging the negative elongation factors and allowing transcription to continue. Therefore, by inhibiting CDK9, transcription can be blocked, leading to apoptosis of tumor cells (Nat. Rev. Cancer 2013, 13, 299-314). Therefore, the development of CDK9 inhibitors has become one of the most important targets for the industry to expand the scope of CDK family inhibitors.

[0003] Activity-based protein profiling (ABPP) is an emerging technology platform for systematically identifying proteins with specific functions in complex biological systems. It has been widely applied in protein functional analysis, identification of active small molecule target proteins, and screening for small molecule inhibitors. The core of ABPP technology lies in the use of chemical probes to label functionally active proteins in the proteome (Chem. Soc. Rev. 2010, 39, 1302). Chemical probes primarily consist of an "active group" that selectively binds to specific proteins and a "bioorthogonal group" that can be attached to biotin or fluorescent groups using "click chemistry." Active molecular probes that bind non-covalently incorporate photocrosslinking groups. UV irradiation generates free radicals that crosslink with nearby amino acids, achieving covalent labeling of the probe with the target protein. When applying ABPP to the screening and optimization of small molecule compounds targeting proteins, the binding ability of the small molecule to the target can be determined by competitively blocking the chemical probe's signal, which changes the labeling signal.

[0004] Compared with traditional enzyme activity determination methods, screening methods based on active molecular probes have the following advantages: (1) Traditional enzyme activity experiments require the purification of target proteins and cannot be directly labeled in the proteome; (2) Enzyme activity experiments need to detect the conversion of substrate molecules to product molecules based on the specific catalytic function of the target protein. ABPP is based on the labeling of enzyme activity centers by active molecular probes and can be performed without knowing the substrates and products of the target protein; (3) Traditional enzyme activity experiments can only screen a single target protein, and thus cannot guarantee the specificity of the small molecules screened. Active molecular probes can be studied in the proteome, so the potency and specificity of the inhibitor can be monitored simultaneously through the labeling results, helping to optimize the acquisition of small molecule compounds with strong inhibitory potency and high specificity.

[0005] Therefore, we designed and synthesized a photoaffinity-active chemical probe targeting CDK9 and verified its target CDK9, providing a new means for the screening of CDK9 inhibitors and the identification of CDK9 target proteins. Summary of the Invention

[0006] In order to screen CDK9 inhibitors and identify CDK9 target proteins, the present application provides a photoaffinity probe targeting cyclin-dependent kinase 9, and a preparation method and application thereof.

[0007] The embodiment of the present application is implemented as follows:

[0008] The present invention provides a photoaffinity probe targeting cyclin-dependent kinase 9, which has the following structure:

[0009]

[0010] The present application also provides a method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9, which is used to prepare the photoaffinity probe targeting cyclin-dependent kinase 9, comprising the following steps:

[0011] (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid, N,N-dimethylformamide, N,N'-carbonyldiimidazole and ammonium acetate are reacted under first preset reaction conditions, and after the reaction is completed, a first reaction solution is obtained;

[0012] The first reaction solution is post-treated and purified to obtain compound I;

[0013] 4-fluoro-2-methoxyphenylborane, dioxane, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) are reacted under second preset conditions to obtain a second reaction solution after the reaction is completed;

[0014] The second reaction solution is post-treated and purified to obtain compound II;

[0015] Taking the compound I, the compound II, dioxane, a catalyst, tetrakis(triphenylphosphine palladium) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene, reacting under third preset conditions, and obtaining a third reaction solution after the reaction is completed;

[0016] The third reaction solution is post-treated and purified to obtain compound III;

[0017] The compound III and trifluoroacetic acid are reacted under fourth preset conditions, and after the reaction is completed, a fourth reaction solution is obtained;

[0018] The fourth reaction solution is post-treated and purified to obtain compound IV;

[0019] The compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are reacted under the fifth preset conditions. After the reaction is completed, a fifth reaction solution is obtained.

[0020] Post-treating the fifth reaction solution and purifying it to obtain compound V, a photoaffinity probe targeting cyclin-dependent kinase 9;

[0021]

[0022]

[0023] In some embodiments, when the reaction is carried out under the first preset reaction conditions, the molar ratio of (1S,3R)-3-(tert-butyloxycarbonylamino)cyclohexanecarboxylic acid, N,N'-carbonyldiimidazole and ammonium acetate is 1:2.8-3.2:6.8-7.2;

[0024] When the reaction is carried out under the second preset reaction conditions, the molar ratio of the 4-fluoro-2-methoxyphenylboronic acid, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) is 1:0.8-1.2:0.03-0.07;

[0025] When the reaction is carried out under the third preset reaction conditions, the molar ratio of compound I, compound II, cesium carbonate, tetrakis(triphenylphosphine palladium) and 4,5-diphenylphosphine-9,9-dimethylxanthene is 1:0.8-1.2:0.03-0.07-0.08-0.12;

[0026] When the reaction is carried out under the fifth preset reaction conditions, the molar ratio of compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.0~1.4:1.3~1.7:2.3~2.7:1.3~1.7.

[0027] In some embodiments, the first reaction solution is post-treated and purified to obtain Compound I, comprising:

[0028] Add ice water to the first reaction solution, stir evenly, and then filter to obtain a filter cake;

[0029] The filter cake is washed with water and dried in sequence to obtain the compound I.

[0030] In some embodiments, the second reaction solution is post-treated and purified to obtain Compound II, comprising:

[0031] Adding pure water to the second reaction solution, then adding dichloromethane for extraction and combining the first organic phase;

[0032] The first organic phase is dried and concentrated in sequence to obtain a crude compound II;

[0033] The crude compound II is purified by column chromatography to obtain the compound II.

[0034] In some embodiments, the third reaction solution is post-treated and purified to obtain compound III, comprising:

[0035] Adding pure water to the third reaction solution, then adding ethyl acetate for extraction and combining the second organic phase;

[0036] The second organic phase was dried over anhydrous sodium sulfate to obtain a crude compound III;

[0037] The crude compound III is purified by column chromatography to obtain the compound III.

[0038] In some embodiments, the fourth reaction solution is post-treated and purified to obtain compound IV, comprising:

[0039] adding dichloromethane to the fourth reaction solution, and then adding a saturated sodium carbonate aqueous solution for washing to obtain a third organic phase;

[0040] The third organic phase is dried and concentrated in sequence to obtain the compound IV.

[0041] In some embodiments, the fifth reaction solution is post-treated and purified to obtain Compound V, a photoaffinity probe targeting cyclin-dependent kinase 9, comprising:

[0042] Adding pure water to the fifth reaction solution, then adding dichloromethane for extraction and combining the fourth organic phase;

[0043] The fourth organic phase is dried and concentrated in sequence to obtain a crude compound V;

[0044] The crude compound V is purified by column chromatography to obtain the compound V, which is the photoaffinity probe targeting cyclin-dependent kinase 9.

[0045] In some embodiments, the first preset reaction conditions include a reaction temperature of 20°C to 25°C and a reaction time of 10 hours to 14 hours;

[0046] The second preset reaction conditions include a reaction temperature of 100° C. to 120° C., a reaction time of 10 hours to 14 hours, and reflux under nitrogen protection;

[0047] The third preset reaction conditions include a reaction temperature of 100° C. to 120° C. and a reaction time of 10 hours to 14 hours;

[0048] The fourth preset reaction conditions include a reaction temperature of 20°C to 25°C and a reaction time of 10 hours to 14 hours;

[0049] The fifth preset reaction conditions include a reaction temperature of 20° C. to 25° C. and a reaction time of 10 hours to 14 hours.

[0050] The embodiments of the present application also provide an application of a photoaffinity probe targeting cyclin-dependent kinase 9, including the application of the photoaffinity probe targeting cyclin-dependent kinase 9 in screening CDK9 inhibitors and identifying CDK9 target proteins.

[0051] Beneficial effects of the present application: The photoaffinity probe targeting cyclin-dependent kinase 9 provided by the present application includes an active group, a photocrosslinking group and a bioorthogonal group.

[0052] Among them, the CDK9 inhibitor structure serves as an active group, enabling the photoaffinity probe molecule to enter the active center of the CDK9 target protein so as to perform subsequent photoaffinity reactions using the photocrosslinking group;

[0053] The photocrosslinking group, namely the diazirine structure, can generate free radicals under the excitation of ultraviolet light, which undergo insertion crosslinking reactions with its adjacent CH or CC bonds, thereby covalently capturing the CDK9 target protein. The diazirine structure is small in size, avoiding the shortcomings of the photoaffinity probe molecule, such as poor solubility and poor cell permeability.

[0054] The bioorthogonal group, i.e., the alkyne group, can be used for subsequent coupling with biotin or fluorescein to separate and enrich proteins labeled with photoaffinity probes or perform fluorescence imaging, thereby identifying the signal labeled by the photoaffinity probes;

[0055] Based on the above, the photoaffinity probe targeting cyclin-dependent kinase 9 provided in the present application can achieve the purpose of screening CDK9 inhibitors and identifying CDK9 target proteins.

[0056] The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 provided in the present application has mild reaction conditions and a simple process, and can quickly prepare a photoaffinity probe targeting cyclin-dependent kinase 9. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0058] Figure 1 The photoaffinity probe targeting cyclin-dependent kinase 9 prepared in the embodiment of the present application 1 H NMR spectrum;

[0059] Figure 2 The photoaffinity probe targeting cyclin-dependent kinase 9 prepared in the embodiment of the present application 13 C NMR spectrum;

[0060] Figure 3 Results of in-gel fluorescence analysis of a photoaffinity probe targeting cyclin-dependent kinase 9;

[0061] Figure 4 This is the validation result of the target of CDK9 Probe based on western blotting. DETAILED DESCRIPTION

[0062] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0063] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0064] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0065] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0066] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0067] The following examples were prepared using conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art. In the present specification and the following examples, unless otherwise specified, "%" indicates percentage by weight, "part" indicates parts by weight, and ratios are expressed by weight.

[0068] Example 1

[0069] The present application provides a photoaffinity probe targeting cyclin-dependent kinase 9, the structural formula of which is:

[0070]

[0071] Example 2 Preparation of a Photoaffinity Probe Targeting Cyclin-Dependent Kinase 9

[0072] This embodiment provides a method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9, comprising the following steps:

[0073] Synthesis of compound Ⅰ:

[0074] To a 50 mL three-necked flask charged with (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid (1.00 g, 4.11 mmol, 1.00 eq), 10 mL of N,N-dimethylformamide (DMF) was added, followed by the addition of N,N'-carbonyldiimidazole (CDI, 2.00 g, 12.33 mmol, 3.00 eq). The mixture was stirred at room temperature for 4 hours. After the reaction solution in the three-necked flask was cooled to 0°C, ammonium acetate (2.20 g, 28.77 mmol, 7.00 eq) and 0.7 mL of dichloromethane were added. The mixture was stirred at 20°C to 25°C for 12 hours. After the reaction was completed, a first reaction solution was obtained; wherein the molar ratio of (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid, N,N'-carbonyldiimidazole, and ammonium acetate was 1:3:7.

[0075] Add 50 mL of ice water to the first reaction solution, stir evenly, and filter to obtain a filter cake;

[0076] The filter cake was washed with water and dried in sequence to obtain 745 mg of a white solid, Compound I. The yield of Compound I was calculated to be 74.80%.

[0077]

[0078] Synthesis of compound II:

[0079] To an eggplant-shaped flask containing 4-fluoro-2-methoxyphenylboronic acid (4.40 g, 25.89 mmol, 1.00 eq), 100 mL of dioxane, 2-chloro-4-bromopyridine (5.00 g, 25.89 mmol, 1.00 eq) and 32 mL of saturated aqueous sodium carbonate solution were added, and under the protection of replacement nitrogen, tetrakis(triphenylphosphine palladium) (chemical formula: Pd(PPh3)4, 1.50 g, 1.30 mmol, 0.05 eq) was added, and the mixture was reacted at 110°C with reflux for 12 hours. After the reaction, a second reaction solution was obtained; wherein, the molar ratio of 4-fluoro-2-methoxyphenylboronic acid, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) was 1:1:0.05.

[0080] 50 mL of pure water was added to the second reaction solution, and then dichloromethane was added for extraction and the first organic phase was combined. The extraction was performed three times, with 30 mL of dichloromethane used for each extraction.

[0081] The first organic phase is dried and concentrated in sequence to obtain a crude compound II, wherein the organic phase is dried using anhydrous sodium sulfate;

[0082] The crude compound II was purified by column chromatography to obtain 4.52 g of a white solid, namely compound II. The yield of compound II was calculated to be 73.74%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 93:7.

[0083] Synthesis of compound III:

[0084]

[0085] To an eggplant-shaped flask containing compound I (500 mg, 2.06 mmol, 1.00 eq) were added 10 mL of dioxane, compound II (490 mg, 2.06 mmol, 1.00 eq), and catalyst cesium carbonate (chemical formula Cs2CO3, amount 1.40 g, 4.30 mmol, 2.00 eq), and then under nitrogen protection, tetrakis(triphenylphosphine palladium) (chemical formula Pd(PPh3)4, amount 119 mg, 0.10 mmol, 0.05eq) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (abbreviated as Xant-Phos, the amount is 119 mg, 0.21 mmol, 0.10eq), and the reaction is carried out at 120°C with reflux for 12 hours. After the reaction is completed, a third reaction solution is obtained; wherein the molar ratio of compound I, compound II, cesium carbonate, tetrakis(triphenylphosphine palladium) and 4,5-diphenylphosphine-9,9-dimethylxanthene is 1:1:0.05:0.1.

[0086] 20 mL of pure water was added to the third reaction solution, and then ethyl acetate was added for extraction and the second organic phases were combined. The extraction was performed three times, with 10 mL of ethyl acetate used for each extraction.

[0087] The second organic phase was dried over anhydrous sodium sulfate to obtain a crude compound III;

[0088] The crude compound III was purified by column chromatography to obtain 450 mg of a light yellow solid, namely compound III. The yield of compound III was calculated to be 49.13%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 73:27.

[0089]

[0090] Synthesis of compound IV:

[0091] To an eggplant-shaped flask containing compound III (300 mg, 0.68 mmol, 1.00 eq), 6 mL of dichloromethane (DCM) and 3 mL of trifluoroacetic acid (TFA) were added, and the mixture was stirred at 20°C to 25°C for 3 h. After the reaction, a fourth reaction solution was obtained.

[0092] 8 mL of dichloromethane was added to the fourth reaction solution, and then a saturated aqueous sodium carbonate solution was added for washing to obtain a third organic phase;

[0093] The third organic phase was dried and concentrated in sequence to obtain 223 mg of a light yellow solid, Compound IV. The yield of Compound IV was calculated to be 96.00%. The third organic phase was dried over anhydrous sodium sulfate.

[0094]

[0095] Synthesis of photoaffinity probe targeting cyclin-dependent kinase 9:

[0096] To an eggplant flask containing compound IV (223 mg, 0.65 mmol, 1.00 eq) were added 4 mL of tetrahydrofuran, 2-(3-butynylaziridin-3-yl)acetic acid (119 mg, 0.78 mmol, 1.20 eq), 1-hydroxybenzotriazole (abbreviated as HOBT, amount used is 132 mg, 0.97 mmol, 1.50 eq) and N, N-diisopropylethylamine (abbreviated as DIEA, amount used is 210 mg, 1.62 mmol, 2.50 eq) and the mixture was cooled in an ice-water bath with stirring for 5 minutes. After 10 minutes, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 187 mg, 0.97 mmol, 1.50 eq) was added, and the reaction was carried out at 20°C to 25°C for 12 hours. After the reaction, a fifth reaction solution was obtained; wherein the molar ratio of compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 1:1.2:1.5:2.5:1.5.

[0097] 10 mL of pure water was added to the fifth reaction solution, and then dichloromethane was added for extraction and the fourth organic phase was combined. The extraction was performed three times, with 5 mL of dichloromethane used for each extraction.

[0098] The fourth organic phase is dried and concentrated in sequence to obtain a crude compound V, wherein the fourth organic phase is dried using anhydrous sodium sulfate, and vacuum rotary evaporation can be used to concentrate the fourth organic phase;

[0099] The crude compound V was purified by column chromatography to obtain 450 mg of the target product as a pale yellow solid. The yield of the target product was calculated to be 51.61%. The eluent used for column chromatography separation and purification was a 3:1 volume ratio of petroleum ether and ethyl acetate. The target product was characterized.

[0100]

[0101] The target product1 H NMR spectrum Figure 1 As shown, 13 C NMR spectrum Figure 2 shown.

[0102] like Figure 1 As shown, the hydrogen spectrum data of the target product is: 1 H NMR (400MHz, CDCl3) δ8.90(s,1H),8.35(s,1H),8.28(d,J=5.3Hz,1H),7.35(dd,J=8.3,6.7Hz,1H ),7.24(dd,J=5.2,1.6Hz,1H),6.73(ddd,J=13.2,7.5,4.0Hz,2H),6.01(d,J=8.3Hz,1H),3.89(dq ,J=7.7,3.8Hz,1H),3.84(s,3H),2.49(t,J=11.3Hz,1H),2.26(s,3H),2.06(ddd,J=11.9,8.4,3.6 Hz,3H),1.94(q,J=14.4Hz,3H),1.76(t,J=7.3Hz,2H),1.51(q,J=11.9Hz,2H),1.33–1.14(m,2H).

[0103] pass 1 H NMR spectra and 13 The C NMR spectrum can confirm that the structure of the target product is as shown in Compound V, that is, a photoaffinity probe targeting cyclin-dependent kinase 9 is synthesized by the preparation method provided in this application.

[0104] Example 3 Preparation of a Photoaffinity Probe Targeting Cyclin-Dependent Kinase 9

[0105] This embodiment provides another method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9, comprising the following steps:

[0106] Synthesis of compound Ⅰ:

[0107] To a 50 mL three-necked flask charged with (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid (1.20 g, 4.93 mmol, 1.00 eq), 10 mL of N,N-dimethylformamide (DMF) was added, followed by the addition of N,N'-carbonyldiimidazole (CDI, 2.24 g, 13.80 mmol, 2.80 eq). The mixture was stirred at room temperature for 4 hours. After the reaction solution in the three-necked flask was cooled to 0°C, ammonium acetate (2.56 g, 33.52 mmol, 6.80 eq) and 0.5 mL of dichloromethane were added. The mixture was stirred at 20°C to 25°C for 10 hours. After the reaction was completed, a first reaction solution was obtained; wherein the molar ratio of (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid, N,N'-carbonyldiimidazole, and ammonium acetate was 1:2.8:6.8.

[0108] Add 60 mL of ice water to the first reaction solution, stir evenly, and filter to obtain a filter cake;

[0109] The filter cake was washed with water and dried in sequence to obtain 710 mg of a white solid, Compound I. The yield of Compound I was calculated to be 71.29%.

[0110] Synthesis of compound II:

[0111] To an eggplant-shaped flask containing 4-fluoro-2-methoxyphenylboronic acid (4.50 g, 26.48 mmol, 1.00 eq), 100 mL of dioxane, 2-chloro-4-bromopyridine (4.08 g, 21.18 mmol, 0.80 eq) and 32 mL of saturated sodium carbonate aqueous solution were added, and under the protection of replacement nitrogen, tetrakis(triphenylphosphine palladium) (chemical formula: Pd(PPh3)4, 0.92 g, 0.80 mmol, 0.03 eq) was added, and the reaction was carried out at 110°C with reflux for 12 hours. After the reaction, a second reaction solution was obtained; wherein, the molar ratio of 4-fluoro-2-methoxyphenylboronic acid, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) was 1:0.8:0.03.

[0112] 50 mL of pure water was added to the second reaction solution, and then dichloromethane was added for extraction and the first organic phase was combined. The extraction was performed three times, with 30 mL of dichloromethane used for each extraction.

[0113] The first organic phase is dried and concentrated in sequence to obtain a crude compound II, wherein the organic phase is dried using anhydrous sodium sulfate;

[0114] The crude compound II was purified by column chromatography to obtain 4.30 g of a white solid, namely compound II. The yield of compound II was calculated to be 70.15%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 93:7.

[0115] Synthesis of compound III:

[0116] To an eggplant-shaped flask containing compound I (500 mg, 2.06 mmol, 1.00 eq) were added 10 mL of dioxane, compound II (392 mg, 1.65 mmol, 0.80 eq), and catalyst cesium carbonate (chemical formula Cs2CO3, amount 1.21 mg, 3.71 mmol, 1.80 eq), and then under nitrogen protection, tetrakis(triphenylphosphine palladium) (chemical formula Pd(PPh3)4, amount 71 mg, 0.0 6mmol, 0.03eq) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (abbreviated as Xant-Phos, amount is 91mg, 0.16mmol, 0.08eq), and react at 120°C with reflux for 12 hours. After the reaction, a third reaction solution is obtained; wherein the molar ratio of compound I, compound II, tetrakis(triphenylphosphine palladium) and 4,5-diphenylphosphine-9,9-dimethylxanthene is 1:0.8:0.03:0.08.

[0117] 20 mL of pure water was added to the third reaction solution, and then ethyl acetate was added for extraction and the second organic phases were combined. The extraction was performed three times, with 10 mL of ethyl acetate used for each extraction.

[0118] The second organic phase was dried over anhydrous sodium sulfate to obtain a crude compound III;

[0119] The crude compound III was purified by column chromatography to obtain 390 mg of a light yellow solid, namely compound III. The yield of compound III was calculated to be 42.58%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 73:27.

[0120] Synthesis of compound IV:

[0121] To an eggplant-shaped flask containing compound III (300 mg, 0.68 mmol, 1.00 eq), 6 mL of dichloromethane (DCM) and 3 mL of trifluoroacetic acid (TFA) were added, and the mixture was stirred at 20°C to 25°C for 3 h. After the reaction, a fourth reaction solution was obtained.

[0122] 6 mL of dichloromethane was added to the fourth reaction solution, and then a saturated aqueous sodium carbonate solution was added for washing to obtain a third organic phase;

[0123] The third organic phase was dried and concentrated in sequence to obtain 228 mg of a light yellow solid, namely, Compound IV. The yield of Compound IV was calculated to be 98.15%. The third organic phase was dried over anhydrous sodium sulfate.

[0124] Synthesis of photoaffinity probe targeting cyclin-dependent kinase 9:

[0125] To an eggplant-shaped flask containing compound IV (228 mg, 0.66 mmol, 1.00 eq) were added 4 mL of tetrahydrofuran, 2-(3-butynylaziridin-3-yl)acetic acid (101 mg, 0.66 mmol, 1.00 eq), 1-hydroxybenzotriazole (abbreviated as HOBT, amount used is 117 mg, 0.86 mmol, 1.30 eq) and N,N-diisopropylethylamine (abbreviated as DIEA, amount used is 197 mg, 1.52 mmol, 2.30 eq), and the mixture was cooled in an ice-water bath with stirring for 5 min. After 10 minutes, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 166 mg, 0.86 mmol, 1.30 eq) was added, and the reaction was carried out at 20°C to 25°C for 10 hours. After the reaction, a fifth reaction solution was obtained; wherein, the molar ratio of compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 1:1:1.3:2.3:1.3.

[0126] 6 mL of pure water was added to the fifth reaction solution, and then dichloromethane was added for extraction and the fourth organic phase was combined. The extraction was performed three times, with 5 mL of dichloromethane used for each extraction.

[0127] The fourth organic phase is dried and concentrated in sequence to obtain a crude compound V, wherein the fourth organic phase is dried using anhydrous sodium sulfate, and vacuum rotary evaporation can be used to concentrate the fourth organic phase;

[0128] The crude compound V was purified by column chromatography to obtain 454 mg of a light yellow solid, i.e., the target product. The yield of the target product was calculated to be 51.30%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0129] Example 4 Preparation of a Photoaffinity Probe Targeting Cyclin-Dependent Kinase 9

[0130] This embodiment provides another method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9, comprising the following steps:

[0131] Synthesis of compound Ⅰ:

[0132] To a 50 mL three-necked flask charged with (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid (1.00 g, 4.11 mmol, 1.00 eq), 10 mL of N,N-dimethylformamide (DMF) was added, followed by the addition of N,N'-carbonyldiimidazole (CDI, 2.13 g, 13.15 mmol, 3.20 eq). The mixture was stirred at room temperature for 4 hours. After the reaction solution in the three-necked flask was cooled to 0°C, ammonium acetate (2.26 g, 29.60 mmol, 7.20 eq) and 0.7 mL of dichloromethane were added. The mixture was stirred at 20°C to 25°C for 10 hours. After the reaction was completed, a first reaction solution was obtained; wherein the molar ratio of (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid, N,N'-carbonyldiimidazole, and ammonium acetate was 1:3.2:7.2.

[0133] Add 80 mL of ice water to the first reaction solution, stir evenly, and filter to obtain a filter cake;

[0134] The filter cake was washed with water and dried in sequence to obtain 769 mg of a white solid, Compound I. The yield of Compound I was calculated to be 77.21%.

[0135] Synthesis of compound II:

[0136] To an eggplant-shaped flask containing 4-fluoro-2-methoxyphenylboronic acid (4.40 g, 25.89 mmol, 1.00 eq), 100 mL of dioxane, 2-chloro-4-bromopyridine (6.00 g, 31.07 mmol, 1.20 eq) and 34 mL of saturated aqueous sodium carbonate solution were added, and under the protection of replacement nitrogen, tetrakis(triphenylphosphine palladium) (chemical formula: Pd(PPh3)4, 2.09 g, 1.81 mmol, 0.07 eq) was added, and the mixture was reacted at 120°C with reflux for 14 hours. After the reaction, a second reaction solution was obtained; wherein, the molar ratio of 4-fluoro-2-methoxyphenylboronic acid, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) was 1:1.2:0.07.

[0137] 60 mL of pure water was added to the second reaction solution, and then dichloromethane was added for extraction and the first organic phase was combined. The extraction was performed three times, with 30 mL of dichloromethane used for each extraction.

[0138] The first organic phase is dried and concentrated in sequence to obtain a crude compound II, wherein the organic phase is dried using anhydrous sodium sulfate;

[0139] The crude compound II was purified by column chromatography to obtain 4.73 g of a white solid, namely compound II. The yield of compound II was calculated to be 76.84%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 93:7.

[0140] Synthesis of compound III:

[0141] To an eggplant-shaped flask containing compound I (500 mg, 2.06 mmol, 1.00 eq) were added 10 mL of dioxane, compound II (588 mg, 2.47 mmol, 1.20 eq), and catalyst cesium carbonate (chemical formula Cs2CO3, amount 1.47 mg, 4.53 mmol, 2.20 eq), and then under nitrogen protection, tetrakis(triphenylphosphine palladium) (chemical formula Pd(PPh3)4, amount 117 mg, 0.1 4mmol, 0.07eq) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (abbreviated as Xant-Phos, amount is 142mg, 0.25mmol, 0.12eq), and react at 120°C with reflux for 12 hours. After the reaction, a third reaction solution is obtained; wherein the molar ratio of compound I, compound II, tetrakis(triphenylphosphine palladium) and 4,5-diphenylphosphine-9,9-dimethylxanthene is 1:1.2:0.07:0.12.

[0142] 30 mL of pure water was added to the third reaction solution, and ethyl acetate was added for extraction and the second organic phases were combined. The extraction was performed three times, with 10 mL of ethyl acetate used for each extraction.

[0143] The second organic phase was dried over anhydrous sodium sulfate to obtain a crude compound III;

[0144] The crude compound III was purified by column chromatography to obtain 433 mg of a light yellow solid, namely compound III. The yield of compound III was calculated to be 47.27%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 73:27.

[0145] Synthesis of compound IV:

[0146] To an eggplant-shaped flask containing compound III (300 mg, 0.68 mmol, 1.00 eq), 8 mL of dichloromethane (DCM) and 3 mL of trifluoroacetic acid (TFA) were added, and the mixture was stirred at 20°C to 25°C for 3 h. After the reaction, a fourth reaction solution was obtained.

[0147] 8 mL of dichloromethane was added to the fourth reaction solution, and then a saturated aqueous sodium carbonate solution was added for washing to obtain a third organic phase;

[0148] The third organic phase was dried and concentrated in sequence to obtain 221 mg of a light yellow solid, namely Compound IV. The yield of Compound IV was calculated to be 95.26%. The third organic phase was dried over anhydrous sodium sulfate.

[0149] Synthesis of photoaffinity probe targeting cyclin-dependent kinase 9:

[0150] To an eggplant-shaped flask containing compound IV (221 mg, 0.64 mmol, 1.00 eq) were added 4 mL of tetrahydrofuran, 2-(3-butynylaziridin-3-yl)acetic acid (137 mg, 0.90 mmol, 1.40 eq), 1-hydroxybenzotriazole (abbreviated as HOBT, amount used is 148 mg, 1.09 mmol, 1.70 eq) and N, N-diisopropylethylamine (abbreviated as DIEA, amount used is 224 mg, 1.73 mmol, 2.70 eq), and the mixture was cooled in an ice-water bath with stirring for 5 minutes. After 10 minutes, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 210 mg, 1.09 mmol, 1.70 eq) was added, and the reaction was carried out at 20°C to 25°C for 14 hours. After the reaction, a fifth reaction solution was obtained; wherein the molar ratio of compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 1:1.4:1.7:2.7:1.7.

[0151] 8 mL of pure water was added to the fifth reaction solution, and then dichloromethane was added for extraction and the fourth organic phase was combined. The extraction was performed three times, with 5 mL of dichloromethane used for each extraction.

[0152] The fourth organic phase is dried and concentrated in sequence to obtain a crude compound V, wherein the fourth organic phase is dried using anhydrous sodium sulfate, and vacuum rotary evaporation can be used to concentrate the fourth organic phase;

[0153] The crude compound V was purified by column chromatography to obtain 455 mg of a light yellow solid, namely the target product. The yield of the target product was calculated to be 52.97%. The eluent used for column chromatography separation and purification was petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0154] Example 5 In-gel fluorescence analysis experiment of a photoaffinity probe targeting cyclin-dependent kinase 9 (hereinafter referred to as CDK9 Probe)

[0155] The in-gel fluorescence analysis experiment based on the photoaffinity probe targeting cyclin-dependent kinase 9 includes the following steps:

[0156] HCC1806 cells (i.e., human breast squamous cell carcinoma cells) were cultured in 6 cm culture dishes until the density reached more than 85% (cell number 1.2×10 6 , 24 hours later, the number of cells was about 2×10 6 ), the culture medium was aspirated, and the cells were washed twice with 5 mL PBS and once with serum-free culture medium.

[0157] 2 mL of serum-free culture medium containing different concentrations (0-25 μM, specifically 0 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 25 μM) of CDK9 Probe was added to the cells, and the cells were placed in a cell culture incubator and incubated for 1 hour. Then, the cell culture dish was placed on ice, the culture dish lid was removed, and the cells were irradiated with 365 nm ultraviolet light under a UV crosslinker. During irradiation, the cell surface was 5-10 cm away from the lamp tube and irradiated for 15 minutes.

[0158] After irradiation, the cells were gently scraped off with a cell scraper and collected into a 1.5 ml centrifuge tube, and then washed twice with cold PBS.

[0159] The mixture was centrifuged at 3000 rpm for 3 minutes at 4°C, and the upper PBS buffer was removed. Cell lysis buffer [the cell lysis buffer includes PBS (ThermoFisher Scientific), 1% IGEPAL-CA-630 (Sigma-Aldrich), 0.2% SDS (Sigma-Aldrich), 1% EDTA-free protease inhibitor mixture (Sigma-Aldrich), 0.1% Benzonase (Beyotime)] was added to the cells. After ultrasonic disruption, the cells were centrifuged at 20000 r / min at 4°C using a desktop centrifuge for 30 minutes.

[0160] After centrifugation, the supernatant was collected and the protein solution concentration was determined on a microplate reader using the BCA protein assay. The protein solution concentration was adjusted to 2 mg / mL with cell lysis buffer. Then, 50 μL of the protein solution was added with a final concentration of 200 μM fluorescein azide (such as rhodamine azide), 2.5 mM sodium ascorbate (Sigma-Aldrich), 25 mM BTTAA (Confluore), and 12.5 mM CuSO4 (Innochem) at room temperature and reacted for 1 hour.

[0161] After the reaction, the samples were separated by SDS-PAGE and electrophoresed at 150 V for about 1 hour. The gel was scanned using ChemiDoc imaging to display fluorescent bands, and then the gel was stained with Coomassie blue.

[0162] The results of in-gel fluorescence analysis of photoaffinity probe targeting cyclin-dependent kinase 9 are as follows Figure 3 shown.

[0163] Depend on Figure 3From the rhodamine fluorescence signal, it can be seen that significant fluorescent bands appeared at 35kDa and 42kDa, and the fluorescent labels at other places were relatively weak. Combined with the literature data (Nat. Commun. 2019, 10, 1832), it is speculated that the fluorescent band at 35kDa is the off-target protein VDAC2 (voltage-dependent anion channel protein 2) of the photoaffinity group. The fluorescent band appearing at 42kDa is consistent with the molecular weight of CDK9. At the same time, from Figure 3 The fluorescence signal intensity at 42 kDa on the gel image was quantitatively analyzed. When the concentration of CDK9 Probe was 25 μM, the signal intensity value of the band at 42 kDa was set to 1.00; when the concentration of CDK9 Probe was 0 or 0.1 μM, the quantitative value of the signal intensity of the band at 42 kDa was 0; when the concentration of CDK9 Probe was 1 μM, the quantitative value of the signal intensity of the band at 42 kDa was 0.06; when the concentration of CDK9 Probe was 5 μM, the quantitative value of the signal intensity of the band at 42 kDa was 0.19; when the concentration of CDK9 Probe was 10 μM, the quantitative value of the signal intensity of the band at 42 kDa was 0.26. Therefore, a concentration of 25 μM CDK9 Probe was used to verify the direct binding of CDK9 Probe to CDK9.

[0164] Depend on Figure 3 As can be seen from the Coomassie Brilliant Blue signal, which reflects the total proteome content, the software's quantification tool was used to analyze the three relatively independent bands at corresponding positions in each lane and calculate the average fluorescence signal intensity of these bands. The average fluorescence signal intensity was set to 1.00 when the CDK9 probe concentration was 25 μM; when the CDK9 probe concentration was 0 μM, the fluorescence signal intensity was 0.92; when the CDK9 probe concentration was 0.1 μM, the fluorescence signal intensity was 0.86; when the CDK9 probe concentration was 1 μM, the fluorescence signal intensity was 0.95; when the CDK9 probe concentration was 5 μM, the fluorescence signal intensity was 0.94; and when the CDK9 probe concentration was 10 μM, the fluorescence signal intensity was 0.86. The above data reflect that the sample loading amount in each lane was basically consistent.

[0165] Example 6 Verification of the Target of CDK9 Probe Based on Western Blot

[0166] Western Blot analysis was used to verify the target of CDK9 Probe, including the following steps:

[0167] HCC1806 cells were cultured in 15 cm culture dishes to a density of more than 85% (2 × 10 7 The culture time was 24 hours and the number of cells was about 3×10 7 ), the culture medium was aspirated, and the cells were washed twice with 15 mL PBS and once with serum-free culture medium.

[0168] Then, 15 mL of serum-free culture medium containing CDK9 Probe at a concentration of 25 μM was added to the cells, and the cells were placed in a cell culture incubator and incubated for 1 hour. After that, the cell culture dish was placed on ice, the culture dish cover was removed, and the cells were irradiated with 365 nm ultraviolet light under a UV crosslinker. During irradiation, the cell surface was 5-10 cm away from the lamp tube, and the irradiation time was 15 minutes.

[0169] After irradiation, the cells were gently scraped off with a cell scraper and collected into a 1.5 ml centrifuge tube, and then washed twice with cold PBS.

[0170] After centrifugation at 3000 rpm at 4°C for 3 minutes, the upper PBS layer was removed and the resulting cells were added to a cell lysis buffer [cell lysis buffer comprising PBS (ThermoFisher Scientific), 1% IGEPAL-CA-630 (Sigma-Aldrich), 0.2% SDS (Sigma-Aldrich), 1% EDTA-free protease inhibitor mixture (Sigma-Aldrich), and 0.1% Benzonase (Beyotime)], and then ultrasonically disrupted and high-speed centrifuged at 20,000 rpm in a 4°C tabletop centrifuge for 30 minutes.

[0171] After centrifugation, the supernatant was collected and the protein concentration was determined on a microplate reader using the BCA protein assay method. The protein concentration was then adjusted to 2 mg / mL using cell lysis buffer.

[0172] Take 500 μL of protein solution and add biotin azide, 200 μM biotin, 2.5 mM sodium ascorbate (Sigma-Aldrich), 25 mM BTTAA (CONFLUORE) and 12.5 mM CuSO4 (Innochem) at room temperature to a final concentration of 200 μM. The reaction was carried out for 1 hour. After the reaction, the protein solution was transferred to a 15 mL centrifuge tube and 4 volumes of methanol were added in sequence. Vortex and mix thoroughly, 1 volume of chloroform was added, vortex and mix thoroughly, and 3 volumes of water were added in sequence. Vortex and mix thoroughly. Centrifuge at 3000 rpm for 10 minutes at 4°C to retain the protein precipitate (the protein precipitate is in the form of discs).

[0173] Add 1 mL of pre-chilled methanol to the protein precipitate. Disintegrate the precipitate by vortexing or sonicating (for approximately 5 seconds). Transfer the precipitate to a 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 3 minutes at 4°C. Remove the supernatant and retain the protein precipitate. Repeat this step once. Finally, air-dry the protein precipitate for 2 minutes to completely remove the methanol.

[0174] The protein precipitate was resuspended in 3 mL of 0.05% SDS / PBS, and 200 μL of streptavidin-coupled magnetic beads (ThermoFisher Scientific) were added and incubated with rotation at room temperature for 3 h. After enrichment, the proteins were separated by SDS-PAGE and transferred to nitrocellulose (NC) membrane.

[0175] After transfer, the NC membrane was soaked in Ponceau staining solution and the Ponceau signal was read. The NC membrane was then incubated in 10 mL of 5% BSA / TBST at room temperature for 1 hour. The membrane was then incubated with the target protein, CDK9 Polyclonal Antibody (Proteintech), overnight at 4°C. Finally, the membrane was incubated with a secondary antibody (Alexa Fluor 488-labeled Goat Anti-Rabbit IgG, Beyotime) for 1 hour. The secondary antibody fluorescence signal was scanned using ChemiDoc imaging.

[0176] It can be seen that in this embodiment, the protein is labeled with the active molecular probe CDK9 Probe in living cells (i.e., HCC1806 cells), and then the biotin reporter group is coupled through a bioorthogonal reaction, and then the labeled protein is enriched using streptavidin magnetic beads. Proteins of different molecular weights are separated by SDS-PAGE and then transferred to the solid phase carrier NC membrane, and incubated with the target protein CDK9 antibody and the corresponding secondary antibody with fluorescein, and the binding of CDK9 Probe to the target is reflected by the fluorescent bands. During the experiment, we also set up DMSO-treated cells as a blank control to reduce false positive results. The verification results of the target of CDK9 Probe based on protein immunoblotting are as follows. Figure 4 shown.

[0177] Figure 4 The Ponceau red signal reflects the total protein content on the nitrocellulose membrane after transfer; Figure 4 The AlexaFluor 488 signal is the signal after the CDK9 Probe labeled protein is enriched and incubated with CDK9 antibody and the corresponding secondary antibody with fluorescein. There is a clear band near 42kDa. It can be seen that the target protein CDK9 appears in the band of the experimental group treated with CDK9 Probe, which proves that CDK9 Probe can successfully label CDK9. It should be noted that Figure 4 Input and eluate represent the total protein sample and magnetic bead eluted protein sample, respectively.

[0178] The photoaffinity probe targeting cyclin-dependent kinase 9 provided in the present application includes an active group, a photocrosslinking group and a bioorthogonal group.

[0179] Among them, the CDK9 inhibitor structure serves as an active group, which enables the photoaffinity probe molecule to enter the active center of the CDK9 target protein so as to use the photocrosslinking group to carry out subsequent photoaffinity reaction.

[0180] The photocrosslinking group, namely the diazirine structure, can generate free radicals under the excitation of ultraviolet light, which undergo insertion crosslinking reactions with its adjacent CH or CC bonds, thereby covalently capturing the CDK9 target protein. The diazirine structure is small in size, avoiding the shortcomings of the photoaffinity probe molecule, such as poor solubility and poor cell permeability.

[0181] The bioorthogonal group, i.e., the alkyne group, can be used for subsequent coupling with biotin or fluorescein to separate and enrich proteins labeled with photoaffinity probes or perform fluorescence imaging, thereby identifying the signal labeled by the photoaffinity probes;

[0182] Based on the above, the photoaffinity probe targeting cyclin-dependent kinase 9 provided in the present application can be used in screening CDK9 inhibitors and identifying CDK9 target proteins.

[0183] The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 provided in the present application has mild reaction conditions and a simple process, and can quickly prepare a photoaffinity probe targeting cyclin-dependent kinase 9.

[0184] This application designs and synthesizes a photoaffinity probe targeting CDK9 based on a CDK9 inhibitor, and verifies the protein binding of the CDK9 probe using fluorescent gel and Western blot. The CDK9 probe in this application can not only label the CDK9 protein but also be specifically linked to an active molecular probe with a corresponding reporter group according to subsequent experimental methods. More importantly, it expands its applications in functional analysis of the CDK9 protein and screening of small molecule inhibitors.

[0185] For ease of explanation, the above description has been made in conjunction with specific embodiments. However, the above discussion of some embodiments is not intended to be exhaustive or to limit the embodiments to the specific forms disclosed above. Based on the above teachings, various modifications and variations can be obtained. The selection and description of the above embodiments are intended to better explain the principles and practical applications, so that those skilled in the art can better use the embodiments and various different variations of the embodiments suitable for specific use considerations.

Claims

1. A photoaffinity probe targeting cyclin-dependent kinase 9, characterized in that: Has the following structure:

2. A method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9, for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 1, characterized in that: The following steps are involved: (1S,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid, N,N-dimethylformamide, N,N'-carbonyldiimidazole and ammonium acetate are reacted under first preset reaction conditions, and after the reaction is completed, a first reaction solution is obtained; The first reaction solution is post-treated and purified to obtain compound I; 4-fluoro-2-methoxyphenylboronic acid, dioxane, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) are reacted under second preset conditions, and after completion of the reaction, a second reaction solution is obtained; The second reaction solution is post-treated and purified to obtain compound II; Taking the compound I, the compound II, dioxane, a catalyst, tetrakis(triphenylphosphine palladium) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene, reacting under third preset conditions, and obtaining a third reaction solution after the reaction is completed; The third reaction solution is post-treated and purified to obtain compound III; The compound III and trifluoroacetic acid are reacted under fourth preset conditions, and after the reaction is completed, a fourth reaction solution is obtained; The fourth reaction solution is post-treated and purified to obtain compound IV; The compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are reacted under the fifth preset conditions. After the reaction is completed, a fifth reaction solution is obtained. Post-treating the fifth reaction solution and purifying it to obtain compound V, a photoaffinity probe targeting cyclin-dependent kinase 9; 3. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: When the reaction is carried out under the first preset reaction conditions, the molar ratio of the (1S,3R)-3-(tert-butyloxycarbonylamino)cyclohexanecarboxylic acid, N,N'-carbonyldiimidazole and ammonium acetate is 1:2.8-3.2:6.8-7.2; When the reaction is carried out under the second preset reaction conditions, the molar ratio of the 4-fluoro-2-methoxyphenylboronic acid, 2-chloro-4-bromopyridine and tetrakis(triphenylphosphine palladium) is 1:0.8-1.2:0.03-0.07; When the reaction is carried out under the third preset reaction conditions, the molar ratio of compound I, compound II, catalyst, tetrakis(triphenylphosphine palladium) and 4,5-bisphenylphosphine-9,9-dimethylxanthene is 1:0.8-1.2:0.03-0.07:0.08-0.12; When the reaction is carried out under the fifth preset reaction conditions, the molar ratio of compound IV, 2-(3-butynylaziridin-3-yl)acetic acid, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.0~1.4:1.3~1.7:2.3~2.7:1.3~1.

7.

4. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: The first reaction solution is post-treated and purified to obtain compound I, comprising: Add ice water to the first reaction solution, stir evenly, and then filter to obtain a filter cake; The filter cake is washed with water and dried in sequence to obtain the compound I.

5. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: The second reaction solution is post-treated and purified to obtain compound II, comprising: Adding pure water to the second reaction solution, then adding dichloromethane for extraction and combining the first organic phase; The first organic phase is dried and concentrated in sequence to obtain a crude compound II; The crude compound II is purified by column chromatography to obtain the compound II.

6. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: The third reaction solution is post-treated and purified to obtain compound III, comprising: Adding pure water to the third reaction solution, then adding ethyl acetate for extraction and combining the second organic phase; The second organic phase was dried over anhydrous sodium sulfate to obtain a crude compound III; The crude compound III is purified by column chromatography to obtain the compound III.

7. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: The fourth reaction solution is post-treated and purified to obtain compound IV, comprising: adding dichloromethane to the fourth reaction solution, and then adding a saturated sodium carbonate aqueous solution for washing to obtain a third organic phase; The third organic phase is dried and concentrated in sequence to obtain the compound IV.

8. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: The fifth reaction solution is post-treated and purified to obtain compound V, a photoaffinity probe targeting cyclin-dependent kinase 9, comprising: Adding pure water to the fifth reaction solution, then adding dichloromethane for extraction and combining the fourth organic phase; The fourth organic phase is dried and concentrated in sequence to obtain a crude compound V; The crude compound V is purified by column chromatography to obtain the compound V, which is the photoaffinity probe targeting cyclin-dependent kinase 9.

9. The method for preparing a photoaffinity probe targeting cyclin-dependent kinase 9 according to claim 2, characterized in that: The first preset reaction conditions include a reaction temperature of 20°C to 25°C and a reaction time of 10 hours to 14 hours; The second preset reaction conditions include a reaction temperature of 100° C. to 120° C., a reaction time of 10 hours to 14 hours, and reflux under nitrogen protection; The third preset reaction conditions include a reaction temperature of 100° C. to 120° C. and a reaction time of 10 hours to 14 hours; The fourth preset reaction conditions include a reaction temperature of 20°C to 25°C and a reaction time of 10 hours to 14 hours; The fifth preset reaction conditions include a reaction temperature of 20° C. to 25° C. and a reaction time of 10 hours to 14 hours.

10. Use of a photoaffinity probe targeting cyclin-dependent kinase 9, characterized in that: The invention also includes the use of the photoaffinity probe targeting cyclin-dependent kinase 9 as claimed in claim 1 in screening CDK9 inhibitors and identifying CDK9 target proteins, wherein the application does not involve the diagnosis and treatment of diseases.

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