Novel fluorescent compound targeting ROR gamma normal site as well as preparation method and application of novel fluorescent compound

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly discloses a novel fluorescent compound capable of being combined with a retinoic acid receptor related orphan receptor gamma normal site and application of the novel fluorescent compound serving as a fluorescent probe to inhibitor screening, imaging and the like. Compounds P1-P11 conforming to the general formula (I) are synthesized, a novel ROR gamma normal inhibitor screening platform can be established on the basis of a fluorescence polarization probe system constructed by the compounds according to a competitive ligand replacement principle, and the technical scheme breaks through the limitation of a traditional activity detection method and has a wide application prospect. An activity evaluation tool with economical efficiency, timeliness and reliability is provided for research and development of drugs targeting ROR gamma normal sites, and the lead compound discovery and structure optimization process can be accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and specifically includes novel fluorescent compounds that can bind to the orthosteric site of retinoic acid receptor-related orphan receptor gamma and their applications as fluorescent probes for inhibitor screening, imaging, and the like. Background Art

[0002] The immune system is a crucial line of defense for maintaining human health. However, autoimmune diseases such as psoriasis, inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis exhibit pathological characteristics of abnormal immune system activation. These diseases arise from the immune system's mistaken attack on its own tissues, posing significant challenges to clinical drug development. Psoriasis, for example, is characterized by a vicious cycle of epidermal hyperplasia and immune dysfunction, leading to its nickname, "immortal cancer," due to its stubbornness and recurring nature. Currently, the efficacy and safety of traditional psoriasis treatments are insufficient to meet the needs of the new era. Recent studies have shown that abnormally elevated levels of interleukin-17 (IL-17) are closely associated with the pathological progression of autoimmune diseases. Consequently, biological agents and small molecule drugs that target and inhibit IL-17 production have rapidly developed and demonstrated significant efficacy in the clinical treatment of diseases such as psoriasis and ankylosing spondylitis.

[0003] Retinoic acid receptor-related orphan receptor γ (RORγ), as a transcriptional regulator in the nuclear receptor family, plays a key role in the pathological process of psoriasis by regulating the differentiation and gene expression of helper cells 17 (Th17 cells) that produce the pro-inflammatory cytokine IL-17. This receptor has typical nuclear receptor structural characteristics: structurally, RORγ consists of a nitrogen-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (Hinge), a ligand-binding domain (LBD) and a carbon-terminal domain (CTD). Among them, the LBD is a key domain for transcriptional regulation, which is composed of twelve α-helices (H1-H12). The carbon-terminal H12 can recruit co-transcription factors, promote the binding of DBD to target genes, and regulate downstream gene expression (Int J Mol Sci 2020, 21, 5329-5346). It is worth noting that the conserved orthosteric binding site within the LBD has a dual regulatory function, that is, endogenous ligands can enhance transcriptional activity by stabilizing the H12 conformation, while exogenous small molecule antagonists inhibit RORγ function by destroying this conformation. At present, significant progress has been made in the development of drugs targeting this target, and a number of orthosteric site inhibitors have entered the clinical evaluation stage, providing a new intervention strategy for the treatment of psoriasis. At present, there are no drugs on the market in the direction of RORγ inhibitors. As a very potential autoimmune target, the discovery of RORγ inhibitors with new skeletons is of great significance. Accurate, stable and reliable RORγ orthosteric site activity detection methods are an important basis for the discovery and optimization of new RORγ inhibitors. They can provide technical support for the discovery of new RORγ orthosteric inhibitors, and improve the drugability characteristics of candidate molecules by guiding structural optimization, thereby promoting the development of psoriasis treatment drugs targeting RORγ.

[0004] Chinese Patent No. 2023106508312 discloses a novel small-molecule fluorescent probe targeting RORγ, its preparation method, and its application. The small-molecule fluorescent probe disclosed in this patent can bind to the allosteric site of the retinoic acid receptor-related orphan receptor γ. Currently, no patents disclose novel fluorescent compounds that can bind to the orthosteric site of the retinoic acid receptor-related orphan receptor γ.

[0005] Based on this, the present invention discloses a class of fluorescent probes targeting the RORγ-LBD orthosteric binding pocket and a RORγ orthosteric inhibitor activity detection system based on the principle of fluorescence polarization. In addition, the probe can also be used for imaging RORγ in cells and living bodies. Summary of the Invention

[0006] The general formula (I) of the small molecule fluorescent probe disclosed in the present invention is as follows:

[0007]

[0008] or a pharmaceutically acceptable salt thereof, in the general formula (I):

[0009] L stands for

[0010]

[0011] The present invention also discloses a method for preparing the RORγ small molecule fluorescent probe, comprising the following steps:

[0012]

[0013] S1: Using 4-bromo-2-fluoro-1-nitrobenzene as the starting material, 1-(5-bromo-2-nitrophenyl)piperidine is prepared by nucleophilic substitution with piperidine hydrochloride; the nitro group is then reduced to an amino group under the catalysis of stannous dichloride to obtain 4-bromo-2-(piperidin-1-yl)aniline; this is then subjected to an amide condensation reaction with 2-chloro-6-fluorobenzoyl chloride to construct the N-(4-bromo-2-(piperidin-1-yl)phenyl)-2-chloro-6-fluorobenzamide intermediate; aminomethylation is achieved by the action of a methylating agent to obtain N-(4-bromo-2-(piperidin-1-yl)phenyl)-2-chloro-6-fluoro-N-methylbenzamide; finally, a palladium-catalyzed Suzuki coupling reaction is performed to couple with 5-carboxy-2-chlorophenylboronic acid to complete the synthesis of the key intermediate BI-1. The synthesis of intermediate BI-1 can also be obtained by referring to the literature Bioorg MedChem Lett 2015, 25, 2991-2997.

[0014] BI-1 was used as the raw material to react with HATU and different amine compounds at room temperature under alkaline conditions to obtain the intermediate BI-2-L. 1～9,11 .

[0015] Preferably, in step S1, the diamine compound used in the reaction is a mono-Boc-protected diamine.

[0016] Preferably, in step S1, the organic solvent used in the reaction is dichloromethane (DCM); the base used in the reaction is N,N-diisopropylethylamine (DIPEA); and the reaction temperature is 20-25°C.

[0017]

[0018] S2: React the raw material BI-1 with HATU and benzyl (5-[(2-aminoethyl)amino]-5-oxopentyl) carbamate under alkaline conditions at room temperature to obtain the intermediate BI-2-L 10；

[0019] Preferably, in step S2, the organic solvent used in the reaction is dichloromethane (DCM); the base used in the reaction is N,N-diisopropylethylamine (DIPEA); and the reaction temperature is 20-25°C.

[0020]

[0021] S3: Raw material BI-2-L 1～9,11 Dissolved in dichloromethane, reacted at room temperature under acidic conditions, and removed the tert-butyloxycarbonyl (Boc) group to obtain compound BI-3-L 1～9,11 ;

[0022] Preferably, in step S3, the acidic reagent is trifluoroacetic acid or dioxane hydrochloride solution.

[0023]

[0024] S4: Raw material BI-2-L 10 Dissolved in methanol, reacted at room temperature under hydrogen atmosphere, and removed the benzyloxycarbonyl (Cbz) to obtain compound BI-3-L 10；

[0025]

[0026] S5: Intermediate BI-3-L 1～11 Condensation reaction with fluorescein isothiocyanate (FITC) under alkaline conditions to obtain final products P1 to P11;

[0027] Preferably, in step S5, the alkaline condition used in the condensation reaction is N,N-diisopropylethylamine (DIPEA), the solvent used is N,N-dimethylformamide (DMF), and the reaction conditions are room temperature and nitrogen protection.

[0028] The present invention also discloses an affinity detection method system for RORγ orthosteric inhibitors constructed using the fluorescent probe of formula (I) to test the binding ability of other compounds to the RORγ orthosteric site. The specific steps are as follows:

[0029] S1: Mix the RORγ small molecule fluorescent probe of formula (I) (concentration 40-1000 nM), RORγ protein (concentration 40-1000 nM) and the test compound in a certain ratio to obtain a test system;

[0030] S2: Fluorescence polarization technology is used to measure the fluorescence polarization value of the test system using a multifunctional microplate reader. Based on the fluorescence polarization value, it is determined whether the test compound can bind to the orthosteric site of RORγ, and the IC is calculated by fitting. 50 and Ki value.

[0031] Beneficial Effects: The compounds developed in this invention exhibit significant fluorescence performance advantages over conventional technologies, enabling highly selective targeting of the orthosteric binding site of the RORγ receptor ligand-binding domain. A fluorescence polarization probe system constructed based on these compounds, utilizing the principle of competitive ligand displacement, enables the establishment of a novel RORγ orthosteric inhibitor screening platform. This technical solution overcomes the limitations of traditional activity assays, providing an economical, timely, and reliable activity evaluation tool for drug development targeting the RORγ orthosteric site, accelerating the discovery and structural optimization of lead compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the curve of the fluorescence polarization signal of the probe changing with the concentration of fluorescent probes P1-P11.

[0033] Figure 2 The curve of the fluorescence polarization signal of the probe changing with the concentration of RORγ-LBD protein

[0034] Figure 3 To test the affinity of the reported RORγ orthosteric inhibitors T0901317, SR1001, Ursolic acid and the RORγ allosteric inhibitor MRL-871 for the RORγ orthosteric site using P5

[0035] Figure 4 The fluorescence polarization assay performance of probe P5 was evaluated by Z' factor statistical experiment.

[0036] Figure 5 This is the effect of DMSO content on the binding of fluorescent probe P5 to protein RORγ-LBD.

[0037] Figure 6 This is an imaging study of RORγ using probe P5 in A375 cells. DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to the examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions in the art or the conditions recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are all commercially available from conventional markets. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention fall within the scope of protection claimed in the present invention.

[0039] Example 1:

[0040] Synthesis of probe P1:

[0041]

[0042] To a mixture of BI-1 (800 mg, 1.60 mmol, 1.0 equiv.), HATU (911.7 mg, 2.40 mmol, 1.5 equiv.), and DIEPA (620.3 mg, 4.79 mmol, 3.0 equiv.) was added DCM (20 mL) under an ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of N-Boc-1,2-ethylenediamine (384.2 mg, 2.4 mmol, 1.5 equiv.) dissolved in DCM (10 mL) under an ice-water bath. The mixture was allowed to react overnight under nitrogen at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 × 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 940 mg of BI-2-L1 as a white crystalline solid; yield: 91.5%. 1 HNMR(500MHz,DMSO-d6)δ8.60(t,J=5.9Hz,1H),7.99-7.90(m,1H),7.89-7.82(m,1H),7.68( d,J=8.3Hz,1H),7.63-7.57(m,1H),7.52(d,J=8.1Hz,1H),7.45(t,J=8.6Hz,1H),7.37-7.29 (m,1H),7.21(d,J=8.3Hz,2H),6.91(p,J=5.8,5.0Hz,1H),3.32-3.27(m,2H),3.18(s,3H),3 .11(q,J=6.6Hz,2H),2.70(s,4H),1.66(q,J=6.1Hz,4H),1.54(p,J=5.6Hz,2H),1.36(s,9H).

[0043] To a solution of BI-2-L1 (940 mg, 1.47 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 789 mg of BI-3-L1 as a white solid (98.9% yield). 1H NMR (400MHz, DMSO-d6) δ8.76(t,J=5.7Hz,1H),7.96(s,1H),7.89(d,J=8.4Hz,1H),7.84(s,1H),7.7 2(d,J=8.3Hz,1H),7.60(q,J=8.0,7.4Hz,1H),7.53(d,J=8.1Hz,1H),7.45(t,J=8.6Hz,1H),7.35(d ,J=8.3Hz,1H),7.20(d,J=9.9Hz,2H),3.52(q,J=6.2Hz,2H),3.18(s,3H),3.00(t,J=6.3Hz,2H),2. 84(d,J=22.0Hz,2H),2.69(d,J=2.2Hz,2H),1.80-1.71(m,2H),1.67(s,4H),1.54(t,J=6.2Hz,2H).

[0044] BI-3-L1 (789 mg, 1.73 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (8 mL). Fluorescein isothiocyanate (631.1 mg, 1.62 mmol, 1.0 equiv.) and DIPEA (142 mg, 1.077 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P1, as an orange-yellow solid (532 mg, 38.7%). 1H NMR(400MHz,DMSO-d6)δ10.64(s,1H),10.24(s,2H),8.85(s,1H),8.61(s,1H),8.33(s,1H),8.01(s,1H),7.95-7.88(m, 1H),7.81(d,J=8.8Hz,1H),7.67(d,J=8.1Hz,1H),7.59(q,J=7.5Hz,1H),7.51(d,J=8.1Hz,1H),7.44(t,J=8.5Hz,1H),7 .32(d,J=7.6Hz,1H),7.23(d,J=9.9Hz,2H),7.14(d,J=8.5Hz,1H),6.70(s,2H),6.63-6.52(m,4H),3.73(t,J=6.4Hz,2H ),3.53(q,J=6.4Hz,2H),3.43(s,2H),3.16(s,3H),2.91-2.72(m,2H),1.77-1.56(m,4H),1.51(t,J=6.3Hz,2H).HPLC:t R =11.256min,95.08% purity.

[0045] Example 2:

[0046] Synthesis of probe P2:

[0047]

[0048] To a mixture of BI-1 (650 mg, 1.30 mmol, 1.0 equiv.), HATU (741.9 mg, 1.95 mmol, 1.5 equiv.), and DIEPA (503.2 mg, 3.90 mmol, 3.0 equiv.) was added DCM (20 mL) under an ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of N-Boc-1,4-butanediamine (367.3 mg, 1.95 mmol, 1.5 equiv.) in DCM (10 mL) under an ice-water bath. The mixture was allowed to react overnight under nitrogen at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 658 mg of BI-2-L2 as a white crystalline solid; yield 75.5%. 1H NMR (500MHz, DMSO-d6) δ8.59(t,J=5.6Hz,1H),7.94(d,J=2.3Hz,1H),7.87(dd,J=8.4,2.2Hz,1H),7.6 8(d,J=8.4Hz,1H),7.63-7.57(m,1H),7.52(d,J=8.1Hz,1H),7.45(t,J=8.6Hz,1H),7.36-7.30(m,1H) ,7.25-7.18(m,2H),6.79(t,J=5.8Hz,1H),3.26(q,J=6.5Hz,2H),3.18(s,3H),2.93(q,J=6.4Hz,2H), 2.70(s,4H),1.75-1.59(m,4H),1.51(dq,J=14.3,7.0,6.6Hz,4H),1.41(t,J=7.4Hz,2H),1.36(s,9H).

[0049] To a solution of BI-2-L2 (650 mg, 0.97 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 490 mg of BI-3-L2 as a white solid (90.2% yield). 1 H NMR (400MHz, DMSO-d6) δ8.67(d,J=6.5Hz,1H),7.94(s,1H),7.87(d,J=8.5Hz,1H) ,7.77-7.72(m,2H),7.71-7.67(m,1H),7.60(q,J=7.7Hz,1H),7.52(d,J=8.2Hz,1H ),7.45(t,J=8.6Hz,1H),7.35(d,J=8.5Hz,1H),7.27-7.15(m,2H),3.30(d,J=5.8H z,2H),3.18(s,3H),3.15-2.65(m,6H),1.80-1.61(m,4H),1.55(d,J=16.6Hz,6H).

[0050] BI-3-L2 (200 mg, 0.351 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (3 mL). Fluorescein isothiocyanate (150.1 mg, 0.386 mmol, 1.0 equiv.) and DIPEA (150 mg, 1.160 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P2, as an orange-yellow solid (120 mg, 35.7%). 1 H NMR (400MHz, DMSO-d6) δ10.23(d,J=80.4Hz,2H),8.66(t,J=5.9Hz,1H),8.30(d,J=26.9Hz,2H),7.96(d,J=2.2Hz,1H),7.88(dd,J =8.3,2.2Hz,1H),7.74(d,J=7.3Hz,1H),7.67(d,J=8.3Hz,1H),7.59(dd,J=14.5,8.1Hz,1H),7.51(d,J=8.0Hz,1H),7.44(t,J=8.6 Hz,1H),7.34(q,J=6.4,5.8Hz,1H),7.21(d,J=8.4Hz,2H),7.14(d,J=8.3Hz,1H),6.66-6.61(m,4H),6.55(dd,J=8.6,2.1Hz,2H),3 .54(s,2H),3.44(s,2H),3.34-3.31(m,2H),3.17(s,3H),2.91-2.73(m,2H),1.71-1.56(m,8H),1.52(q,J=6.3,5.8Hz,2H).HPLC:t R =12.688min,96.18% purity.

[0051] Example 3:

[0052] Synthesis of probe P3:

[0053]

[0054] To a mixture of BI-1 (620 mg, 1.237 mmol, 1.0 equiv.), HATU (702.5 mg, 1.850 mmol, 1.5 equiv.), and DIEPA (480.1 mg, 3.70 mmol, 3.0 equiv.) was added DCM (20 mL) under an ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of N-Boc-1,6-hexanediamine (348.3 mg, 1.85 mmol, 1.5 equiv.) in DCM (10 mL) under an ice-water bath. The mixture was allowed to react overnight under nitrogen at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 × 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 557 mg of BI-2-L3 as a white crystalline solid; yield: 64.5%. 1 H NMR (400MHz, DMSO-d6) δ8.57(d,J=6.3Hz,1H),7.95(d,J=3.0Hz,1H),7.87(d,J=8.3Hz,1H),7.66(dd,J=8.4,2.3 Hz,1H),7.58(t,J=7.2Hz,1H),7.51(d,J=8.1Hz,1H),7.44(t,J=8.7Hz,1H),7.34(d,J=7.3Hz,1H),7.24-7.18(m ,2H),6.76(d,J=6.1Hz,1H),3.35(d,J=2.2Hz,4H),3.25(t,J=7.0Hz,2H),3.17(d,J=2.2Hz,3H),2.90(q,J=6.8H z,2H),1.76(p,J=6.5Hz,2H),1.69-1.57(m,4H),1.52(t,J=7.5Hz,4H),1.36(d,J=2.2Hz,9H),1.30-1.25(m,4H).

[0055] To a solution of BI-2-L3 (557 mg, 0.80 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. The reaction was complete by TLC. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 457 mg of BI-3-L3 as a white solid (95.5% yield). 1H NMR (400MHz, DMSO-d6) δ8.60(d,J=6.3Hz,1H),7.93(d,J=3.2Hz,1H),7.87(d,J=8.5Hz,1H),7.70-7. 68(m,1H),7.67(d,J=2.6Hz,1H),7.60(q,J=7.0,6.4Hz,1H),7.52(d,J=8.1Hz,1H),7.44(t,J=8.6Hz, 1H),7.35-7.33(m,1H),7.23-7.21(m,2H),7.19(s,1H),3.26(t,J=7.0Hz,2H),3.18(d,J=2.5Hz,3H) ,2.79(t,J=7.0Hz,2H),2.69(d,J=2.5Hz,6H),1.67(s,4H),1.52(q,J=7.0Hz,6H),1.37-1.27(m,4H).

[0056] BI-3-L3 (200 mg, 0.334 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (3 mL). Fluorescein isothiocyanate (130.4 mg, 0.334 mmol, 1.0 equiv.) and DIPEA (130 mg, 1.002 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P3, as an orange-yellow solid (164 mg, 49.8%). 1H NMR (400MHz, DMSO-d6) δ10.14(s,2H),9.89(s,1H),8.60(d,J=6.3Hz,1H),8.24(s,1H),8.11(s,1H),7.95(s,1H),7.87( d,J=8.3Hz,1H),7.78-7.70(m,1H),7.67(d,J=8.3Hz,1H),7.59(q,J=7.2Hz,1H),7.51(d,J=8.1Hz,1H),7.44(t,J=8.7Hz ,1H),7.33(t,J=7.4Hz,1H),7.21(d,J=8.5Hz,2H),7.17(d,J=8.3Hz,1H),6.68(s,2H),6.62-6.55(m,4H),3.51(s,2H), 3.42(d,J=22.2Hz,2H),3.31-3.25(m,2H),3.17(s,3H),2.84(d,J=23.7Hz,2H),1.72-1.50(m,10H),1.37(s,4H).HPLC:t R =10.035min,95.26% purity.

[0057] Example 4

[0058] Synthesis of probe P4:

[0059]

[0060] To a mixture of BI-1 (680 mg, 1.36 mmol, 1.0 equiv.), HATU (776.3 mg, 2.04 mmol, 1.5 equiv.), and DIEPA (775.7 mg, 4.08 mmol, 3.0 equiv.) was added DCM (20 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of N-BOC-4-(4-piperidinylmethyl)piperazine (577.8 mg, 2.04 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath. The mixture was allowed to react overnight under nitrogen atmosphere at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 819 mg of white crystalline solid BI-2-L4; the yield was 78.6%. 1H NMR (500MHz, DMSO-d6) δ7.65(d,J=8.2Hz,1H),7.62-7.56(m,1H),7.52(d,J=8.1Hz,1H),7.45(dt,J=14.8,5. 2Hz,3H),7.31(dd,J=14.2,8.0Hz,1H),7.21(d,J=7.8Hz,2H),3.91(d,J=12.8Hz,2H),3.61(s,2H),3.42(d,J= 33.5Hz,2H),3.17(s,3H),2.91-2.76(m,2H),2.68(q,J=7.5,6.4Hz,2H),2.37(d,J=23.7Hz,4H),2.15(d,J=6 .6Hz,2H),1.67(d,J=12.2Hz,8H),1.56-1.50(m,2H),1.39(s,9H),1.26-1.23(m,1H),0.94(d,J=10.8Hz,2H).

[0061] To a solution of BI-2-L4 (819 mg, 1.07 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 637 mg of BI-3-L4 as a white solid (yield 89.4%). 1 H NMR (400MHz, DMSO-d6) δ7.64(d,J=8.1Hz,1H),7.61-7.55(m,1H),7.51(d,J=8.0Hz,1H),7.47-7.43(m,2H),7.42-7.39(m,1H),7.35-7.29(m,1H) ,7.23-7.18(m,2H),3.17(s,3H),3.03-2.75(m,4H),2.44(q,J=7.1Hz,4 H), 2.26 (s, 4H), 2.10 (d, J = 6.8Hz, 2H), 1.85-1.48 (m, 11H), 1.09 (s, 1H).

[0062] BI-3-L4 (90 mg, 0.135 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (2 mL). Fluorescein isothiocyanate (53.2 mg, 0.135 mmol, 1.0 equiv.) and DIPEA (53 mg, 0.405 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P4, as an orange-yellow solid (67 mg, 47.0%). 1 H NMR (400MHz, DMSO-d6) δ10.12(s,2H),9.65(s,1H),7.95(d,J=1.9Hz,1H),7.77(dd,J=8.4,2.0Hz,1H),7.65(d,J =8.1Hz,1H),7.59(td,J=8.2,6.1Hz,1H),7.52(d,J=8.0Hz,1H),7.49-7.40(m,3H),7.37-7.30(m,1H),7.22(d,J= 7.1Hz,2H),7.16(d,J=8.3Hz,1H),6.69(d,J=1.8Hz,2H),6.59(d,J=2.1Hz,4H),3.94(s,4H),3.56-3.38(m,2H),3 .18(s,3H),3.11(d,J=7.5Hz,2H),2.96-2.62(m,4H),2.46(s,4H),2.27-2.15(m,2H),1.96-1.40(m,11H).HPLC:t R =10.908min,97.86% purity.

[0063] Example 5

[0064] Synthesis of probe P5:

[0065]

[0066] To a mixture of BI-1 (500 mg, 0.997 mmol, 1.0 equiv.), HATU (568.6 mg, 1.496 mmol, 1.5 equiv.), and DIEPA (386.3 mg, 2.992 mmol, 3.0 equiv.) was added DCM (20 mL) under an ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of 1-BOC-4-(2-aminoethyl)piperazine (345.1 mg, 1.496 mmol, 1.5 equiv.) in DCM (10 mL) under an ice-water bath. The mixture was allowed to react overnight at room temperature under nitrogen. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 480 mg of white crystalline solid BI-2-L5; the yield was 67.6%. 1 H NMR (400MHz, DMSO-d6) δ8.58-8.52(m,1H),7.93(d,J=2.2Hz,1H),7.86(dd,J=8.4,2.2Hz,1H),7. 68(d,J=8.3Hz,1H),7.60(dd,J=14.5,8.2Hz,1H),7.52(d,J=8.0Hz,1H),7.45(t,J=8.6Hz,1H),7 .37-7.30(m,1H),7.22(s,2H),3.40(q,J=6.5Hz,2H),3.30(t,J=5.0Hz,4H),3.18(s,3H),3.14-2 .64(m,4H),2.47(s,2H),2.38(t,J=4.6Hz,4H),1.67(s,4H),1.54(d,J=6.9Hz,2H),1.39(s,9H).

[0067] To a solution of BI-2-L5 (480 mg, 0.674 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 401 mg of BI-3-L5 as a white solid (97.1% yield). 1H NMR (400MHz, DMSO-d6) δ8.54(t,J=5.6Hz,1H),7.93(d,J=2.2Hz,1H),7.86(dd,J=8.4,2.2Hz,1H),7.68(d ,J=8.4Hz,1H),7.63-7.56(m,1H),7.52(d,J=8.1Hz,1H),7.45(t,J=8.6Hz,1H),7.33(dt,J=11.4,6.1Hz, 1H),7.21(d,J=7.6Hz,2H),3.38(q,J=6.6Hz,3H),3.18(s,3H),3.13-2.75(m,4H),2.68(t,J=4.8Hz,4H), 2.42(q,J=7.0Hz,4H),2.34(t,J=4.9Hz,4H),1.74-1.60(m,4H),1.53(p,J=6.5,5.8Hz,2H),1.23(s,1H).

[0068] BI-3-L5 (200 mg, 0.326 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (3 mL). Fluorescein isothiocyanate (127 mg, 0.326 mmol, 1.0 equiv.) and DIPEA (127 mg, 0.979 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P5, as an orange-yellow solid (131 mg, 40.1%). 1H NMR (400MHz, DMSO-d6) δ10.11(s,2H),9.64(s,1H),8.60(t,J=5.8Hz,1H),8.07-7.92(m,2H),7.88(dd,J=8.4,2.1H z,1H),7.80-7.74(m,1H),7.70(d,J=8.4Hz,1H),7.64-7.56(m,1H),7.52(d,J=8.1Hz,1H),7.44(t,J=8.5Hz,1H),7. 38-7.29(m,1H),7.23(d,J=7.9Hz,2H),7.19-7.13(m,1H),6.69(s,2H),6.59(s,4H),3.96(s,4H),3.46(dtd,J=17. 2,12.0,10.7,6.8Hz,4H),3.18(s,3H),2.85(d,J=20.4Hz,2H),2.57(s,6H),1.67(s,4H),1.58-1.49(m,2H).HPLC:t R =10.944min,97.61% purity.

[0069] Example 6

[0070] Synthesis of probe P6:

[0071]

[0072] To a mixture of BI-1 (500 mg, 0.997 mmol, 1.0 equiv.), HATU (569.0 mg, 1.496 mmol, 1.5 equiv.), and DIEPA (387.2 mg, 2.992 mmol, 3.0 equiv.) was added DCM (20 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of 1-(N-Boc-aminoethyl)piperazine (343.8 mg, 1.496 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath and allowed to react overnight at room temperature under nitrogen. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 600 mg of white crystalline solid BI-2-L6; the yield was 84.4%. 1H NMR(400MHz,DMSO-d6)δ7.65(d,J=8.2Hz,1H),7.58(dd,J=8.2,6.3Hz,1H),7.52 (d,J=8.0Hz,1H),7.49-7.39(m,3H),7.31(t,J=10.4Hz,1H),7.21(d,J=7.3Hz,2 H),6.65(t,J=5.7Hz,1H),3.60(s,2H),3.42(d,J=27.1Hz,2H),3.17(s,3H),3.1 4-2.65(m,6H),2.48-2.31(m,6H),1.66(s,4H),1.57-1.49(m,2H),1.37(s,9H).

[0073] To a solution of BI-2-L6 (600 mg, 0.842 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 473 mg of BI-3-L6 as a white solid (91.7% yield). 1 H NMR (400MHz, DMSO-d6) δ7.65(d,J=8.2Hz,1H),7.58(dd,J=8.3,6.3Hz,1H),7.52(d,J=8.0 Hz,1H),7.48-7.40(m,3H),7.35-7.29(m,1H),7.21(d,J=7.7Hz,2H),3.62(s,2H),3.44-3. 34(m,2H),3.17(s,3H),3.08-2.75(m,4H),2.65(t,J=6.5Hz,2H),2.42(q,J=7.1Hz,4H),2. 35(t,J=6.3Hz,2H),1.66(d,J=13.3Hz,4H),1.53(p,J=6.4,5.9Hz,2H),1.28-1.21(m,2H).

[0074] BI-3-L6 (200 mg, 0.326 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (3 mL). Fluorescein isothiocyanate (127 mg, 0.326 mmol, 1.0 equiv.) and DIPEA (127 mg, 0.979 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P6, as an orange-yellow solid (175 mg, 53.6%). 1 H NMR (400MHz, DMSO-d6) δ10.11(s,2H),10.07(s,1H),8.26(d,J=2.0Hz,1H),8.00(s,1H),7.74(d,J=8.4Hz ,1H),7.66(d,J=8.2Hz,1H),7.59(td,J=8.2,6.0Hz,1H),7.51(d,J=8.1Hz,1H),7.49-7.41(m,3H),7.34- 7.29(m,1H),7.24-7.18(m,3H),6.68(d,J=2.3Hz,2H),6.63-6.55(m,4H),3.66(s,4H),3.41(d,J=28.8Hz ,4H),3.17(s,3H),2.92-2.74(m,2H),2.65-2.51(m,6H),1.65(s,4H),1.52(p,J=6.4,5.6Hz,2H).HPLC:t R =10.877min,99.08% purity.

[0075] Example 7

[0076] Synthesis of probe P7:

[0077]

[0078] To a mixture of BI-1 (500 mg, 0.997 mmol, 1.0 equiv.), HATU (570.1 mg, 1.496 mmol, 1.5 equiv.), and DIEPA (388.2 mg, 2.992 mmol, 3.0 equiv.) was added DCM (20 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (383.1 mg, 1.496 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath. The mixture was allowed to react overnight under nitrogen at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 643 mg of white crystalline solid BI-2-L7; the yield was 87.4%. 1 H NMR(500MHz,DMSO-d6)δ7.65(d,J=8.2Hz,1H),7.62-7.56(m,1H),7.52(d,J=8 .1Hz,1H),7.48-7.40(m,3H),7.35-7.29(m,1H),7.21(d,J=6.7Hz,2H),3.60( s,2H),3.33-3.33(m,2H),3.30(d,J=9.6Hz,4H),3.17(s,3H),3.13-2.65(m,4 H),1.72-1.59(m,4H),1.55-1.48(m,4H),1.42(t,J=5.8Hz,6H),1.39(s,9H).

[0079] To a solution of BI-2-L7 (643 mg, 0.872 mmol, 1.0 equiv.) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 520 mg of BI-3-L7 as a white solid (93.6% yield). 1H NMR (400MHz, DMSO-d6) δ7.65(d,J=8.2Hz,1H),7.59(dd,J=8.2,6.2Hz,1H),7.52(d,J=8.0Hz,1H),7.48-7.40(m,3H),7.34-7.28(m,1H),7.21 (d,J=7.0Hz,2H),3.69(s,2H),3.38(q,J=7.2Hz,2H),3.17(s,3H),2.98-2.65(m,8H),1.66(d,J=13.7Hz,4H),1.54(dd,J=12.5,6.2Hz,11H).

[0080] BI-3-L7 (200 mg, 0.314 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (3 mL). Fluorescein isothiocyanate (122.1 mg, 0.314 mmol, 1.0 equiv.) and DIPEA (122 mg, 0.901 mmol, 3.0 equiv.) were added. After the addition was complete, the mixture was incubated at room temperature under a nitrogen atmosphere in the dark for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P7, as an orange-yellow solid (97 mg, 30.1%). 1 H NMR (400MHz, DMSO-d6) δ9.55(s,1H),7.94(d,J=1.9Hz,1H),7.76(dd,J=8.3,2.0Hz,1H),7.66(d,J=8.2H z,1H),7.59(td,J=8.3,6.2Hz,1H),7.52(d,J=8.1Hz,1H),7.49(d,J=2.0Hz,1H),7.47-7.38(m,2H),7.35 -7.30(m,1H),7.22(d,J=7.1Hz,2H),7.15(d,J=8.2Hz,1H),6.68(d,J=2.0Hz,2H),6.59(d,J=2.8Hz,4H) ,3.95(s,4H),3.66(s,2H),3.45(s,2H),3.18(s,3H),2.85(d,J=24.0Hz,4H),1.76-1.45(m,14H).HPLC:t R =13.025min,96.32% purity.

[0081] Example 8

[0082] Synthesis of probe P8:

[0083]

[0084] To a mixture of BI-1 (250.0 mg, 0.499 mmol, 1.0 equiv.), HATU (284.4 mg, 0.748 mmol, 1.5 equiv.), and DIEPA (193.9 mg, 1.500 mmol, 3.0 equiv.) was added DCM (10 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (185.7 mg, 0.748 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath. The mixture was allowed to react overnight under nitrogen at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 292.3 mg of white solid BI-2-L8; the yield was 81.7%. 1 H NMR(500MHz,DMSO-d6)δ8.76(dd,J=4.4,1.4Hz,1H),8.67(t,J=5.7Hz,1H),8.53(dd,J=8.5,1.5Hz,1H),7.9 5(d,J=2.3Hz,1H),7.87(dd,J=8.4,2.3Hz,1H),7.69(d,J=8.3Hz,1H),7.60(td,J=8.1,6.0Hz,1H),7.55-7.4 9(m,2H),7.45(t,J=8.6Hz,1H),7.37-7.31(m,1H),7.24-7.18(m,2H),6.74(d,J=6.3Hz,1H),3.56-3.41(m,1 0H),3.36(t,J=6.2Hz,4H),3.18(s,3H),3.04(q,J=6.1Hz,2H),1.67(s,4H),1.57-1.51(m,2H),1.36(s,9H).

[0085] To a solution of BI-2-L8 (280.0 mg, 0.390 mmol, 1.0 equiv.) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. The reaction was complete as determined by TLC. The solvent was evaporated under reduced pressure and the product was used directly in the next step without purification.

[0086] The crude BI-3-L8 product was dissolved in anhydrous DMF (3 mL), and fluorescein isothiocyanate (151.7 mg, 0.390 mmol) and DIPEA (151.2 mg, 1.170 mmol) were added. After the addition was complete, the mixture was protected from light and nitrogen atmosphere was maintained at room temperature for 4 h. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P8, as an orange-yellow solid (67.2 mg, 17.1%). 1 H NMR (500MHz, DMSO-d6) δ10.11(s,2H),9.64(s,1H),8.76(dd,J=4.4,1.4Hz,1H),8.67(t,J=5.7Hz,1H),8.5 3(dd,J=8.5,1.5Hz,1H),7.95(d,J=2.3Hz,1H),7.87(dd,J=8.4,2.3Hz,1H),7.69(d,J=8.3Hz,1H),7.60(t d,J=8.1,6.0Hz,1H),7.55-7.49(m,2H),7.45(t,J=8.6Hz,1H),7.37-7.31(m,1H),7.24-7.18(m,2H),3.56 -3.41(m,10H),3.36(t,J=6.2Hz,4H),3.18(s,3H),3.04(q,J=6.1Hz,2H),1.67(s,4H),1.57-1.51(m,2H).

[0087] Example 9

[0088] Synthesis of probe P9:

[0089]

[0090] To a mixture of BI-1 (250.0 mg, 0.499 mmol, 1.0 equiv.), HATU (284.4 mg, 0.748 mmol, 1.5 equiv.), and DIEPA (193.9 mg, 1.500 mmol, 3.0 equiv.) was added DCM (10 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of tert-butyl (2-(5-aminopentanamido)ethyl)carbamate (193.9 mg, 0.748 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath. The mixture was allowed to react overnight under nitrogen atmosphere at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), followed by washing the organic layer with saturated brine (20 mL) and drying over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 279.4 mg of white solid BI-2-L9; the yield was 75.4%. 1 H NMR (500MHz, DMSO-d6) δ8.60(t,J=5.7Hz,1H),7.94(d,J=2.3Hz,1H),7.87(dd,J=8.4,2.2Hz,1H),7.80(t,J=5.7 Hz,1H),7.68(d,J=8.4Hz,1H),7.60(td,J=8.2,6.1Hz,1H),7.53(d,J=8.1Hz,1H),7.46(t,J=8.6Hz,1H),7.33(d d,J=12.7,7.6Hz,1H),7.24-7.18(m,2H),6.78(t,J=5.7Hz,1H),3.26(q,J=6.3Hz,2H),3.18(s,3H),3.05(q,J=6 .3Hz,2H),2.95(q,J=6.4Hz,2H),2.08(q,J=5.6,4.6Hz,2H),1.77-1.59(m,4H),1.57-1.46(m,7H),1.36(s,9H).

[0091] To a solution of BI-2-L9 (270.0 mg, 0.872 mmol, 1.0 equiv.) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. The reaction was complete by TLC. The solvent was evaporated under reduced pressure and the product was used directly in the next step without purification.

[0092] The crude BI-3-L9 product was dissolved in anhydrous DMF (3 mL), and fluorescein isothiocyanate (156.4 mg, 0.402 mmol) and DIPEA (155.7 mg, 1.205 mmol) were added. After the addition was complete, the mixture was protected from light and nitrogen atmosphere was maintained at room temperature for 4 h. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic layers were purified by silica gel column chromatography to yield the final product, P9, as an orange-yellow solid (107.2 mg, 25.8%). 1 H NMR (500MHz, DMSO-d6) δ10.72(s,2H),10.21(s,1H),8.61(t,J=6.2Hz,1H),8.29(d,J=30.2Hz,1H),8.22(s,1H),8.02(s, 1H),7.94(s,1H),7.87(d,J=8.4Hz,1H),7.77-7.71(m,1H),7.67(d,J=8.3Hz,1H),7.59(q,J=7.5Hz,1H),7.52(d,J=8.2Hz ,1H),7.45(t,J=8.6Hz,1H),7.34(d,J=7.7Hz,1H),7.21(d,J=9.1Hz,2H),7.17(d,J=8.3Hz,1H),6.68(s,2H),6.63-6.55( m,4H),3.58(s,2H),3.27(dq,J=13.0,5.9Hz,4H),3.17(s,3H),3.14-2.72(m,4H),2.16-2.10(m,2H),1.73-1.48(m,10H).

[0093] Example 10

[0094] Synthesis of probe P10:

[0095]

[0096] To a mixture of BI-1 (250.0 mg, 0.499 mmol, 1.0 equiv.), HATU (284.4 mg, 0.748 mmol, 1.5 equiv.), and DIEPA (193.9 mg, 1.500 mmol, 3.0 equiv.) was added DCM (10 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of benzyl (5-((2-aminoethyl)amino)-5-oxopentyl)carbamate (219.4 mg, 0.748 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath. The mixture was allowed to react overnight under nitrogen atmosphere at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 276.3 mg of white solid BI-2-L 10 ; Yield 71.3%. 1 H NMR (500MHz, DMSO-d6) δ8.64(t,J=5.6Hz,1H),7.97-7.89(m,2H),7.86(dd,J=8.3,2.2Hz,1H),7.68(d,J=8.4Hz,1H) ,7.60(td,J=8.4,6.2Hz,1H),7.53(d,J=8.1Hz,1H),7.45(t,J=8.6Hz,1H),7.40-7.26(m,7H),7.22(s,2H),5.00(d,J =7.0Hz,2H),3.30(t,J=6.1Hz,2H),3.22(t,J=6.2Hz,2H),3.18(s,3H),2.96(p,J=6.5,5.8Hz,2H),2.91-2.74(m,2H ),2.09-2.02(m,2H),1.66(d,J=6.3Hz,4H),1.53(p,J=7.3,6.2Hz,2H),1.47(q,J=7.4Hz,2H),1.36(p,J=7.0Hz,2H).

[0097] BI-2-L 10 The product (270.0 mg, 0.872 mmol, 1.0 equiv.) was dissolved in methanol (10 mL) and reacted under a H₂ atmosphere and Pd / C at room temperature for 2 h. The reaction was complete as determined by TLC. The solvent was evaporated under reduced pressure and the product was used directly in the next step without purification.

[0098] BI-3-L 10The crude product was dissolved in anhydrous DMF (3 mL), and fluorescein isothiocyanate (120.6 mg, 0.310 mmol) and DIPEA (120.2 mg, 0.930 mmol) were added. After the addition was complete, the mixture was protected from light and nitrogen atmosphere was maintained at room temperature for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic phases were purified by silica gel column chromatography to yield the final product, P10, as an orange-yellow solid (73 mg, 22.9%). 1 H NMR(500MHz,DMSO-d6)δ10.68(s,2H),10.25(s,1H),8.66(s,1H),8.41(s,1H),8.29(s,1H),8.11(s ,1H),8.01(d,J=14.1Hz,2H),7.89-7.69(m,3H),7.57(s,2H),7.51(d,J=8.4Hz,1H),7.42(d,J=8.3H z,2H),7.32(s,1H),7.15(d,J=8.3Hz,1H),6.67(s,2H),6.58(q,J=9.0Hz,4H),3.45(d,J=30.0Hz,4 H),3.27(s,2H),3.16(s,3H),3.10-2.70(m,4H),2.14(s,2H),1.89(s,2H),1.61(d,J=54.9Hz,10H).

[0099] Example 11

[0100] Synthesis of probe P11:

[0101]

[0102] To a mixture of BI-1 (250.0 mg, 0.499 mmol, 1.0 equiv.), HATU (284.4 mg, 0.748 mmol, 1.5 equiv.), and DIEPA (193.9 mg, 1.500 mmol, 3.0 equiv.) was added DCM (15 mL) under ice-water bath and stirred at room temperature for 30 min. To this mixture was added a solution of tert-butyl (3-(4-aminobutyrylamino)propyl)carbamate (193.9 mg, 0.748 mmol, 1.5 equiv.) in DCM (10 mL) under ice-water bath. The mixture was allowed to react overnight under nitrogen atmosphere at room temperature. TLC confirmed the reaction was complete. The reaction was washed with water (3 x 20 mL), and the organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 288.2 mg of white solid BI-2-L 11 ; Yield 77.8%. 1 H NMR(500MHz,DMSO-d6)δ8.63(t,J=5.6Hz,1H),7.94(d,J=2.3Hz,1H),7.87(dd,J=8.4,2.3Hz,1H),7.79(t,J=5.7Hz,1H), 7.69(d,J=8.4Hz,1H),7.63-7.58(m,1H),7.53(d,J=8.1Hz,1H),7.46(t,J=8.6Hz,1H),7.37-7.31(m,1H),7.22(d,J=8.3H z,2H),6.76(t,J=5.7Hz,1H),3.26(q,J=6.5Hz,2H),3.18(s,3H),3.01(q,J=6.6Hz,2H),2.90(q,J=6.7Hz,2H),2.12(t,J =7.5Hz,2H),1.75(p,J=7.5Hz,4H),1.66(d,J=5.6Hz,2H),1.54(q,J=6.0,5.5Hz,2H),1.48(q,J=6.9Hz,2H),1.37(s,9H).

[0103] To BI-2-L 11 To a solution of the compound (275.0 mg, 0.872 mmol, 1.0 equiv.) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL). The mixture was allowed to react at room temperature under nitrogen for 2 h. After TLC analysis, the reaction was complete. The solvent was evaporated under reduced pressure and the compound was used directly in the next step without purification.

[0104] BI-3-L 11The crude product was dissolved in anhydrous DMF (3 mL), and fluorescein isothiocyanate (138.6 mg, 0.479 mmol) and DIPEA (185.9 mg, 1.439 mmol) were added. After the addition was complete, the mixture was protected from light and nitrogen atmosphere was maintained at room temperature for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The combined organic phases were purified by silica gel column chromatography to yield the final product, P11, as an orange-yellow solid (123.0 mg, 25.8%). 1 H NMR (500MHz, DMSO-d6) δ11.12-10.32(m,2H),10.12(s,1H),8.63(t,J=5.8Hz,1H),8.22(d,J=18.0Hz,2H),7.93(d,J=10.8Hz,2H),7.87( d,J=8.3Hz,1H),7.79-7.72(m,1H),7.68(d,J=8.2Hz,1H),7.59(q,J=7.7Hz,1H),7.51(d,J=8.1Hz,1H),7.44(t,J=8.6Hz,1H),7.32(dd,J =15.3,7.6Hz,1H),7.21(d,J=9.3Hz,2H),7.17(d,J=8.3Hz,1H),6.68(s,2H),6.64-6.54(m,4H),3.51(s,2H),3.28(q,J=6.6Hz,2H),3.17 (s,3H),3.11(q,J=6.9Hz,2H),2.90-2.74(m,2H),2.15(t,J=7.5Hz,2H),1.77(p,J=7.1Hz,2H),1.73-1.58(m,6H),1.52(d,J=8.5Hz,2H).

[0105] Example 12 Optical Properties of Fluorescent Probes in Tris Buffer Solution

[0106] Fluorescent probes P1 to P11 were diluted from a stock solution (10 mM) to 100 μM using a Tris buffer solution (20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0). A multifunctional microplate reader (SpectraMax M5) was used to set the excitation wavelength to 490 nm and scan the corresponding fluorescence excitation intensity of the probe to be tested in the range of 400 to 550 nm to obtain the fluorescence excitation spectrum of the probe. A multifunctional microplate reader (SpectraMax M5) was used to set the emission wavelength to 525 nm and scan the corresponding fluorescence emission intensity value of the probe to be tested at 500 to 600 nm to obtain the fluorescence emission spectrum of the probe. 1 μM fluorescein was prepared in water, and the integrated fluorescence intensity of 1 μM, 10 μM, and 100 μM fluorescein and the maximum absorbance value at the excitation wavelength (490 nm) were measured. The integrated fluorescence intensity of the 100 μM fluorescent probe and the maximum absorbance value at the excitation wavelength (490 nm) were measured. Substitute the obtained parameters into the quantum yield calculation formula to calculate the fluorescence quantum yield of the fluorescent probe solution. The calculation formula is:

[0107]

[0108] Where Y U is the quantum yield of the unknown sample to be measured; Y S is the fluorescence quantum yield of the standard substance; F U 、F S A is the integrated fluorescence intensity of the sample to be tested and the dilute solution of the standard; U 、A S It is the maximum absorbance value of the sample and standard at the excitation wavelength.

[0109] The experimental results are shown in Table 1. The small molecule fluorescent probes described in the present invention can all emit fluorescence under excitation light, with the maximum emission wavelength being around 520 nm. Some probes have a high fluorescence quantum yield.

[0110] Table 1 Maximum excitation and emission wavelengths and fluorescence quantum yields of the fluorescent probes of the present invention

[0111]

[0112]

[0113] Example 13 Protein thermal shift technique to detect the affinity of the probe to RORγ-LBD

[0114] In a 96-well PCR plate, 5 μM RORγ-LBD, 100 μM compound (BI-2-L 1～11) and protein thermal transfer dye (4461146, Thermo Fisher). Four replicates were tested for each concentration. The PCR plate was centrifuged in the dark and placed in a StepOnePlus qPCR instrument (Thermo Fisher Scientific) to determine the protein melting curve. The data were analyzed using PTS software v1.3 (Thermo Fisher Scientific), and the derivative curve was calculated to determine the protein melting curve. PTS software v1.3 (Thermo Fisher Scientific) was used to analyze the data and calculate the derivative curve to obtain the corresponding Tm value. The Tm value of the test well was subtracted from the Tm value of the blank well to obtain the ΔTm value of the test compound. The higher the ΔTm, the stronger the affinity of the compound to the protein. It can be seen from Table 2 that the probe compound precursors disclosed in the present invention all have good affinity with RORγ, which indirectly shows that the probes P1-P11 disclosed in the present invention have good binding to RORγ.

[0115] Table 2 Protein thermal migration technique to detect the affinity of the fluorescent probe of the present invention to RORγ-LBD

[0116] Compound ΔTm(℃) <![CDATA[BI-2-L1]]> 5.3 <![CDATA[BI-2-L2]]> 6.1 <![CDATA[BI-2-L3]]> 5.4 <![CDATA[BI-2-L4]]> 2.9 <![CDATA[BI-2-L5]]> 5.7 <![CDATA[BI-2-L6]]> 4.7 <![CDATA[BI-2-L7]]> 1.7 <![CDATA[BI-2-L8]]> 1.1 <![CDATA[BI-2-L9]]> 1.4 <![CDATA[BI-2-L 10 ]]> 1.2 <![CDATA[BI-2-L 11 ]]> 1.4 .

[0117] Example 14: Testing the affinity of compounds for the orthosteric site of RORγ using a fluorescent probe based on fluorescence polarization technology

[0118] Fluorescence polarization (FP) is a phenomenon in which the light emitted by a fluorescent molecule has unequal intensities along different polarization axes. This phenomenon is currently widely used to detect the binding of macromolecules to small molecules, and has the advantages of being fast, stable, and cheap (Methods Appl. Fluoresc. 2016, 4, 022001). In the present invention, when the fluorescent probe binds to the RORγ-LBD protein, its fluorescence polarization signal, millipolarization units (mP), increases compared to when it is not bound. At this point, adding a test compound that competes with the fluorescent probe for binding to the RORγ-LBD orthosteric site to the system will cause a decrease in mP, and the affinity of the test compound to RORγ-LBD is judged based on the degree of decrease.

[0119] The following is an example of using a fluorescent probe to test the inhibitory activity of a compound on the orthosteric site of RORγ

[0120] 1) Test the probe curve to determine the optimal probe working concentration

[0121] 20 μL of fluorescent probes P1-P11 (starting concentration 10 μM) and 40 μL of Tris buffer solution (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0) were added to the test wells of a 384-well black plate (#3575, Corning). The final working concentration of the RORγ-LBD protein was diluted 3-fold starting from 10 μM, with 3 replicates set for each concentration, for a total of 9 concentration gradients. After addition, the mP value of each well was read using the fluorescence polarization module (ex: 490 nm, em: 525 nm) of a multifunctional microplate reader (SpectraMax M5). The data were curve fitted and analyzed using Graph pad Prism 8.0 ( Figure 1 The experimental results showed that probes P1, P2, P10 and P11 had relatively stable fluorescence polarization measurement performance at a probe concentration of 300 nM, while probes P3-P9 had relatively stable fluorescence polarization measurement performance at a probe concentration of 100 nM.

[0122] 2) Test the probe-protein curve to determine the optimal protein working concentration

[0123] 20 μL of fluorescent probes P1-P11 (final concentration 100 nM or 300 nM), 20 μL of Tris buffer solution (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, PH = 8.0) and 20 μL of RORγ-LBD (aa261-518) protein were added to the test wells of a 384-well black board (#3575, Corning). The final working concentration of the RORγ-LBD protein was diluted 3 times starting from 1 μM, with 3 replicate wells set for each concentration, for a total of 8 concentration gradients. After addition, incubate at 4 ° C for 0.5 hours. The mP value of each well was then read using the fluorescence polarization module (ex: 490 nm, em: 525 nm) of a multifunctional microplate reader (SpectraMax M5), and the data were curve fitted and analyzed using Graph pad Prism 8.0 ( Figure 2 The experimental results showed that probes P5, P2, P4, and P6 had relatively suitable detection performance. When the RORγ-LBD protein concentration was 100 nM, the fluorescence polarization signal reached a high platform, with a good detection window. Therefore, 100 nM was selected as the working concentration of RORγ-LBD protein.

[0124] 3) Using probe P5 to test the affinity of the compound to the orthosteric site of RORγ-LBD

[0125] This example tested the reported RORγ orthosteric inhibitors T0901317, SR1001, Ursolic acid and the RORγ allosteric inhibitor MRL-871, and verified the reliability of this method by comparing the activities tested by this method with those reported in the literature.

[0126] 20 μL of fluorescent probe P5 (final concentration 100 nM) was added to the test wells of a 384-well black board (#3575, Corning), along with 20 μL of RORγ-LBD (final concentration 100 nM) protein and 20 μL of the test compound of the corresponding concentration. The concentrations of T0901317 and MRL-871 were diluted threefold from 3 μM to 1.37 nM, and the test concentrations of SR1001 and Ursolic acid were diluted threefold from 10 μM to 4.57 nM, with three replicate wells set for each concentration. After addition, the cells were incubated at 4°C for 0.5 h, and then the mP value of each well was read using the fluorescence polarization module (ex: 490 nm, em: 525 nm) of a multifunctional microplate reader (SpectraMax M5). The data were curve fitted using Graphpad Prism 8.0 to calculate the Ki value of each compound ( Figure 3 ). The experimental results showed that the Ki values ​​of compounds T0901317, SR1001, and Ursolic acid were 61nM, 160nM, and 186nM, respectively. These results are similar to the data reported for these three compounds, indicating that the activity test method using the fluorescent probe P5 of the present invention can better identify whether a compound has affinity for the orthosteric site of RORγ-LBD. At the same time, MRL-871, a positive drug reported to target the allosteric site of the RORγ ligand binding domain, was not detected using this activity detection method, indicating that the activity test system using the fluorescent probe P5 of the present invention is not affected by RORγ allosteric inhibitors.

[0127] Example 15 Evaluation of Fluorescence Polarization Performance and Stability of Fluorescent Probe P5

[0128] The Z-factor is a combination of signal range and variation and is a key parameter for evaluating the quality of a test method. The Z' factor statistical experiment was used to determine the analytical stability of the high signal group (probe P5 bound to the RORγ-LBD protein) and the low signal group (probe P5). The Z' value of the fluorescent probe was calculated by substituting the obtained parameters into the Z' value calculation formula:

[0129] Z′=1-3(σb+σf) / |μb-μf|

[0130] Wherein, σb and σf are the standard deviations of the high signal group and the low signal group, respectively, and μb and μf are the mean values ​​of the high signal group and the low signal group, respectively.

[0131] In the study of small molecule fluorescent probes binding to proteins, DMSO tolerance testing is a crucial quality control step, verifying the effects of DMSO on protein structure, probe fluorescence, and the binding process. In DMSO tolerance experiments, changes in DMSO content affect protein activity and probe fluorescence, leading to variations in Δmp values. The degree of variation is then used to determine the DMSO tolerance of both the probe and protein.

[0132] 1) The following is an example of the performance evaluation of the fluorescence polarization assay of the fluorescent probe P5:

[0133] A Z'-factor statistical experiment was performed to evaluate the fluorescence polarization performance of probe P5. To the test wells of a 384-well black plate (#3575, Corning), 20 μL of fluorescent probe P5 (final concentration 100 nM), 20 μL of Tris buffer (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0), and 20 μL of RORγ-LBD protein (final concentration 100 nM) were added. Correspondingly, to another test well, 20 μL of fluorescent probe P5 (final concentration 100 nM) and 40 μL of Tris buffer (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0) were added. After addition, the cells were incubated at 4°C for 0.5 h, and then the mP value of each well was read using the fluorescence polarization module (ex: 490 nm, em: 525 nm) of a multifunctional microplate reader (SpectraMax M5). The data were curve fitted using Graphpad Prism 8.0 to calculate the Z' value ( Figure 4 The experimental results show that the Z' value of probe P5 is 0.81, which indicates that the activity test method using the fluorescent probe P5 of the present invention has a relatively stable measurement performance.

[0134] 2) The following is an example of a DMSO tolerance experiment between the fluorescent probe P5 and the protein RORγ-LBD:

[0135] To the test wells of a 384-well black plate (#3575, Corning), 20 μL of fluorescent probe P5 (final concentration 100 nM), 20 μL of Tris buffer (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0) containing varying amounts of DMSO, and 20 μL of RORγ-LBD protein (final concentration 100 nM) were added. Correspondingly, to another test well, 20 μL of fluorescent probe P5 (final concentration 100 nM), 20 μL of Tris buffer (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0) containing varying amounts of DMSO, and 20 μL of Tris buffer (20 μL, 20 mM Tris, 150 mM NaCl, 5 mM DTT, pH = 8.0) were added. After addition, the cells were incubated at 4°C for 0.5 h, and then the mP value of each well was read using the fluorescence polarization module (ex: 490 nm, em: 525 nm) of a multifunctional microplate reader (SpectraMax M5). The data were fitted using Graph Pad Prism 8.0 ( Figure 5 The experimental results showed that the probe P5 could maintain a steady state with the protein RORγ-LBD at a DMSO content of 14%, and had a relatively stable binding activity.

[0136] Example 16 Cell Imaging Experiment with Fluorescent Probes

[0137] A375 cells were cultured in two culture dishes using DMEM medium. Culture dish one: fixed with 4% paraformaldehyde for 15 minutes. Incubated with probe P5 (1 μM) for 1.0 hour. Culture dish two: fixed with 4% paraformaldehyde for 15 minutes. Incubated with BI-7 (10 μM) and probe P5 (1 μM) for 1.0 hour. Afterwards, the cells in the two culture dishes were stained with DAPI. The fluorescence imaging results were recorded using the cell fluorescence detection method using Zeiss LSM 800 (Carl Zeiss, Germany). The relevant fluorescence parameters are: FITC (Ex: 494 / Em: 518), DAPI (Ex: 359 / Em: 461). From Figure 6 The results show that P5, as a RORγ probe, clearly stains A375 cells, which highly express RORγ. When the A375 cells were co-incubated with the fluorescent probe and the RORγ orthosteric inhibitor BI-7, the fluorescence intensity was significantly reduced. This indicates that the fluorescent probe P5 specifically binds to the RORγ orthosteric binding pocket and can be used for on-cell imaging of RORγ.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A novel fluorescent compound targeting the orthosteric site of RORγ, characterized in that: The invention includes compounds of the following general formula (I) or pharmaceutically acceptable salts thereof: In the general formula (I): L stands for 2. The method for preparing the fluorescent compound according to claim 1, characterized in that: The following steps are involved: S1: React the raw materials BI-1, HATU and different amine compounds under alkaline conditions at room temperature to obtain the intermediate BI-2-L 1～9,11 ; S2: React the raw material BI-1 with HATU and benzyl (5-[(2-aminoethyl)amino]-5-oxopentyl) carbamate under alkaline conditions at room temperature to obtain the intermediate BI-2-L 10 ; S3: Raw material BI-2-L 1～9,11 Dissolved in dichloromethane, reacted at room temperature under acidic conditions, and removed the tert-butyloxycarbonyl group Boc to obtain compound BI-3-L 1～9,11 ; S4: Raw material BI-2-L 10 Dissolved in methanol, reacted at room temperature under hydrogen atmosphere, and removed the benzyloxycarbonyl Cbz to obtain compound BI-3-L 10 ; S5: Intermediate AI-3-L 1～11 The final products P1 to P11 are obtained by condensation reaction with fluorescein isothiocyanate FITC under alkaline conditions.

3. The method for preparing the fluorescent compound according to claim 2, wherein: In the step S1, the diamine compound used is a mono-Boc protected diamine.

4. The method for preparing the fluorescent compound according to claim 2, wherein: In the step S1, the organic solvent used in the reaction is dichloromethane; the base used in the reaction is N,N-diisopropylethylamine; and the reaction temperature is 20-25°C.

5. The method for preparing the fluorescent compound according to claim 2, wherein: In step S2, the organic solvent used in the reaction is dichloromethane; the base used in the reaction is N,N-diisopropylethylamine; and the reaction temperature is 20-25°C.

6. The method for preparing a fluorescent compound according to claim 2, wherein: In the step S3, the acidic reagent is trifluoroacetic acid or dioxane hydrochloride solution.

7. The method for preparing a fluorescent compound according to claim 2, wherein: In step S5, the alkaline condition used in the condensation reaction is N,N-diisopropylethylamine, the solvent used is N,N-dimethylformamide, and the reaction conditions are room temperature and nitrogen protection.

8. An affinity detection method system for RORγ orthosteric inhibitors constructed using the compound described in claim 1, the system comprising the compound described in claim 1 at a concentration of 40-1000 nM, RORγ protein at a concentration of 10-1000 nM, and a test compound.

9. Use of the fluorescent compound according to claim 1 in testing the affinity of a compound for RORγ.

10. Use of the fluorescent compound according to claim 1 in cell imaging or in vivo imaging.

Images