A small molecule fluorescent probe compound and its preparation method and application

Abstract

The present invention discloses a small molecule fluorescent probe compound and a preparation method and application thereof. The compound is P2Y 14 R is a small molecule fluorescent probe with fluorescent properties for P2Y 14 R has good specific binding, and this compound can be used as a fluorescence polarization probe to achieve P2Y through a competitive method. 14 The screening of R ligands and the evaluation of ligand affinity levels are used in the screening method for subsequent targeting of P2Y 14 It provides a basis for high-throughput screening of R drugs.

Description

Technical Field

[0001] The present invention relates to a fluorescent probe compound, in particular to a small molecule fluorescent probe compound, and also to a preparation method and application of the fluorescent probe compound. Background Art

[0002] P2Y 14 Receptor (P2Y 14 P2Y has been shown to be a potential target for innate immune inflammatory diseases, such as inflammatory bowel disease, diabetes, cystic fibrosis, renal sterile inflammation, acute gouty arthritis and allergic diseases, and is an important drug target in the GPCR family. 14 Currently, only a few types of receptor antagonists have been reported (pyrimidopyridine, 2-naphthoic acid, 3-substituted benzoic acid and pyrazole-3-carboxylic acid), but they are still in the preclinical research stage. Among them, 2-naphthoic acid has the highest activity and selectivity. However, the antagonists of 2-naphthoic acid structure reported so far have defects such as poor solubility, low oral bioavailability, and difficulty in synthesis and purification, which have brought great difficulties to further discussion of structure-activity relationship and biological evaluation. Therefore, it is necessary to find P2Y with more diverse structures and stronger activity. 14 R antagonists still need to be developed.

[0003] In order to find a new type of P2Y 14 Researchers have established some screening methods for P2Y R antagonists. For example, the content test of downstream signal cAMP at the cellular level. However, the screening experiment at the cellular level has many disadvantages such as many influencing factors and long screening cycle, which makes it difficult to achieve rapid and large-scale screening of compounds. However, the radioligand binding experiment has two problems: safety and high cost. Therefore, it is necessary to develop a new test method for high-throughput screening of P2Y 14 R ligand. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a small molecule fluorescent probe compound, and also to provide a preparation method of the above compound and its application in targeting P2Y 14 Application in high-throughput screening of R drugs.

[0005] Technical solution: The present invention discloses a P2Y 14 R small molecule fluorescent probe compound, the compound is P2Y 14 R is a small molecule fluorescent probe compound, the structure of which is shown in Formula I:

[0006] .

[0007] The preparation method of the above compound comprises the following steps:

[0008] (1) Compound T1 is subjected to a deprotection reaction and then subjected to an amide condensation reaction with N-Boc-γ-aminobutyric acid to obtain intermediate T2;

[0009] (2) The intermediate T2 is subjected to a deprotection reaction and then subjected to an amide condensation reaction with N-(tert-butyloxycarbonyl)-5-aminopentanoic acid to obtain the intermediate T3;

[0010] (3) Deprotecting the intermediate T3 to obtain the intermediate T4;

[0011] (4) The intermediate T4 is subjected to a nucleophilic addition reaction with fluorescein isothiocyanate FITC to obtain a compound of formula I; the general reaction formula is as follows:

[0012] .

[0013] Wherein, in step (1), the molar ratio of the compound T1 to N-Boc-γ-aminobutyric acid is 1:1 to 1:2, the reaction temperature is room temperature, and the reaction time is 12 to 24 hours.

[0014] Wherein, in steps (1) and (2), the amide condensation reaction uses one or more of EDCI, HOAT, HATU or BOP as the condensing agent, one or more of DIPEA or TEA as the base, and one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide as the organic solvent.

[0015] Wherein, in step (2), the deprotection reaction reagent is trifluoroacetic acid or hydrogen chloride ethyl acetate solution; the molar ratio of the compound T2 and N-(tert-butyloxycarbonyl)-5-aminopentanoic acid is 1:1 to 1:2; the reaction temperature is room temperature, and the reaction time is 12 to 24 hours.

[0016] Wherein, in step (3), the base used in the deprotection reaction is one or more of sodium hydroxide, potassium hydroxide or lithium hydroxide, and the solvent is two or more of water, tetrahydrofuran, methanol or ethanol; the reaction temperature is room temperature, and the reaction time is 4-24h.

[0017] Wherein, in step (4), the solvent used in the nucleophilic addition reaction is one or more of tetrahydrofuran, 1,4-dioxane or N,N-dimethylformamide, and the base is one or both of DIPEA and TEA; the reaction temperature is room temperature, and the reaction time is 12-24 hours.

[0018] Wherein, in step (4), the molar ratio of the compound T4 to fluorescein isothiocyanate is 1:1 to 1:2.

[0019] The above compounds were used as fluorescence polarization probes in P2Y 14Application in R ligand screening.

[0020] The application is specifically: the compound of the present invention, P2Y 14 R protein and the test compound are mixed to obtain a mixture; the polarization value of the mixture is measured using fluorescence polarization technology, and the test compound is confirmed to be P2Y according to the polarization value 14 R ligand; in general, when the polarization value decreases by more than 25% of the detection window, it can be determined to be P2Y 14 R's ligand.

[0021] Principle of the invention: P2Y 14 R small molecule fluorescent probe compound, based on the reported high activity and high selectivity of P2Y 14 R receptor antagonist PPTN. Through structural analysis of PPTN, it was found that it is mainly composed of four parts, namely the carboxyl group bound to the hydrogen bond network, the 4-trifluoromethylphenyl group bound to the hydrophobic pocket, the phenylpiperidine group pointing to the extracellular solvation area, and the naphthalene ring connecting ring that maintains the appropriate spatial orientation of the above three. Therefore, the phenylpiperidine pointing to the extracellular area is a very suitable connection site for connecting fluorescein without affecting its overall connection with P2Y 14 Binding of R protein.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The P2Y 14 R small molecule fluorescent probe compound has fluorescent properties and is effective for P2Y 14 R protein; (2) the compound is used as a fluorescence polarization probe, and the fluorescence polarization technology can be used to achieve P2Y 14 Screening of R ligands and evaluation of ligand affinity levels; A P2Y 14 The R ligand high-throughput screening scheme can be used for high-throughput screening of drugs and has the characteristics of being cheap, stable, rapid and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the curve of the fluorescence intensity of the fluorescence polarization probe in the buffer solution versus concentration;

[0024] Figure 2 Fluorescence polarization probe and P2Y 14 Saturation curve of R;

[0025] Figure 3 Fluorescence polarization probe and P2Y 14 Competition curves of R protein in the presence of different concentrations of positive compounds. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0027] Example 1

[0028] P2Y 14 The synthetic route of R small molecule fluorescent probe compound is as follows:

[0029] ;

[0030] The specific synthesis method comprises the following steps:

[0031] (1) Preparation of ethyl 4-(4-(1-(4-((tert-butyloxycarbonyl)amino)butanoyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (Intermediate T2):

[0032] Compound T1 (200 mg, 0.331 mmol) was dissolved in HCl / EtOAc solution (5 mL) at room temperature and stirred for 8 hours. The resulting reaction mixture was concentrated under reduced pressure to give the corresponding amino intermediate (white solid). EDCI (76.2 mg, 0.398 mmol), HOBT (53.7 mg, 0.398 mm) and TEA (80.5 mg, 0.795 mmol) were added to a solution of N-Boc-γ-aminobutyric acid (67.3 mg, 0.331 mmol) in anhydrous DMF (5 mL) at 0 °C and stirred for 30 minutes. The amino intermediate was then added to the reaction mixture at room temperature and stirred until complete conversion by TLC analysis. The mixture was diluted with water (50 mL) and extracted with EtOAc (3×30 mL). The combined organics were washed with brine and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain intermediate T2 as a white solid (201 mg, yield: 88%) 1 H NMR (300 MHz, DMSO- d 6 ) 1 H NMR (300 MHz, DMSO- d 6 ) δ (ppm) 8.79 (d, J = 1.5 Hz, 1H), 8.71 (d, J = 1.9Hz, 1H), 8.08 (t, J = 8.0 Hz, 3H), 7.98 (d, J= 8.9 Hz, 1H), 7.95 – 7.86 (m, 3H), 7.49 (s, 4H), 6.89 (t, J = 5.5 Hz, 1H), 4.62 (d, J = 12.6 Hz, 1H), 4.42 (q, J = 7.1Hz, 2H), 4.03 (d, J = 13.2 Hz, 1H), 3.16 (t, J = 12.7 Hz, 1H), 3.02 – 2.85 (m,3H), 2.64 (d, J = 12.3 Hz, 1H), 2.38 (d, J = 4.2 Hz, 2H), 1.91 (s, 2H), 1.72 –1.53 (m, 4H), 1.42 – 1.37 (m, 12H).

[0033] (2) Preparation of ethyl 4-(4-(1-(4-(5-(tert-butyloxycarbonyl)amino)pentanoyl)butyryl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (Intermediate T3):

[0034] Compound T2 (200 mg, 0.290 mmol) was dissolved in HCl / EtOAc solution (5 mL) at room temperature and stirred for 8 hours. The resulting reaction mixture was concentrated under reduced pressure to give the corresponding amino intermediate (white solid). EDCI (66.8 mg, 0.348 mmol), HOBT (47.1 mg, 0.348 mm) and TEA (70.5 mg, 0.696.9 mmol) were added to a solution of N-(tert-butyloxycarbonyl)-5-aminopentanoic acid (63.1 mg, 0.290 mmol) in anhydrous DMF (5 mL) at 0°C and stirred for 30 minutes. The amino intermediate was then added to the reaction mixture at room temperature and stirred until complete conversion by TLC analysis. The mixture was diluted with water (50 mL) and extracted with EtOAc (3×30 mL). The combined organics were washed with brine and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain intermediate T3 as a white solid (185 mg, yield: 82%) 1 H NMR (300 MHz, Chloroform- d ) δ 8.74 (s, 1H), 8.29 (d, J= 1.9 Hz, 1H), 8.10 (dd, J =5.2, 3.5 Hz, 2H), 7.93 – 7.77 (m, 5H), 7.55 (d, J = 7.7 Hz, 2H), 7.42 (d, J = 7.8Hz, 2H), 6.53 (s, 1H), 4.91 (d, J = 13.2 Hz, 1H), 4.76 (s, 1H), 4.52 (q, J = 7.1Hz, 2H), 4.09 (d, J = 13.5 Hz, 1H), 3.45 – 3.13 (m, 5H), 2.94 (s, 1H), 2.77 (t, J = 12.8 Hz, 1H), 2.55 (t, J = 6.9 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.12 (s,1H), 2.08 – 1.92 (m, 5H), 1.77 (dp, J = 22.1, 8.8, 7.2 Hz, 5H), 1.65 – 1.48 (m,12H).

[0035] (3) Preparation of 4-(4-(1-(4-(5-(tert-butyloxycarbonyl)amino)pentanoyl)butyryl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid (Intermediate T4):

[0036] Potassium hydroxide (1M aqueous solution, 1 mL, 1 mmol) was added to a solution of intermediate T3 (150 mg, 0.194 mmol) in MeOH (1 mL) and THF (1 mL) at room temperature until complete conversion by TLC analysis. The solvent was concentrated in vacuo and the residue was acidified to pH = 1 with 1M hydrochloric acid. The resulting precipitate was filtered to give intermediate T4 (128 mg, yield: 88.5%) as a white solid. 1 H NMR (300 MHz, DMSO- d 6 ) δ 8.79 – 8.73 (m, 1H), 8.69 – 8.62 (m, 1H), 8.13 – 7.77 (m, 9H), 7.49 (d, J = 2.4 Hz, 5H), 4.62 (d, J= 12.6 Hz, 1H), 4.02(d, J = 13.2 Hz, 1H), 3.24 – 3.03 (m, 4H), 2.91 (q, J = 7.6, 6.8 Hz, 3H), 2.66(t, J = 12.5 Hz, 1H), 2.44 – 2.33 (m, 2H), 2.07 (t, J = 7.2 Hz, 2H), 1.92 (s,1H), 1.82 (s, 1H), 1.67 (p, J = 8.1, 7.4 Hz, 3H), 1.50 (q, J = 7.5, 6.8 Hz, 3H),1.37 (s, 9H).

[0037] (4) Preparation of 4-(4-(1-(5-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthenyl]-5-yl)thiourea)pentanamido)butyryl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid (target compound):

[0038] Intermediate T4 (100 mg, 0.134 mmol) was dissolved in HCl / EtOAc solution (3 mL) at room temperature and stirred for 8 h. The resulting reaction mixture was concentrated under reduced pressure to obtain the corresponding amino intermediate as a white solid. TEA (22.3 mg, 0.22 mmol) and FITC (28.5 mg, 0.073) were added to a solution of the amino intermediate (50 mg, 0.073 mmol) in anhydrous DMF (1 mL). The mixture was stirred for 12 h at room temperature in the dark. The mixture was diluted with water (10 mL) and acidified to pH = 4 with 1 M hydrochloric acid. The resulting precipitate was filtered to obtain a crude product, which was recrystallized from a mixed solvent of methanol and water to obtain the target compound (67.2 mg, 44.3%) as an orange solid. HPLC purity: 95.03%. mp 291 °C (decomposed). 1 H NMR (300 MHz, DMSO- d 6 ) δ (ppm) 10.38 (s, 2H), 8.73 (s, 1H), 8.62 (s, 2H), 8.31 (s, 1H),8.07 (d, J = 8.1 Hz, 2H), 8.01 – 7.85 (m, 6H), 7.80 (d,J = 8.5 Hz, 1H), 7.45 (q, J = 7.8 Hz, 4H), 7.18 (d, J = 8.3 Hz, 1H), 6.66 (d, J = 17.3 Hz, 3H), 6.59 (d, J =6.2 Hz, 3H), 4.65 – 4.55 (m, 1H), 4.04 – 3.95 (m, 1H), 3.14 (q, J = 7.8 Hz,4H), 2.89 (d, J = 13.3 Hz, 1H), 2.70 – 2.57 (m, 1H), 2.38 (d, J = 7.6 Hz, 2H),2.20 (d, J = 7.2 Hz, 2H), 1.86 (d, J = 8.2 Hz, 4H), 1.73 – 1.66 (m, 2H), 1.52 (s,2H), 1.25 (d, J = 6.3 Hz, 4H), 0.92 – 0.84 (m, 1H). 13 C NMR (75 MHz, DMSO) δ(ppm) 179.96, 171.23, 169.52, 168.09, 158.99, 151.40, 146.46, 144.72, 143.07,142.88, 141.07, 138.92, 136.69, 135.85, 132.72, 131.62, 129.78, 129.78,129.25, 128.54, 127.52, 127.42, 127.28, 126.55, 126.37, 125.82, 125.44,125.41, 123.47, 115.94, 112.08, 109.24, 101.73, 82.55, 44.95, 41.09, 37.79,32.80, 32.45, 32.21, 29.35, 28.53, 24.49, 24.26.HRMS (ESI-TOF) m / z calc’d forC 58 H 49 F 3 N 4 O 9 S [M+H]+ 1049.3402, found 1049.3411.

[0039] Example 2

[0040] P2Y 14 Preparation of R protein:

[0041] Stably expressing P2Y 14 HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% nonessential amino acids, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, and 3.7 g / L NaHCO3. Human THP-1 cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY), 10% FBS, and 1% penicillin / streptomycin. All cells were cultured at 37°C, 5% CO 2 and 95% air in a humidified atmosphere. The cell pellet was scraped from the culture dish, washed with PBS, and centrifuged at 200 g for 10 minutes at 4°C. The pellet was resuspended in 20 ml of buffer I (10 mM HEPES + 10 mM EDTA, pH = 7.4), mixed for 50-60 seconds, and then centrifuged at 39,000 g for 45 minutes at 4°C. After resuspension and re-centrifugation as described above, the resulting pellet was resuspended in 5 ml of storage buffer and stored at -80°C. The membrane protein concentration was determined by Bradford protein assay (Coomassie Plus The better Bradford Assaytm kit, Prod # 23236, Thermo).

[0042] Example 3

[0043] Establishment of fluorescence polarization probe FP method:

[0044] (1) Determination of probe concentration: In order to determine the optimal probe concentration for the FP method, the fluorescence polarization values ​​of different concentrations of fluorescent probes in buffer II (50 mM Tris-HCl, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, pH = 7.4) were detected to determine the probe curve. Figure 1 As shown, the concentration of 16 nM close to the lower platform was selected as the optimal probe concentration for the FP method;

[0045] (2) Saturation assay: using different concentrations of P2Y 14The R protein was mixed with the above-identified fluorescence polarization probe and incubated at room temperature for 30 minutes, and then the fluorescence polarization value of the system was measured; the implementation scheme of the above saturation experiment is as follows: P2Y was diluted with buffer I 14 After the R protein was mixed with 16 nM fluorescence polarization probe, the 384-well plate was covered and incubated with shaking at room temperature for 30 minutes, and the fluorescence polarization value was measured and a saturation curve was drawn for analysis. Figure 2 As shown, the concentration of 0.3 mg / mL close to the upper platform was selected as the optimal protein concentration for the FP method;

[0046] (3) Competition assay: used to determine the affinity between ligand and receptor. 14 After the R protein and the fluorescence polarization probe are mixed with different concentrations of the test compound, the fluorescence polarization value of the system is measured. The compound competes with the fluorescence polarization probe, causing the fluorescence polarization value to decrease as the concentration of the compound increases.

[0047] The implementation scheme of the above competition experiment is as follows: P2Y 14 The positive compounds PPTN, LR-13 and LR-22 of R protein were diluted with buffer II, and 10 μL of diluted compound, 10 μL of probe solution and 10 μL of protein solution were added to each test well, recorded as mPtest. Two replicate wells were set for each compound concentration, and the experiment set up a blank control (20 μL probe + 40 μL buffer, recorded as mPmin) and a negative control (10 μL protein + 10 μL probe + 10 μL buffer, recorded as mPmax). Cover the plate and shake and incubate for 30 minutes at room temperature, and use a microplate reader to detect the mP value at excitation wavelengths and emission wavelengths of 485 nm and 535 nm, respectively. The IC was calculated using nonlinear fitting in GraphpadPrism8.0 software. 50 value.

[0048] Among them, the structures of PPTN, LR-13 and LR-22 are: .

[0049] The results are as follows Figure 3 As shown in the test results, the IC values ​​of the positive compounds PPTN, LR-13 and LR-22 tested by the FP method were 50 The fluorescence probe compounds of the present invention have fluorescence properties and are effective for P2Y 14 R protein has good specific binding and can be used as a tool molecule to establish P2Y 14 A high-throughput screening method for R protein ligands targeting P2Y 14High-throughput screening of R protein ligand compounds is cheap, stable, rapid and efficient.

Claims

1. A small molecule fluorescent probe compound, characterized in that: The compound is P2Y 14 R is a small molecule fluorescent probe, the structure of which is shown in Formula I: 。 2. A method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) Compound T1 is subjected to a deprotection reaction and then subjected to an amide condensation reaction with N-Boc-γ-aminobutyric acid to obtain intermediate T2; (2) intermediate T2 is subjected to a deprotection reaction and then subjected to an amide condensation reaction with N-(tert-butyloxycarbonyl)-5-aminopentanoic acid to obtain intermediate T3; (3) intermediate T3 is subjected to a deprotection reaction to obtain intermediate T4; (4) The intermediate T4 is subjected to a nucleophilic addition reaction with fluorescein isothiocyanate FITC to obtain a compound of formula I; the general reaction formula is as follows: 。 3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of the compound T1 to N-Boc-γ-aminobutyric acid is 1:1 to 1:

2.

4. The preparation method according to claim 2, characterized in that: In steps (1) and (2), the amide condensation reaction uses one or more of EDCI, HOAT, HATU or BOP as the condensing agent, one or both of DIPEA and TEA as the base, and one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide as the organic solvent.

5. The preparation method according to claim 2, characterized in that: In step (2), the molar ratio of the compound T2 to N-(tert-butyloxycarbonyl)-5-aminopentanoic acid is 1:1 to 1:

2.

6. The preparation method according to claim 2, characterized in that: In step (3), the deprotection reaction uses a base selected from one or more of sodium hydroxide, potassium hydroxide or lithium hydroxide, and a solvent selected from two or more of water, tetrahydrofuran, methanol or ethanol.

7. The preparation method according to claim 2, characterized in that: In step (4), the solvent used in the nucleophilic addition reaction is one or more of tetrahydrofuran, 1,4-dioxane or N,N-dimethylformamide, and the base is one or both of DIPEA and TEA.

8. The preparation method according to claim 2, characterized in that: In step (4), the molar ratio of the compound T4 to fluorescein isothiocyanate is 1:1 to 1:

2.

9. A small molecule fluorescent probe compound as claimed in claim 1 as a fluorescence polarization probe in P2Y 14 Application in R ligand screening.

10. The use according to claim 9, characterized in that: The application is specifically: the compound according to claim 1, P2Y 14 R protein and the test compound are mixed to obtain a mixture; the polarization value of the mixture is measured using fluorescence polarization technology, and the test compound is confirmed to be P2Y according to the polarization value 14 R's ligand.

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