Bcl6 small molecule fluorescent probe and preparation method and application thereof

Abstract

The application discloses a BCL6 small-molecule fluorescent probe and a preparation method and application thereof. The compound provided by the application has fluorescent characteristics and has good specific binding to BCL6. The compound is used as a fluorescent polarization probe, and through a competition method, screening of a BCL6 ligand and evaluation of a ligand affinity level can be realized. The BCL6 small-molecule fluorescent probe provided by the application provides a basis for subsequent high-throughput screening of a drug design targeting BCL6-BTB.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a BCL6 small molecule fluorescent probe, its preparation method, and its application. Background Technology

[0002] B-cell lymphokine 6 (BCL6) is an important transcriptional repressor in humoral immune responses. The human BCL6 gene is approximately 24 kb, encoding a BCL6 protein of about 95 kDa. The BCL6 protein is a member of the POZ / BTB / Zinc finger protein family and is mainly composed of three parts: 1) The N-terminal POZ / BTB domain—the main functional region for transcriptional repression. For BCL6 to exert its transcriptional repression function, the BTB domain needs to spontaneously form a dimer. Three major transcriptional co-repressors, SMRT, BCOR, or NCOR, competitively bind to the BTB binding site, jointly exerting transcriptional repression and participating in the early transcriptional regulation of germinal centers (GC) formation. 2) The middle region (three PEST domains, also known as the RD2 domain), which mainly recruits cofactors such as MTA3 or CTBP1, playing a role in protein stabilization. 3) The C-terminal zinc finger domain, composed of six identical zinc finger structures, mainly functions to bind DNA and is a prerequisite for BCL6 to exert its transcriptional repression function.

[0003] BCL6 is a transcriptional repressor crucial for GC development and maintenance. In the GC response, chromosomal translocations and point mutations of BCL6 lead to persistently high expression of the BCL6 protein, promoting malignant B-cell proliferation and ultimately resulting in B-cell lymphoma. Most B-cell non-Hodgkin lymphomas (B-NHL) originate from GC, with diffuse large B-cell lymphoma (DLBCL) being the most common subtype. BCL6 is widely recognized as a carcinogenic driver of DLBCL. Numerous preclinical studies have also demonstrated that blocking the interaction between the BCL6-BTB domain and its transcriptional co-repressors can inhibit GC formation and DLBCL cell proliferation, representing an effective and safe treatment strategy for DLBCL without causing toxic side effects or macrophage-driven inflammatory responses.

[0004] Several small molecule inhibitors of BCL6 have been reported, such as BI-3802. However, no BCL6 small molecule inhibitor has yet entered the clinical stage, and the development of novel BCL6 small molecule inhibitors remains crucial. Therefore, in vitro inhibitory activity assays and high-throughput screening methods are essential for the development of BCL6 small molecule inhibitors. Accurate, stable, and reliable activity assays can help discover novel lead compounds, guide the structural optimization of lead compounds, and obtain candidate drug molecules with better activity, laying the foundation for the development of drugs targeting DLBCL.

[0005] Fluorescence polarization is a method for detecting intermolecular interactions. By labeling ligands with fluorescent groups and conducting a competition experiment, the binding affinity of an analyte to an acceptor can be detected. The basic principle is that smaller molecules rotate faster in solution than larger molecules; therefore, fluorescent compounds that do not bind to larger molecules produce lower polarization values. Conversely, when a fluorescent compound binds to a larger molecule, its rotation slows down, resulting in a higher polarization value. Therefore, fluorescence polarization is a detection method that can be implemented on microplates, requires minimal reagents, and enables high-throughput screening.

[0006] Today, the use of small-molecule fluorescent probes as an adjunct tool has revealed various interaction modes between small molecules and biomacromolecules, information that is of great significance for rational drug design. For example, the discovery of new drug targets or a new elucidation of the function of a known protein is particularly important for elucidating the occurrence, development, and treatment of diseases at the molecular level. Using small-molecule fluorescent probes as an adjunct tool, these targets can be developed into efficient high-throughput screening models, randomly screening a large number of active compounds in a short time to discover more active small-molecule compounds as lead compounds for further drug development.

[0007] For BCL6, detection methods or commercial kits based on fluorescence polarization competition have been developed. However, there are no reports on using fluorescence polarization methods with small molecule fluorescent probes to test the binding ability of compounds to BCL6. Summary of the Invention

[0008] Objective of this invention: One objective of this invention is to provide a small-molecule fluorescent probe for BCL6. The compound provided by this invention exhibits fluorescent properties and good specificity for binding to BCL6. Using this compound as a fluorescent polarization probe, a competitive method can be employed to screen BCL6 ligands and evaluate ligand affinity levels. Based on the screening method established in this invention, one small-molecule compound capable of binding to BCL6 was obtained, providing a foundation for high-throughput screening of subsequent drugs targeting BCL6.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned compound.

[0010] Another object of the present invention is to provide the application of the above-mentioned compound as a fluorescent polarization probe in the screening of BCL6 ligands.

[0011] Another object of the present invention is to provide a method for screening BCL6 ligands.

[0012] Technical solution: The objective of this invention is achieved through the following technical solution:

[0013] This invention provides a compound represented by Formula I:

[0014]

[0015] The present invention also provides a method for preparing the above-mentioned compound, comprising the following steps:

[0016]

[0017] S1: Compound T1 and compound T2 undergo an amide condensation reaction to obtain intermediate T3;

[0018] S2: Intermediate T3 undergoes a deprotection reaction to yield intermediate T4;

[0019] S3: Intermediate T4 undergoes a condensation reaction with fluorescein active ester T5 to obtain the target compound.

[0020] Preferably, in step S1, the molar ratio of compound T1 to compound T2 is 1:1 to 1:2.

[0021] Preferably, in step S1, the condensing agent for the amide condensation reaction is one or more of HOAT, HOBT, HATU, HBTU, or BOP.

[0022] Preferably, in step S1, the base for the amide condensation reaction is one or both of N,N-diisopropylethylamine (DIPEA) or triethylamine (TEA).

[0023] Preferably, the organic solvent for the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or dimethyl sulfoxide.

[0024] Preferably, in step S1, the temperature of the amide condensation reaction is room temperature, and the reaction time is 12-24 hours.

[0025] Preferably, in step S2, the base for the deprotection reaction is one or both of piperidine and triethylamine.

[0026] Preferably, in step S2, the organic solvent for the deprotection reaction is one or more of N,N-dimethylformamide, dichloromethane, or tetrahydrofuran.

[0027] Preferably, in step S2, the deprotection reaction is carried out at room temperature for 12 to 24 hours.

[0028] Preferably, in step S3, the molar ratio of compound T4 and fluorescein active ester T5 is 1:1 to 1:2.

[0029] Preferably, in step S3, the base for the condensation reaction is one or both of N,N-diisopropylethylamine or triethylamine; and the organic solvent for the condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide.

[0030] Preferably, in step S3, the condensation reaction is carried out at room temperature for 12 to 24 hours.

[0031] This invention also provides the application of the above-mentioned compounds as fluorescent polarization probes in BCL6 ligand screening.

[0032] This invention also provides a method for screening BCL6 ligands, comprising the following steps:

[0033] (1) The above-mentioned compound, the protein containing the BCL6 ligand binding domain and the test compound are mixed to obtain a mixture;

[0034] (2) The polarization value of the mixture is determined by fluorescence polarization technology, and the polarization value is used to confirm whether the test compound is a ligand of BCL6.

[0035] Generally, a polarization value decrease of more than 25% of the detection window is sufficient to identify it as a ligand of BCL6.

[0036] Beneficial effects:

[0037] The compound provided by this invention exhibits fluorescent properties and good specificity for binding to BCL6. Using this compound as a fluorescent polarization probe, fluorescence polarization (FP) technology can be employed to screen BCL6 ligands and evaluate ligand affinity levels. A high-throughput screening scheme for BCL6 small molecules established through fluorescence polarization (FP) can be used for high-throughput drug screening, offering advantages such as low cost, stability, speed, and high efficiency. Attached Figure Description

[0038] Figure 1 The curve shows the fluorescence intensity of the fluorescent polarization probe as a function of concentration in 0.01 M PBS buffer solution.

[0039] Figure 2 The saturation curves of the fluorescent polarization probe and BCL6 are shown.

[0040] Figure 3 The curves show the competition between the fluorescent polarization probe and BCL6 protein in different concentrations of the positive compound BI-3802. Detailed Implementation

[0041] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0044] The synthetic route for the target compound of this invention is as follows:

[0045]

[0046] Example 1: Preparation of (9H-phospha-9-yl)methyl(2-(2-(2-(1-(5-chloro-4-((1-methyl-3-(2-(formamide)2-carbonylethoxy)2-one-1,2-dihydroquinolin-6-yl)amino)pyrimidin-2-yl)piperidin-4-carboxamido)ethoxy)ethoxy)ethyl)carbamate (intermediate T3)

[0047] T1 (100 mg, 0.20 mmol) was dissolved in 5 mL of DMF solution, and HATU (114 mg, 0.30 mmol), DIPEA (129 mg, 1.0 mmol), and T2 (148 mg, 0.40 mmol) were added. The reaction mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, equal volumes of ethyl acetate and water were added, and the mixture was extracted three times. The organic layers were combined and concentrated under low pressure to remove the solvent. The residue was purified by column chromatography (elution system: dichloromethane:methanol = 20:1, v / v) to give intermediate T3 as a pale yellow solid (139 mg, yield: 81.8%).

[0048] 1H NMR(300MHz,DMSO-d6)δ(ppm)8.87(s,1H),8.04(s,1H),8.00-7.93(m,1H),7.91-7.84(m,4H),7.75(dd, J=9.1,2.4Hz,1H),7.67(d,J=7.5Hz,2H),7.48-7.36(m,3H),7.31(m,3H),4.56(s,2H),4.46(d,J=13.1Hz ,2H),4.28(d,J=6.9Hz,2H),4.20(d,J=6.6Hz,1H),3.66(s,3H),3.41-3.38(m,4H),3.20-3.10(m,4H),2. 85(t,J=12.3Hz,2H),2.65(d,J=4.6Hz,3H),2.40(s,1H),1.68(d,J=12.6Hz,2H),1.47(d,J=12.3Hz,2H).

[0049] Example 2: Preparation of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)1-(5-chloro-4-((1-methyl-3-(2-(formamido)2-carbonylethoxy)2-one-1,2-dihydroquinoline-6-yl)amino)pyrimidin-2-yl)piperidine-4-carboxamide (intermediate T4)

[0050] T3 (139 mg, 0.16 mmol) was dissolved in 5 mL of methanol solution, and 2 mL of piperidine solution was added. The reaction mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, the mixture was concentrated under reduced pressure to remove piperidine and methanol solution, 20 mL of dichloromethane solution was added, the mixture was sonicated, filtered, washed with dichloromethane, and dried to give intermediate T4, a pale yellow solid (71 mg, yield: 71.0%).

[0051] 1 H NMR (300MHz, DMSO-d6) δ (ppm) 8.88 (s, 1H), 8.05 (m, 2H), 7.93 (m, 2H), 7.49 (d, J = 9.0Hz, 1H), 7.13 (s, 1H), 4.59 (s, 2H), 4.48 (d ,J=12.4Hz,2H),3.69(s,3H),3.51(s,4H),3.20(s,3H),2.87(s,3H),2.67(s,4H),1.70(d,J=12.4Hz,2H),1.57-1.39(m,4H).

[0052] Example 3 Preparation of 1-(5-chloro-4-((1-methyl-3-(2-(formamido)2-carbonylethoxy)2-one-1,2-dihydroquinolin-6-yl)amino)pyrimidin-2-yl)-N-(2-(2-(2-(3',6'-dihydroxy-3-one-3H-spiro[isobenzofuran-1,9'oxanthracene]5-formylamino)ethoxy)ethoxy)ethyl)piperidine-4-carboxamide (target compound)

[0053] T4 (10.0 mg, 0.016 mmol) was dissolved in 2 mL of DMSO solution, followed by the addition of DIPEA (8.3 mL, 0.048 mmol) and then T5 (7.5 mg, 0.016 mmol). The reaction mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, an equal volume of dichloromethane and water was added, and the mixture was extracted three times. The organic layers were combined and concentrated under low pressure to remove the solvent. The residue was purified by column chromatography (elution system: dichloromethane:methanol = 20:1, v / v) to obtain the target compound as an orange-yellow final product (2.5 mg, yield: 16.1%, purity 98.15%).

[0054] 1 H NMR (300MHz, DMSO-d6) δ (ppm) 10.08 (s, 2H), 8.92 (t, J = 5.4Hz, 1H), 8.87 (s, 1H), 8.48 (d, J = 1.5Hz, 1H), 8.26 (dd, J = 8.1, 1.6Hz, 1H), 8.06 (s, 1H),7.99(d,J=4.9Hz,1H),7.92(d,J=2.4Hz,1H),7.78(m,1H),7.49(d,J=9.1Hz,1H),7.42(d,J=8.6Hz,1H),7.38(d,J=2.2Hz,1H),7.29(d,J =2.2Hz,1H),7.26(d,J=2.7Hz,1H),7.13(s,1H),6.71(d,J=2.1Hz,2H),6.59(s,1H),6.57(d,J=2.2Hz,1H),4.59(s,2H),4.48(d,J=13.0Hz,2 H),3.68(s,3H),3.61-3.53(m,8H),3.43(d,J=6.1Hz,4H),3.19(q,J=5.8Hz,3H),2.67(d,J=4.6Hz,3H),1.70(d,J=12.3Hz,2H),1.50(s,4H).

[0055] HRMS(ESI): m / z, calculated value C 50 H 49 ClN8O 12 [M+Na]+ , 1011.3023; Measured value: 1011.3051.

[0056] Example 4 Expression and purification of BCL6-BTB domain protein

[0057] Expression of the BCL6-BTB domain protein was performed using the pET-28b(+) vector (GenScript Biotechnology) in Escherichia coli BL21(DE3) (ThermoFisher Scientific).

[0058] The above-mentioned Escherichia coli were placed in LB medium (each 2000 mL of LB medium contains 20 g tryptone, 10 g yeast extract and 20 g NaCl), shaken at 37°C and 220 rpm for 1 hour, centrifuged at 3000 rpm for 5 minutes, most of the supernatant was discarded, and the remaining liquid was spread on solid medium containing kanamycin (homemade, the above LB medium was supplemented with 2% agar), and incubated overnight at 37°C. The following day, select single colonies with smooth edges and inoculate them into 5 mL of LB medium containing 1‰ kanamycin. Incubate overnight at 37°C and 220 rpm. Transfer to 500 mL of LB medium and continue incubation for 12-16 hours until the OD600 value is between 0.6 and 0.8. Add the inducer isopropyl-β-d-thiogalactoside (IPTG) to a final concentration of 1 mM and express for 12 hours at 16°C and 180 rpm. Centrifuge at 4000 rpm for 15 minutes at 4°C, discard the supernatant, weigh the small pieces, and freeze at -80°C for 12 hours. Add lysis buffer (Tris 1.21g, NaCl 8.7g, Tween-20 500μL, β-mercaptoethanol 690μL, ultrapure water to a final volume of 500mL) at a ratio of 10mL / g bacteria. Add 1‰ of the protease inhibitor phenylmethanesulfonylfluoride (PMSF). Sonicate the mixture under ice bath conditions (4 seconds on, 6 seconds off, for a total of 40 minutes, 80% power) for 40 minutes. Then centrifuge at 12000rpm for 20 minutes at 4℃, collect the supernatant, and use... Proteins were extracted and purified using the Pure 25 (GE Healthcare, LifeSciences) automated purification system (washed with 25 mM Tris, pH 7.5, 200 mM NaCl, and 10 mM imidazole buffer, followed by elution with 25 mM Tris, pH 7.5, 200 mM NaCl, and 300 mM imidazole buffer). The purified protein was identified by SDS-PAGE electrophoresis and then dialyzed overnight (dialysis buffer: Tris 25 mM, NaCl 150 mM). The following day, the protein was collected, and its concentration was determined using the BCA method before being stored at -80°C for later use.

[0059] Through the above experiments, 400 μL of 30 μM BCL BTB domain protein can be obtained from every 1 L of E. coli culture medium.

[0060] Example 5: Establishment of the Fluorescent Polarization Probe (FP) Method

[0061] Fluorescence polarization measurements were performed using a SpectraMax Multi-Mode Microplate Reader (Molecular Devices) microplate reader. Experiments were conducted using 384-well, black round-bottom, NBS-surfaced, polystyrene microplates (corning).

[0062] (1) Determination of probe concentration: To determine the optimal probe concentration for the FP method, the fluorescence polarization values ​​of fluorescent probes (the target compound prepared in Example 3) at different concentrations (starting at 10 μM, with 13 concentrations obtained through a 3-fold gradient dilution) in PBS buffer solution were measured, and the probe curves were determined. The results are as follows: Figure 1 As shown, we selected a concentration of 11 nM, which is close to the lower plateau, as the optimal probe concentration for the FP method.

[0063] (2) Saturation assay: BCL6 protein (obtained from Example 4) at different concentrations (starting probe concentration of 25 μM, 14 concentrations of 3-fold gradient dilution) was mixed with the fluorescent polarization probe determined above and incubated at room temperature for 30 minutes, and then the fluorescence polarization value of the system was measured.

[0064] The implementation scheme for the saturation experiment in this invention is as follows: BCL6 is diluted with PBS buffer, mixed with an 11 nM fluorescent polarization probe, covered with a plate at room temperature and incubated with shaking for 30 minutes, the fluorescence polarization value is measured, and a saturation curve is plotted for analysis. The results are as follows: Figure 2 As shown, we selected a concentration of 133 nM, which is close to the upper plateau, as the optimal protein concentration for the FP method.

[0065] (3) Competition assay: This assay is used to determine the affinity between ligands and receptors. A fixed concentration of BCL6 and a fluorescent polarization probe are mixed with different concentrations of the analyte, and the fluorescence polarization value of the system is measured. The compounds compete with the fluorescent polarization probe, causing the fluorescence polarization value to decrease with increasing compound concentration.

[0066] The implementation scheme of the competition experiment in this invention is as follows: The positive compound BI-3802 of BCL6 (purchased from MCE) was diluted 3-fold in analytical buffer (0.01M PBS) to create 10 concentration gradients. 20 μL of the diluted compound, 20 μL of probe solution, and 20 μL of protein solution were added to each well, denoted as mP. test Two replicates were set up for each compound concentration, and a blank control (20 μL probe + 40 μL buffer, denoted as mP) was included in the experiment. min ) and negative control (20 μL protein + 20 μL probe + 20 μL buffer, denoted as mP) max Cover the plate with the enzyme and incubate with shaking for 30 minutes at room temperature. Detect the mP value using a microplate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

[0067] The formula for calculating the inhibition rate is: Inhibition rate (%) = [1 - (mP)] test -mP min ) / (mP max -mP min IC was calculated using nonlinear fitting in Graphpad Prism 8.0 software, with a value of 100% (100%). 50 value.

[0068] The results are as follows Figure 3 As shown, the test results indicate that the IC50 of the positive compound BI-3802, tested using the FP method, is [missing information]. 50 For 23.6 nM (as reported in the literature (Cell Rep 20(12):2860-2875), the LUMIER method was used, IC 50 =43nM), which is almost consistent with the reported small molecule activity.

[0069] As can be seen, this invention uses fluorescent polarization probes as tool molecules to establish a high-throughput screening method for BCL6 small molecules through fluorescence polarization (FP) method. This scheme can be used for high-throughput screening of compounds targeting BCL6 ligands, and it has the characteristics of being inexpensive, stable, fast, and efficient.

[0070] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A compound represented by Formula I:

2. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: S1: Compound T1 and compound T2 undergo an amide condensation reaction to obtain intermediate T3; S2: Intermediate T3 undergoes Fmoc deprotection reaction to yield intermediate T4; S3: Intermediate T4 undergoes a condensation reaction with fluorescein active ester T5 to obtain the target compound.

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of compound T1 to compound T2 is 1:1 to 1:

2.

4. The preparation method according to claim 2, characterized in that, In step S1, a condensing agent is used in the amide condensation reaction, which is one or more of HOAT, HOBT, HATU, HBTU, or BOP; a base is also used in the amide condensation reaction, which is one or two of N,N-diisopropylethylamine (DIPEA) or triethylamine (TEA); and the organic solvent used in the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or dimethyl sulfoxide.

5. The preparation method according to claim 2, characterized in that, In step S2, a base is used in the deprotection reaction, and the base is one or both of piperidine and triethylamine; the organic solvent used in the deprotection reaction is one or more of N,N-dimethylformamide, dichloromethane or tetrahydrofuran.

6. The preparation method according to claim 2, characterized in that, In step S3, the molar ratio of compound T4 and fluorescein active ester T5 is 1:1 to 1:

2.

7. The preparation method according to claim 2, characterized in that, In step S3, a base is used in the condensation reaction, and the base is one or two of N,N-diisopropylethylamine or triethylamine; the organic solvent used in the condensation reaction is one or more of 1,4-dioxane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide.

8. The preparation method according to claim 2, characterized in that, In step S1, the temperature of the amide condensation reaction is room temperature, and the reaction time is 12-24 hours; in step S2, the temperature of the deprotection reaction is room temperature, and the reaction time is 12-24 hours; in step S3, the temperature of the condensation reaction is room temperature, and the reaction time is 12-24 hours.

9. The use of the compound of claim 1 as a fluorescent polarization probe in the preparation of BCL6 ligand screening drugs.

10. A method for screening BCL6 ligands, characterized in that, Includes the following steps: (1) A mixture is prepared by mixing the compound of claim 1, the protein containing the BCL6 ligand binding domain, and the test compound; (2) The polarization value of the mixture was determined by fluorescence polarization technology, and the compound to be tested was confirmed to be a ligand of BCL6 based on the polarization value.

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