Diagnosis and treatment integrated boron compound for targeting RET positive thyroid cancer and preparation method of diagnosis and treatment integrated boron compound

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a diagnosis and treatment integrated boron compound for targeting RET positive thyroid cancer and a preparation method of the diagnosis and treatment integrated boron compound. According to the present invention, by using the targeting property of Platinib on RET, and the high efficiency and the target accuracy of boron atoms, the selectivity and the targeting property on RET-positive refractory thyroid cancer are significantly improved, and the good antitumor effect is provided. In a cell experiment, the boron compound disclosed by the invention shows a remarkable proliferation inhibition effect on a human thyroid cancer cell line TT (the IC50 value is 5.56 nM). Animal experiment results show that the boron compound can effectively inhibit tumor growth. In addition, in acute and subacute toxicity tests, mice in an administration group do not have obvious poisoning symptoms, which indicates that the boron compound has safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a boron compound for integrated diagnosis and treatment of RET-positive thyroid cancer and a preparation method thereof. Background Art

[0002] BNCT is a promising treatment method that relies on the ability of the boron-10 isotope to capture neutrons and achieve selective destruction of tumor tissues by releasing alpha particles and Li-7 nuclear reaction products. However, effective BNCT requires a specific carrier that can accurately deliver the boron-10 isotope to tumor tissues. Currently, although second-generation boron drugs such as borophenylalanine (BPA) and BSH have been used, they perform poorly in terms of selectivity and targeting between healthy cells and tumor cells, and fail to meet the standards of efficient boron delivery agents. In addition, studies have also shown that these boron drugs have poor metabolic stability in the body and a fast metabolic rate, resulting in insufficient retention time in cells and low bioavailability. During the BNCT process, this type of boron drug relies on transporters to enter cells and lacks specificity in biodistribution for RET-positive thyroid cancer patients, which may damage normal tissues and organs in the body. Therefore, finding a new method that can overcome the limitations of BPA or BSH drugs and use their ability to specifically bind to RET for BNCT to treat refractory thyroid cancer has become a technical problem that needs to be solved urgently.

[0003] Pralsetinib, as a rearranged during transfection kinase (RET) inhibitor, can effectively block RET kinase and curb cancer cell growth. It has been used clinically to treat a variety of solid tumors, such as RET gene fusion-positive locally advanced or metastatic non-small cell lung cancer, advanced or metastatic RET mutant medullary thyroid cancer, and advanced or metastatic RET fusion-positive thyroid cancer. Pralsetinib exhibits efficient and highly selective RET targeting capabilities, and has the advantages of oral convenience, significant and lasting efficacy. However, pralsetinib is also accompanied by significant toxic side effects in clinical practice, and some patients will develop resistance to the drug. Despite this, these advantageous properties of pralsetinib make it meet the requirements as a boron drug carrier. Therefore, the present invention proposes a boron compound that targets RET-positive thyroid cancer with integrated diagnosis and treatment, uses pralsetinib as a specific carrier, introduces boron atoms, and is expected to be used in combination with BNCT to achieve better anti-tumor therapeutic effects. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention provides a boron compound for integrated diagnosis and treatment of RET-positive thyroid cancer and a preparation method thereof.

[0005] The present invention adopts the following technical solution:

[0006] The first object of the present invention is to provide a boron compound for integrated diagnosis and treatment of RET-positive thyroid cancer, the structural formula of the boron compound is as follows:

[0007]

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, comprising the following steps:

[0009] (1) Pratinib, 1H-pyrazole-4-carboxaldehyde and LiClO 4 Add to a three-necked flask equipped with a stirring rod, a carbon felt anode and a platinum wire cathode, and under an inert gas atmosphere, add CH 3 CN, ether and acetic acid, electrocatalytic reaction under light, the reaction solution is separated and purified to obtain intermediate 1;

[0010] The structural formula of the pralatinib and intermediate 1 is as follows:

[0011]

[0012] (2) Add 4-aminophenylboronic acid to a sodium bicarbonate solution, then add intermediate 1, stir to react, and separate and purify the reaction solution to obtain intermediate 2; the structural formula of intermediate 2 is as follows:

[0013]

[0014] (3) adding 5-bromo-3,4-dihydroxybenzaldehyde to a mixed solution of acetonitrile and triethylamine, and then adding sodium undecahydromercaptodecaboride, stirring and reacting under an inert gas atmosphere, and separating and purifying the reaction solution to obtain intermediate 3;

[0015] The structural formula of the intermediate 3 is as follows:

[0016]

[0017] (4) KB 3 H 8 Add tetrahydrofuran, then add HCl in ether solution, stir for 15-20 minutes, and filter to collect the THF-B 3 H 7 2-aminopyridine was added to the filtrate to react with stirring, and the reaction solution was separated and purified to obtain intermediate 4; the structural formula of the intermediate 4 is as follows:

[0018]

[0019] (5) adding intermediate 3 to a sodium bicarbonate solution, then adding intermediate 4, stirring to react, and separating and purifying the reaction solution to obtain intermediate 5; the structural formula of intermediate 5 is as follows:

[0020]

[0021] (6) Mixing the sodium bicarbonate solution containing the intermediate 2 with the sodium bicarbonate solution containing the intermediate 5, stirring to react, and separating and purifying the reaction solution to obtain the boron compound.

[0022] As a preferred technical solution of the present invention, in step (1), prasitinib, 1H-pyrazole-4-carboxaldehyde, LiClO 4 and acetic acid in a molar ratio of 1:(1-2):(6-8)(9-12); the CH 3 The volume ratio of CN to ether is 1:(1-2); the light intensity of the illumination is 23W; and the time of the electrocatalytic reaction is 35-38h.

[0023] As a preferred technical solution of the present invention, the molar ratio of 4-aminophenylboronic acid to intermediate 1 in step (2) is (1-1.5):1; and the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L.

[0024] As a preferred technical solution of the present invention, the molar ratio of 5-bromo-3,4-dihydroxybenzaldehyde, sodium undecahydromercaptododecaboride and triethylamine in step (3) is 1:(1.5-2.0):(30-35).

[0025] As a preferred technical solution of the present invention, the stirring reaction in step (3) is carried out at a temperature of 80-90°C and for a time of 20-24 hours.

[0026] As a preferred technical solution of the present invention, in step (4), KB 3 H 8 The molar ratio of HCl and 2-aminopyridine is 1:(1-1.2):(0.6-0.8); the temperature for adding 2-aminopyridine to the filtrate is -5-0°C; the temperature for stirring the reaction is 25-30°C, and the time is 20-30 minutes.

[0027] As a preferred technical solution of the present invention, the molar ratio of intermediate 3 to intermediate 4 in step (5) is 1:(1-1.2); and the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L.

[0028] As a preferred technical solution of the present invention, the molar ratio of intermediate 2 to intermediate 5 in step (6) is 1:(1-1.2); the sodium bicarbonate solution containing intermediate 2 and the sodium bicarbonate solution containing intermediate 5 are mixed in equal volumes; and the stirring reaction time is (2-4) h.

[0029] The present invention has the following effects compared with the prior art:

[0030] 1. The present invention provides a boron compound, which is prepared from the RET inhibitor pralsetinib and the boron drug sodium undecahydrothiododecaboride as raw materials. By utilizing the targeting of pralsetinib to RET and the high efficiency and target accuracy of boron atoms, the selectivity and targeting of RET-positive refractory thyroid cancer are significantly improved, and it has a good anti-tumor effect. The results of cell experiments showed that the boron compound showed a significant proliferation inhibition effect on the human thyroid cancer cell line TT (IC 50 The value is 5.56nM). Animal experimental results show that boron compounds can effectively inhibit tumor growth.

[0031] 2. The boron compound of the present invention showed low toxicity in acute and subacute toxicity tests, and no obvious poisoning symptoms were observed in the mice in the drug-treated group, which indicates that the boron compound obtained by the present invention is safe and provides a new and effective option for the treatment of RET-positive refractory thyroid cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the growth inhibition curve of the boron compound of the present invention on the human thyroid cancer cell line TT in Experimental Example 1;

[0033] Figure 2 HE staining of important tissues of rats in Experimental Example 3;

[0034] Figure 3 The figure shows the changes in body weight of the four groups of rats in Experimental Example 3. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0036] In the embodiment KB 3 H 8 The preparation method is as follows: 5.85g of potassium metal is cut into pieces of about 3×3×3mm 3 block and compare it with 1MTHF·BH 3The solution (300 mL, 14044-65-6) was reacted for 12 h. After the reaction was completed, KBH 4 Precipitation, removal of THF in the filtrate, and obtaining THF-solvated KB 3 H 8 , add it to toluene to obtain KB 3 H 8 The precipitate was collected by filtration.

[0037] Example 1

[0038] A boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, the structural formula of which is as follows:

[0039]

[0040] This embodiment also provides a method for preparing the above-mentioned boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, comprising the following steps:

[0041] (1) Pralsetinib (1 mmol), 1H-pyrazole-4-carboxaldehyde (CAS: 35344-95-7, 1.5 mmol) and LiClO were added to a three-necked flask equipped with a stirring bar, a carbon felt anode and a platinum wire cathode. 4 (7 mmol), sealed with a rubber septum and paraffin film, flushed with nitrogen for 5 min, and then CH 3 CN (2 mL), ether (2.0 mL) and acetic acid (10 mmol) were added, and the reaction mixture was purged with nitrogen for another 5 min. The solution was stirred under the irradiation of two 23 W CFL bulbs at room temperature, and electrolysis was started at a constant voltage of 2.0 V for 36 h. The reaction mixture was poured into a saturated sodium bicarbonate solution (about 20 mL), and the carbon felt anode was washed with EtOAc (3×5 mL) in an ultrasonic bath. The aqueous layer was separated and extracted with EtOAc (3×10 mL). The combined organic layer and anode extract were washed with brine and dried, and purified by silica gel column chromatography (EtOAc / hexane) to obtain intermediate 1 in 30% yield.

[0042] The specific reaction process is as follows:

[0043]

[0044] The intermediate 1 1 HNMR:(C 31 H 34 FN 11 O 3 ,600MHz,CDCl 3)δ:11.98(s,1H),9.75(s,1H),8.81(s,1H),8.53(s,1H),8.50(s,1H),8.29(s ,1H),8.17-8.15(d,2H),7.95-7.92(m,2H),7.78-7.75(d,1H),6.38(s,1H),6. 30(s,1H),5.83(s,2H),4.99-4.96(q,1H),2.74-2.71(m,1H),2.45(s,3H),2. 32(s,3H),1.94-1.84(m,4H),1.70-1.60(m,4H),1.49-1.46(d,3H); HRMS[M+H] + =C 31 H 35 FN 11 O 3 + : Calculated to be 628.28, found to be 628.28; the above results confirmed that the obtained product was the target product.

[0045] (2) Add 4-aminophenylboronic acid (CAS: 89415-43-0, 12 mmol) to 50 mL of 0.1 M NaHCO 3 The intermediate 1 (10 mmol) was then added to the solution, and the reaction was stirred overnight. The reaction solution was purified by column chromatography to obtain the intermediate 2;

[0046] The specific reaction formula is as follows:

[0047]

[0048] HRMS[M+H] of the above intermediate 2 + =C 37 H 41 BFN 12 O 4 + : Calculated to be 747.34, found to be 747.34; the above results confirmed that the obtained product was the target product.

[0049] (3) 5-bromo-3,4-dihydroxybenzaldehyde (1 mmol) was added to a mixture of acetonitrile (30 mL) and triethylamine (32 mmol), and then sodium undecahydromercaptodecaboride (Na 2 10 B 12 H 12 S, CAS: 103831-41-0, 1.8 mmol), stirred for 22 h under nitrogen atmosphere at 85 °C. After removing the solvent under reduced pressure, the residue was dissolved in H 2 O, with CHCl3 After washing and removing the solvent under reduced pressure, intermediate 3 was obtained.

[0050] The specific reaction formula is as follows:

[0051]

[0052] Intermediate 3 1 HNMR:(C 7 H 16 B 12 Na 2 O 3 S,600MHz,CDCl 3 )δ:9.82(s,1H),9.44(s,1H),8.72(s,1H),6.78-6.75(dd,2H),2.20-2.13(br,11H); HRMS[M+Na] + =C 7 H 16 B 12 O 3 SNa 3 + : Calculated 381.16 Found 381.16; the above results confirmed that the obtained product is the target product.

[0053] (4) KB 3 H 8 (5 mmol) was added to 15 mL THF, and then a 1.0 mol / L HCl solution in ether (5 mL) was added dropwise and stirred for 15 min. Then, the reaction mixture was filtered to remove KCl, and the filtrate was THF·B 3 H 7 2-Aminopyridine (CAS: 504-29-0, 3.5 mmol) was added dropwise to the filtrate at 0°C and stirred at room temperature for 30 min. The reaction solution was concentrated and then purified by column chromatography (PE / DCM=2:1) ​​to obtain intermediate 4.

[0054] The specific reaction formula is as follows:

[0055]

[0056] Intermediate 4 1 HNMR:(C 5 H 13 B 3 N 2 ,600MHz,CDCl 3)δ:9.04(t,1H),8.68(d,1H),8.49-8.46(t,1H),8.46-8.43(d,1H),8.04(s,2H),2.13-2.08(br,7H); HRMS[M+Na] + =C 5 H 13 B 3 N 2 Na + : Calculated 157.14; Found 157.14; The above results confirmed that the obtained product is the target product.

[0057] (5) Add intermediate 3 (10 mmol) to 50 mL of 0.1 M NaHCO 3 To the solution, intermediate 4 (11 mmol) was added, and the mixture was stirred and reacted overnight. The reaction solution was purified by column chromatography to obtain intermediate 5;

[0058] The specific reaction formula is as follows:

[0059]

[0060] Intermediate 5 1 HNMR:(C 12 H 27 B 15 N 2 Na 2 O 2 S,600MHz,CDCl 3 )δ:9.48(s,1H),8.99(s,1H),8.78(s,1H),8.53(d,1H),7.58(t,1H),7.11 (t,1H),7.01(s,1H),6.93-6.90(m,2H),2.15-2.03(br,18H); HRMS[M+Na] + =C 12 H 27 B 15 N 2 Na 3 O 2 S + : Calculated 474.29; Found 474.29; The above results confirmed that the obtained product is the target product.

[0061] (6) Add intermediate 2 (10 mmol) to 50 mL of 0.1 M NaHCO 3 In the solution, intermediate 5 (11 mmol) was added to 50 mL of 0.1 M NaHCO 3In the solution, the two were mixed in equal volumes, stirred at room temperature for 2 hours, and the reaction solution was separated and purified by column chromatography to obtain the final product boron compound.

[0062] The above boron compounds 1 HNMR:(C 49 H 63 B 16 FN 14 O 4 SNa 2 ,600MHz,CDCl 3 )δ:11.93(s,1H),9.01,(s,1H),8.89(s,1H),8.78(s,1H),8.59,(d,1H),8.53(d,1H),8.47(s,1H),8.27 (s,1H),8.15(d,2H),7.94-7.92(m,2H),7.77-7.75(m,3H),7.58(t,1H),7.31(t,1H),7.14-7.11(d,2H) ,6.93-6.86(m,3H),6.38(s,1H),6.30(s,1H),5.83(s,2H),4.99-4.96(q,1H),2.74-2.70(m,1H),2.43( s,3H),2.34(s,3H),2.14-2.04(br,18H),1.95-1.84(m,4H),1.70-1.60(m,4H),1.48(d,3H); HRMS[M+Na] + =C 49 H 63 B 16 FN 14 O 4 SNa 3 + : Calculated as 1207.60; Found as 1207.60; The above results confirm that the obtained product is the target product.

[0063] Example 2

[0064] A boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, whose structural formula is the same as that of Example 1.

[0065] This embodiment also provides a method for preparing the above-mentioned boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, comprising the following steps:

[0066] (1) Pralsetinib (1 mmol), 1H-pyrazole-4-carboxaldehyde (1 mmol) and LiClO were added to a three-necked flask equipped with a stirring bar, a carbon felt anode and a platinum wire cathode. 4(6 mmol), sealed with a rubber septum and paraffin film, flushed with nitrogen for 5 min, and then CH 3 CN (2 mL), ether (3.0 mL) and acetic acid (9 mmol) were added, and the reaction mixture was purged with nitrogen for another 5 min. The solution was stirred under the irradiation of two 23 W CFL bulbs at room temperature, and electrolysis was started at a constant voltage of 2.0 V for 35 h. The reaction mixture was poured into a saturated sodium bicarbonate solution (about 20 mL), and the carbon felt anode was washed with EtOAc (3×5 mL) in an ultrasonic bath. The aqueous layer was separated and extracted with EtOAc (3×10 mL). The combined organic layer and the anode extract were washed with brine and dried, and purified by silica gel column chromatography (EtOAc / hexane) to obtain intermediate 1. Intermediate 1 1 HNMR was consistent with Example 1.

[0067] (2) Add 4-aminophenylboronic acid (10 mmol) to 50 mL of 0.2 M NaHCO 3 Then, intermediate 1 (10 mmol) was added to the solution, and the reaction was stirred overnight. The reaction solution was purified by column chromatography to obtain intermediate 2. The HRMS of intermediate 2 was consistent with that of Example 1.

[0068] (3) 5-Bromo-3,4-dihydroxybenzaldehyde (1 mmol) was added to a mixture of acetonitrile (30 mL) and triethylamine (30 mmol), and then sodium undecahydromercaptodecaboride (1.5 mmol) was added. The mixture was stirred at 80° C. under nitrogen atmosphere for 24 h. After the solvent was removed under reduced pressure, the residue was dissolved in H 2 O, with CHCl 3 After washing and removing the solvent under reduced pressure, intermediate 3 is obtained. 1 HNMR and HRMS were consistent with those in Example 1.

[0069] (4) KB 3 H 8 (5 mmol) was added to 15 mL THF, and then a 1.0 mol / L HCl solution in ether (6 mL) was added dropwise and stirred for 20 min. Then, the reaction mixture was filtered to remove KCl, and the filtrate was THF·B 3 H 7 THF solution. 2-Aminopyridine (3 mmol) was added dropwise to the filtrate at -5°C and stirred at 30°C for 20 min. The reaction solution was concentrated and then purified by column chromatography (PE / DCM = 2:1) to obtain intermediate 4. 1 HNMR and HRMS were consistent with those in Example 1.

[0070] (5) Add intermediate 3 (10 mmol) to 50 mL of 0.1 M NaHCO 3 The intermediate 4 (10 mmol) was added to the solution, and the reaction was stirred overnight. The reaction solution was purified by column chromatography to obtain the intermediate 5. 1 HNMR was consistent with Example 1.

[0071] (6) Add intermediate 2 (10 mmol) to 50 mL of 0.1 M NaHCO 3 In the solution, intermediate 5 (10 mmol) was added to 50 mL of 0.1 M NaHCO 3 The two were mixed in equal volumes and stirred at room temperature for 2 h. The reaction solution was separated and purified by column chromatography to obtain the final product, the boron compound. 1 HNMR and HRMS were consistent with those in Example 1.

[0072] Example 3

[0073] A boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, whose structural formula is the same as that of Example 1.

[0074] This embodiment also provides a method for preparing the above-mentioned boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer, comprising the following steps:

[0075] (1) Pralsetinib (1 mmol), 1H-pyrazole-4-carboxaldehyde (2 mmol) and LiClO were added to a three-necked flask equipped with a stirring bar, a carbon felt anode and a platinum wire cathode. 4 (8 mmol), sealed with a rubber septum and paraffin film, flushed with nitrogen for 5 min, and then CH 3 CN (2 mL), ether (4.0 mL) and acetic acid (12 mmol) were added, and the reaction mixture was purged with nitrogen for another 5 min. The solution was stirred under the irradiation of two 23 W CFL bulbs at room temperature, and electrolysis was started at a constant voltage of 2.0 V for 38 h. The reaction mixture was poured into a saturated sodium bicarbonate solution (about 20 mL), and the carbon felt anode was washed with EtOAc (3×5 mL) in an ultrasonic bath. The aqueous layer was separated and extracted with EtOAc (3×10 mL). The combined organic layer and the anode extract were washed with brine and dried, and purified by silica gel column chromatography (EtOAc / hexane) to obtain intermediate 1. Intermediate 1 1 HNMR was consistent with Example 1.

[0076] (2) Add 4-aminophenylboronic acid (15 mmol) to 55 mL of 0.1 M NaHCO 3Then, intermediate 1 (10 mmol) was added to the solution, and the reaction was stirred overnight. The reaction solution was purified by column chromatography to obtain intermediate 2. The HRMS of intermediate 2 was consistent with that of Example 1.

[0077] (3) 5-Bromo-3,4-dihydroxybenzaldehyde (1 mmol) was added to a mixture of acetonitrile (40 mL) and triethylamine (35 mmol), and then sodium undecahydromercaptodecaboride (2 mmol) was added. The mixture was stirred at 90° C. under nitrogen atmosphere for 20 h. After the solvent was removed under reduced pressure, the residue was dissolved in H 2 O, with CHCl 3 After washing and removing the solvent under reduced pressure, intermediate 3 is obtained. 1 HNMR and HRMS were consistent with those in Example 1.

[0078] (4) KB 3 H 8 (5 mmol) was added to 15 mL THF, and then a 1.0 mol / L HCl solution in ether (6 mL) was added dropwise and stirred for 15 min. Then, the reaction mixture was filtered to remove KCl, and the filtrate was THF·B 3 H 7 2-Aminopyridine (4 mmol) was added dropwise to the filtrate at 0°C and stirred at room temperature for 30 min. The reaction solution was concentrated and then purified by column chromatography (PE / DCM=2:1) ​​to obtain intermediate 4. 1 HNMR and HRMS were consistent with those in Example 1.

[0079] (5) Add intermediate 3 (10 mmol) to 50 mL of 0.1 M NaHCO 3 The intermediate 4 (12 mmol) was added to the solution, and the reaction was stirred overnight. The reaction solution was purified by column chromatography to obtain the intermediate 5. 1 HNMR was consistent with Example 1.

[0080] (6) Add intermediate 2 (10 mmol) to 50 mL of 0.2 M NaHCO 3 In the solution, intermediate 5 (12 mmol) was added to 50 mL of 0.2 M NaHCO 3 The two were mixed in equal volumes and reacted at room temperature for 4 hours under stirring. The reaction solution was separated and purified by column chromatography to obtain the final product, the boron compound. 1 HNMR and HRMS were consistent with those in Example 1.

[0081] Test Example 1

[0082] Human thyroid cancer cell line TT was inoculated into CMEM medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO 2 The cells were cultured under the same conditions and subcultured regularly to maintain the cells in the logarithmic growth phase. 3 TT cells were seeded in a 96-well plate at a density of 100 μL / well, and 100 μL of culture medium was added to each well. After the cells were cultured for 24 hours, different concentrations of the boron compound obtained in Example 1 were added to the experimental group, and an equal volume of physiological saline was added to the control group, with 3 replicate wells set for each group.

[0083] After 72 hours, the culture medium was removed and 100 μL of 10% trichloroacetic acid (TCA) was added to each well to fix the cells overnight at 4°C. The next day, the cells were washed three times with distilled water and dried naturally. Subsequently, 100 μL of 4 mg / mL SRB dye (dissolved in 1% glacial acetic acid) was added to each well and stained for 15 minutes. After staining, the cells were washed five times with 1% glacial acetic acid until no red dye remained and dried naturally again.

[0084] Finally, add 150 μL of 10 mM Tris-base buffer to each well, shake and mix using a VERSAmax microplate reader, and measure the optical density (OD) value at a wavelength of 560 nm. The proliferation inhibition rate (IR) is calculated as follows: (control group OD value - experimental group OD value) / control group OD value × 100%. According to the inhibition rate at each concentration, the half inhibitory concentration (IC 50 ) and plotted the proliferation inhibition curve. The experimental results were repeated 3 times and the average value was taken. The proliferation inhibition curve can be found in Figure 1 .

[0085] from Figure 1 It can be seen that the boron compound obtained in Example 1 has a significant proliferation inhibition on human thyroid cancer cell line TT at a concentration of 4-20nmol / L, IC 50 The value is 5.56nmol / L.

[0086] Test Example 2

[0087] Experimental animals: BALB / c nude mice, 4-6 weeks old, weighing 20±2g, were housed in a constant temperature (25±1°C), sterile environment with regular lighting (12 h light / 12 h dark), and given ample food and water.

[0088] Experimental process: TT cells of human thyroid cancer cell line in logarithmic growth phase were subcutaneously inoculated with 5×10 6 After the transplanted tumor was formed in the mouse and subcultured 1 to 2 times, the rapidly growing tumor tissue was taken and cut into pieces of about 1.5 mm 3 A small uniform piece of the vaccine was subcutaneously inoculated into the right axilla of the mouse.

[0089] Tumor diameter was monitored by vernier calipers when the tumor volume reached 100 to 200 mm 3 At the same time, the mice were randomly divided into a model group, an experimental group (high, medium and low groups of the boron compound obtained in Example 1) and a positive drug group. The boron compound obtained in Example 1 and pratinib were suspended in 1.0% sodium carboxymethyl cellulose to the required concentration and taken orally once a day for 21 days. The model group was gavaged with an equal amount of solvent, the experimental group was gavaged with high (10 mg / kg), medium (5 mg / kg) and low (1 mg / kg) doses of the boron compound obtained in Example 1, and the positive control group was gavaged with 10 mg / kg pratinib.

[0090] The body weight of mice was monitored daily during the experiment, and the tumor diameter was recorded at the beginning of the experiment (d0) and on day 21. The tumor volume (TV) was calculated according to the formula TV = 1 / 2 (a × b) 2 Calculate (a and b are the length and width of the tumor, respectively), and then derive the relative tumor volume (RTV), RTV = V t / V 0 (V 0 is the tumor volume at the beginning of the experiment, V t The tumor volume was measured on the 21st day. The relative tumor proliferation rate T / C (%) was finally calculated = (RTV of the experimental group / RTV of the model group) × 100% to evaluate the efficacy of the drug. The average value of all mice in each group was taken, and the results are shown in Table 1:

[0091] Table 1

[0092]

[0093]

[0094] As can be seen from Table 1, the boron compound prepared by the present invention has a good inhibitory effect on tumor growth in the TT nude mouse transplanted tumor model. After 21 days of administration, the T / C percentages of the high, medium and low dose groups of Example 1 were 43.50%, 47.41% and 53.65%, respectively. The inhibitory effect of the medium dose group was equivalent to that of the positive drug group, and the inhibitory effect of the high dose group was better than that of the positive drug group. In addition, there was no significant difference in the average body weight of mice in each group, indicating that the mice tolerated the dose.

[0095] Test Example 3

[0096] 1. Acute oral toxicity test

[0097] Twenty 8-week-old SPF Kunming rats were used, half male and half female, with a body weight of (100±10) g. All rats were fed an adaptive diet for 7 days before the experiment. The feeding environment conditions were 20-26°C and 40%-70% humidity. The rats were randomly divided into two groups: a control group and a drug-treated group. The two groups of mice were fasted but not watered for 4 hours before the experiment. The maximum tolerated dose method was used. The drug-treated group was given a one-time oral gavage of 20 mL / kg, and the control group was given an equal volume of distilled water. The experiment lasted for 14 days, during which the survival of the rats was observed. After the experiment, the animals were killed, and important organs such as the heart, liver, and kidney were removed. After fixation with 4% paraformaldehyde, the tissues were embedded in paraffin, and the sections were stained with hematoxylin-eosin (HE). The histopathological changes were observed under an optical microscope.

[0098] Results: After a single oral administration of 171 mg / kg of the boron compound suspension obtained in Example 1 (1.0% sodium carboxymethyl cellulose solution), the rats in the drug group performed well, had normal mental state, smooth and neat hair, were active, ate and drank water normally, and had no obvious symptoms of poisoning. No deaths occurred during the 14-day observation period. Compared with the control group, the appearance of the heart, liver, kidney and other organs of the rats in the drug group did not show any abnormality, and there was no significant change in morphology, color and texture. Figure 2 .

[0099] 2. Subacute toxicity test

[0100] 80 8-week-old SPF-grade Kunming rats were used, half of which were male and half were female, and their body weight was (100±10) g. Adaptive feeding was carried out for 7 days before the experiment. The temperature of the breeding environment was 20-26°C, and the humidity was 40%-70%. The rats were randomly divided into a control group, a low-dose group, a medium-dose group, and a high-dose group, with 20 rats in each group, half of which were male and half were female. The low-dose, medium-dose, and high-dose groups were gavaged with 57, 114, and 171 mg / kg of the boron compound suspension obtained in Example 1 (10, 20, and 30 times the clinical dose of pralsetinib for adults), respectively, once a day for 30 consecutive days, and the control group was given an equal volume of distilled water. During the experiment, the general condition of the rats was observed, and the body weight, food intake, and water intake of the rats were recorded weekly. For changes in body weight of the four groups of rats, see Figure 3 .

[0101] After the experiment, all rats were fasted for 16 hours, and were killed by cervical dislocation under anesthesia. Blood was collected to analyze blood indicators (such as red blood cells, hemoglobin, white blood cells, platelets, etc.), and serum biochemical indicators (such as ALB, ALT, AST, Cr, BUN, Glu, etc.) were detected simultaneously. Subsequently, major organs (heart, liver, kidney, stomach, duodenum) were weighed and organ coefficients were calculated. After fixation, histopathological analysis was performed to observe whether there were abnormal changes. Blood indicators and serum biochemical indicators of 4 groups of rats are shown in Tables 2 and 3, and each group of data is the average value of all rats.

[0102] Table 2 Blood parameters of rats in four groups

[0103] Group Hemoglobin (g / L) Neutrophil % Lymphocyte % <![CDATA[Platelets (×10 9 / L)]]> <![CDATA[Red blood cells (×10 12 / L)]]> Control group 133.51 33.45 59.18 737.51 6.72 Low dose group 130.17 30.81 55.36 728.50 6.90 Medium dose group 121.68 28.54 44.23 850.69 7.22 High dose group 109.56 26.21 32.14 894.34 7.02

[0104] Table 3 Serum biochemical parameters of rats in the four groups

[0105] Group ALB(g / L) ALT(U / L) AST(U / L) Cr(umol / L) BUN (mmol / L) Glu (mmol / L) Control group 33.80 37.45 141.94 41.71 11.51 4.78 Low dose group 34.60 39.35 146.53 39.62 14.17 4.77 Medium dose group 36.31 43.35 154.35 40.63 10.12 5.51 High dose group 35.02 52.09 163.42 35.33 10.83 5.25

[0106] from Figure 2 As can be seen from Table 1 and Table 2, there was no abnormal change in the body weight of rats in each dosage group, and there was no abnormal change in the amount of food and water consumed by the rats. The hemoglobin, neutrophil and lymphocyte counts in the high-dose group decreased, and platelets, ALT and AST increased, which is consistent with the common adverse reactions of pralatrex. However, there were no significant abnormal changes in body weight, blood and serum biochemical indicators in the low-dose and medium-dose groups. The above experimental results show that the subacute toxicity of the boron compound of the present invention is low within the dosage range, and no obvious damage is caused to the physiological functions of rats.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A boron compound for integrated diagnosis and treatment of RET-positive thyroid cancer, characterized in that: The structural formula of the boron compound is as follows:

2. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 1, characterized in that: The following steps are involved: (1) Add pralatinib, 1H-pyrazole-4-carboxaldehyde and LiClO4 to a three-necked flask equipped with a stirring rod, a carbon felt anode and a platinum wire cathode, and add CH3CN, ether and acetic acid in sequence under an inert gas atmosphere, and perform an electrocatalytic reaction under light. After separation and purification, the reaction solution obtains intermediate 1; the structural formula of pralatinib and intermediate 1 is as follows: (2) Add 4-aminophenylboronic acid to a sodium bicarbonate solution, then add intermediate 1, stir to react, and separate and purify the reaction solution to obtain intermediate 2; the structural formula of intermediate 2 is as follows: (3) Add 5-bromo-3,4-dihydroxybenzaldehyde to a mixed solution of acetonitrile and triethylamine, then add sodium undecahydromercaptodecaboride, and stir to react under an inert gas atmosphere. After separation and purification, the reaction solution is separated to obtain an intermediate 3; the structural formula of the intermediate 3 is as follows: (4) KB3H8 was added to tetrahydrofuran, and then HCl in ether solution was added, and the mixture was stirred for 15-20 min. The filtrate containing THF-B3H7 was collected by filtration; 2-aminopyridine was added to the filtrate for stirring reaction, and the reaction solution was separated and purified to obtain intermediate 4; the structural formula of intermediate 4 is as follows: (5) adding intermediate 3 to a sodium bicarbonate solution, then adding intermediate 4, stirring to react, and separating and purifying the reaction solution to obtain intermediate 5; The structural formula of the intermediate 5 is as follows: (6) Mixing the sodium bicarbonate solution containing the intermediate 2 with the sodium bicarbonate solution containing the intermediate 5, stirring to react, and separating and purifying the reaction solution to obtain the boron compound.

3. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: In step (1), the molar ratio of prasetinib, 1H-pyrazole-4-carboxaldehyde, LiClO4 and acetic acid is 1:(1-2):(6-8)(9-12); the volume ratio of CH3CN and ether is 1:(1-2); the light intensity of the illumination is 23W; and the time of the electrocatalytic reaction is 35-38h.

4. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: In step (2), the molar ratio of 4-aminophenylboronic acid to intermediate 1 is (1-1.5):1; and the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L.

5. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: In step (3), the molar ratio of 5-bromo-3,4-dihydroxybenzaldehyde, sodium undecahydromercaptododecaboride and triethylamine is 1:(1.5-2.0):(30-35).

6. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: The stirring reaction in step (3) is carried out at a temperature of 80-90° C. and for a time of 20-24 h.

7. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: In step (4), the molar ratio of KB3H8, HCl and 2-aminopyridine is 1:(1-1.2):(0.6-0.8); the temperature for adding 2-aminopyridine to the filtrate is -5-0°C; the temperature for stirring the reaction is 25-30°C and the time is 20-30 min.

8. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: In step (5), the molar ratio of intermediate 3 to intermediate 4 is 1:(1-1.2); the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L.

9. The method for preparing the boron compound for integrated diagnosis and treatment targeting RET-positive thyroid cancer according to claim 2, characterized in that: In step (6), the molar ratio of intermediate 2 to intermediate 5 is 1:(1-1.2); the sodium bicarbonate solution containing intermediate 2 and the sodium bicarbonate solution containing intermediate 5 are mixed in equal volumes; and the stirring reaction time is (2-4) hours.

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